CN116249559A - Anti-idiotype compositions and methods of use thereof - Google Patents

Abstract

The present disclosure provides methods and compositions comprising polynucleotides, including nucleic acids encoding anti-idiotype polypeptides, as well as polypeptides encoded by the polynucleotides and cells comprising and expressing the polypeptides. The disclosed methods include methods of utilizing an anti-idiotype polypeptide as a safety switch when the anti-idiotype polypeptide is used in combination with an antibody, including approved biological antibodies that include a recognized idiotype. Certain embodiments include an anti-idiotype polypeptide and a nucleotide encoding the anti-idiotype polypeptide, the nucleotide comprising an internal domain. In some embodiments, this internal domain has a functional domain that can induce proliferation or cell death upon binding of the anti-idiotype polypeptide to its target antibody.

Description

Anti-idiotype compositions and methods of use thereof

Cross reference to related applications

The present application claims the following priorities: international application No. PCT/US2021/020922, filed on 3/4 of 2021; international application No. PCT/US2020/048843 filed on 8/31/2020; U.S. provisional application No. 63/200,329, filed on 3/1/2021; and U.S. provisional application No. 63/136,177 filed on day 1 and 11 of 2021; international application PCT/US2021/020922 filed on 3/4/2021 requires U.S. provisional application No. 62/985,741 filed on 5/3/2020; international application No. PCT/US2020/048843 filed on 8/31/2020; U.S. provisional application No. 63/136,177 filed on day 1 and 11 of 2021; and priority of U.S. provisional application No. 63/200,329 filed on 3/1/2021; PCT/US2020/048843 filed on 31 th month 2020 is part of the continuation application of PCT/US2019/049259 filed on 2 nd month 9 of 2019; and U.S. provisional application No. 62/894,849 filed on 1 month 9 of 2019; U.S. provisional application No. 62/894,852 filed on 1/9/2019; U.S. provisional application No. 62/894,853 filed on 1 month 9 of 2019; U.S. provisional application No. 62/894,926, filed on day 2 of 9 in 2019; U.S. provisional application No. 62/943,207 filed on 12/3/2019; and U.S. provisional application No. 62/985,741 filed 3/5 in 2020; international application No. PCT/US2019/049259 is part of the continuation application of international application No. PCT/US2018/051392 filed on date 17 of 2018, 9; and U.S. provisional application No. 62/726,293 filed on 2 days 9 and 2018; U.S. provisional application No. 62/726,294 filed on day 2 of 9 in 2018; U.S. provisional application Ser. No. 62/728,056, filed on 6/9/2018; U.S. provisional application No. 62/732,528 filed on day 17 of 9 in 2018; U.S. provisional application No. 62/821,434 filed on 3/20 in 2019; and U.S. provisional application No. 62/894,853 filed on day 1 of 9 in 2019; international application PCT/US2018/051392 is part of the continuation application of international application PCT/US2018/020818 filed on 3 days of 2018; and U.S. provisional application No. 62/560,176 filed on 18 of 9 of 2017; U.S. provisional application Ser. No. 62/564,253 filed on 27/9/2017; U.S. provisional application Ser. No. 62/564,991 filed on 28 th 9 th 2017; and U.S. provisional application No. 62/728,056 filed on 6 of 9.2018; international application PCT/US2018/020818 is part of the continuation application of international application PCT/US 2017/02112 filed on day 19 of 3/2017; part of the international application PCT/US 2017/04277 filed on 7, 8, 2017; part of U.S. application Ser. No. 15/462,855, filed on day 19 of 3.2017, continues to apply; part of the continuation-in-process application of U.S. application Ser. No. 15/644,778 filed on 7/8 of 2017; and require U.S. provisional application No. 62/467,039 filed on 3 months and 3 days of 2017; U.S. provisional application No. 62/560,176 filed on 18/9/2017; U.S. provisional application Ser. No. 62/564,253 filed on 27/9/2017; and U.S. provisional application No. 62/564,991 filed on 28 of 9 of 2017; international application No. PCT/US 2017/02112 claims U.S. provisional application No. 62/390,093 filed on day 2016, month 3, and day 19; U.S. provisional application No. 62/360,041 filed on 7/8 of 2016; and U.S. provisional application No. 62/467,039 filed 3/2017; international application PCT/US 2017/04277 claims international application PCT/US 2017/02112 filed on 19 days of 3 months of 2017; U.S. patent application Ser. No. 15/462,855, filed on 3/19/2017; U.S. provisional application No. 62/360,041 filed on 7/8 of 2016; and U.S. provisional application No. 62/467,039 filed on 3 months of 2017; U.S. application Ser. No. 15/462,855 claims U.S. provisional application Ser. No. 62/390,093 filed on day 2016, month 3; U.S. provisional application No. 62/360,041 filed on 7/8 of 2016; and U.S. provisional application No. 62/467,039 filed on 3 months of 2017; U.S. application Ser. No. 15/644,778 is part of the continued application of International application No. PCT/US 2017/02112 filed on date 19 at 3 of 2017; and U.S. patent application Ser. No. 15/462,855 filed on day 19 of 3.2017; and claims the benefits of U.S. provisional application No. 62/360,041 filed on day 2016, 7, 8 and U.S. provisional application No. 62/467,039 filed on day 3, 2017. These applications are incorporated herein by reference in their entirety.

Sequence listing

The present application incorporates by reference the materials of the electronic sequence listing filed with the present application. The material in the electronic sequence listing was submitted in the form of a text (.txt) file (whose file size is 729 KB) created at month 8 and 31 of 2021, titled "f1_007_wo_01_sequence_listing" and incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to the field of immunology or more specifically to genetic modification of T lymphocytes or other immune cells, and methods of controlling proliferation of such cells.

Background

Lymphocytes from a subject (e.g., a patient) can be genetically modified ex vivo or in vivo to express synthetic proteins that are capable of repositioning binding to other cells and the environment based on the incorporated genetic program. Examples of such synthetic proteins include engineered T Cell Receptors (TCRs) and Chimeric Antigen Receptors (CARs). Such lymphocytes have become an important therapy (e.g., CAR-T therapy) for the treatment of diseases such as cancer.

While the efficacy of some cell therapies is attractive, even curative, for some patients and some cancers, safety remains a concern for therapies using genetically modified cells such as lymphocytes. For example, there is a risk that genetically modified cells introduced into a subject may become oncogenic, for example, due to the insertion site of a transgene within the target cell genome. For CAR-T, a problem is its safety, for example because CAR-T cells can bind their target (i.e., on-target, off-target) at sites other than the tumor, thus leading to undesirable side effects. In addition, on-target and on-tumor (on-tumor) toxicity may be generated, such as tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome. Some adverse events of CAR-T cells may also include pain and/or dangerous neurotoxicity, including IEC-related neurotoxicity syndrome (ICANS).

Accordingly, genetic safety switches have been developed in an attempt to address these safety issues by providing a method of killing genetically modified cells in patients. However, these safety switches are very time consuming to develop and have limited design flexibility. For example, some safety switches require modification of the natural target against approved antibody biologics that are still capable of specifically binding antibodies. For example, the previously disclosed safety switches include cell surface molecules that are truncated tyrosine kinase receptors. In some of these examples, the truncated tyrosine kinase receptor is a member of the Epidermal Growth Factor Receptor (EGFR) family (e.g., erbB1 (HER 1), erbB2, erbB3, and ErbB 4), for example as disclosed in us patent 8,802,374 or WO 2018226897. Thus, some safety switches are polypeptides recognized by antibodies that recognize the extracellular domain of an EGFR member. Such truncated EGFR polypeptides are sometimes referred to herein as etags. For example, by

Mediated Antibody Dependent Cellular Cytotoxicity (ADCC) pathways, the eTAG was demonstrated to have cell killing potential.

Current safety switches have a number of drawbacks, and they fail to address several long-term challenges of cell therapy. One disadvantage of etags is that they include EGFR family member domains and are therefore structurally limited to EGFR domains only. Furthermore, they may produce adverse side effects when delivered to a subject because they are structurally related to growth factors (e.g., EGFR) found in the subject. Thus, there remains a need for a more flexible system to generate a safety switch that can utilize nearly any antibody (including nearly any approved antibody biologic), can be constructed using established techniques, and does not require the delivery of modified growth factor receptor extracellular domains. Furthermore, there remains a long-standing challenge in cell therapies that use genetically modified cells such as CAR-T to provide a flexible and relatively easy to develop genetic tool that can be used to modify almost any activity of a cell, including but not limited to inducing proliferation or cell death of a genetically modified cell.

Disclosure of Invention

Provided herein are methods, uses, compositions, and kits that help overcome problems associated with the effectiveness and safety of prior art safety switches and methods for performing cell therapies. The anti-idiotype polypeptide safety switches provided herein are more robust and flexible in design and development than prior art safety switches such as eTag. The anti-idiotype polypeptide safety switch can be designed to be recognized by virtually any antibody, including any clinical antibody such as a therapeutic antibody. Exemplary methods for developing anti-idiotype antibodies are known.

Thus, in some aspects, provided herein are methods, compositions, and kits for or comprising or encoding anti-idiotype polypeptides. Further, provided herein are methods for delivering modified lymphocytes (particularly modified and genetically modified T cells and/or NK cells) and/or for modulating transduced, genetically modified and/or modified T cells and/or NK cells. Such methods, compositions, and kits provide improved efficacy and safety compared to current technology, particularly compared to T cells and/or NK cells that express lymphoproliferative elements (e.g., chimeric cytokine receptors), engineered T Cell Receptors (TCRs), and Chimeric Antigen Receptors (CARs), including micro-environmental limited organisms ("MRB") CARs. In illustrative embodiments of delivery from a retroviral (e.g., lentiviral) genome via a retroviral (e.g., lentiviral) particle, transduced and/or modified and in illustrative embodiments genetically modified T cells and/or NK cells produced by and/or used in the methods provided herein include a combination of typical anti-idiotype and other functional groups. Such cells, alone or in combination with other functional groups, anti-idiotype polypeptides or polynucleotides encoding the polypeptides provide improved characteristics (including safety features) and methods of using such cells, such as research methods, commercial production methods, and adoptive cell therapies. For example, if such cells become a safety issue for the subject delivering them, such cells may be controllably killed, and may have improved growth characteristics that may be better regulated.

In one aspect, provided herein is a polynucleotide comprising one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter, wherein the one or more transcriptional units encode a polypeptide comprising:

a) Polynucleotide sequences encoding one or more inhibitory RNA molecules and/or a first engineered signaling polypeptide, and

b) A polynucleotide sequence encoding an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane associated domain, wherein the extracellular recognition domain comprises a domain that recognizes the idiotype of a target antibody or a target antibody mimetic.

In one aspect, provided herein is a method for administering a cell therapy to a mammalian subject, the method comprising administering modified cells to the mammalian subject, wherein the modified cells each comprise a polynucleotide encoding: a) One or more inhibitory RNA molecules and/or a first engineered polypeptide, and b) an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or a target antibody mimetic.

In one aspect, provided herein is a method for administering gene therapy to a mammalian subject, the method comprising administering to the mammalian subject a gene vector, wherein the gene vectors each comprise a polynucleotide encoding: a) One or more inhibitory RNA molecules and/or a first engineered polypeptide, and b) an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or a target antibody mimetic.

In some embodiments, the anti-idiotype polypeptide is capable of binding to the idiotype of a clinically-approved target antibody or antibody mimetic. Such target antibodies or antibody mimics may be capable of, adapted to, configured to, and/or effective in promoting cell death.

In some embodiments, the anti-idiotype polypeptide further comprises one or more intracellular domains. The intracellular signaling domain may activate or inhibit pro-apoptotic or anti-apoptotic pathways and/or pro-survival or anti-survival pathways.

Additional details regarding aspects and embodiments of the present disclosure are provided throughout the present patent application. Chapters and chapter titles are for ease of reading and are not intended to limit the combinations of the present disclosure, such as methods, compositions, and kits, or functional elements therein, throughout the chapters.

Drawings

FIG. 1 shows a non-limiting example of a polynucleotide that is part of a lentiviral vector, wherein the nucleic acid encoding an anti-idiotype polypeptide comprises an anti-idiotype extracellular recognition domain ("anti-idiotype"), a stem, and a Membrane Association Domain (MAD). The vector also includes a promoter ("promoter") positioned to drive expression of nucleic acids encoding different functional polypeptides, including anti-idiotype polypeptides, and includes the various functional domains shown, including various viral functional domains, and encodes an inhibitory RNA molecule within the promoter.

Figures 2A-G show non-limiting examples of polynucleotides comprising nucleic acids encoding one or more anti-idiotype polypeptides in various combinations with other polypeptides. FIG. 2A shows nucleic acids encoding anti-idiotype polypeptides having a Membrane Associated Domain (MAD) and an anti-idiotype extracellular recognition domain (referred to as an "anti-idiotype" in FIG. 2). Figures 2B-D show anti-idiotype polypeptides expressed as part of a single polynucleotide that also encodes a Chimeric Antigen Receptor (CAR) or an engineered T Cell Receptor (TCR) (figure 2B), a lymphoproliferative element (figure 2C), or a cytokine (figure 2D). Figures 2E-G show expression of an anti-idiotype polypeptide as part of a single polynucleotide that also encodes a CAR or TCR, an anti-idiotype polypeptide, and a lymphoproliferative element (figure 2E) or a cytokine (figure 2F) or both a lymphoproliferative element and a cytokine (figure 2G).

Figures 3A-G show non-limiting examples of anti-idiotype polypeptides. FIG. 3A shows an anti-idiotype polypeptide having an extracellular recognition domain (sometimes referred to as an anti-idiotype extracellular recognition domain, an anti-id ERD, or an anti-id ECD, and denoted as "anti-idiotype" in FIGS. 3A-F and 3H), a handle, and a Membrane Association Domain (MAD). Figures 3B-3G show anti-idiotype polypeptides having one or more intracellular domains (sometimes referred to herein as ICD) and a transmembrane domain ("TM" in figures 3B-3G). FIG. 3B shows an anti-idiotype polypeptide having a single intracellular domain. Figures 3C-E show anti-idiotype polypeptides having a P3 domain of a lymphoproliferative element and optionally a P4 domain (figure 3C) or an intracellular domain having a CAR or TCR (figure 3D) or an intracellular domain having a death domain. Figure 3F shows an anti-idiotype polypeptide with anti-id ERD as one of the ASTRs of a bispecific CAR or TCR. FIG. 3G shows an anti-idiotype polypeptide similar to FIG. 3B but having a cleavage site between or as part of the transmembrane domain and ICD.

FIG. 4 shows a non-limiting example of an anti-idiotype polypeptide.

FIGS. 5A and 5B show schematic diagrams of illustrative bicistronic lentiviral genome vectors with divergent transcription units.

FIG. 6A shows the use of Dynabeads ^TM Protein a was subjected to the screening rounds of screening experiment 1, phage input (pfu), bead volume, binding time, number of washes, phage output (pfu) recovery and enrichment.

FIG. 6B shows the use of Dynabeads ^TM M-270 epoxy [/screening round number of screening experiment 1 performed, phage input (pfu), bead volume, binding time, number of washes, phage output (pfu) recovery and enrichment

Figure 7 shows binding of phage output to cetuximab from rounds 1-5 of screening experiment 1 as measured by ELISA. In each screening round, elution buffer was used from Dynabeads ^TM Phage were eluted in M-270 epoxy, except for R5 indicated, where the bound phage was eluted with EGFR.

Figure 8 shows the sequence information of 19 unique clones identified in screening experiment 1 using ab1.1 as bait.

FIGS. 9A-9C show sequence information for 42 unique clones identified in screening experiment 2 using Ab1.2 as a bait

Figure 10 shows the ELISA results of competitive binding assays in which soluble EGFR competes with monoclonal phage clones from screening experiment 2 for binding to cetuximab.

FIG. 11 shows a schematic diagram of an illustrative lentiviral genome vector having a single transcriptional unit encoding a CAR and an anti-idiotype polypeptide separated by a ribosome-hopping sequence.

FIG. 12 shows the expression of 2 different cetuximab anti-idiotype polypeptides on 293T and CHO-S cells as detected by cetuximab and assayed by FAC.

Definition of the definition

As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to engineered receptors that specifically transplant antigen onto cells, such as T cells, NK cells, macrophages and stem cells. The CARs of the invention include at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an Intracellular Activation Domain (IAD), and may include a handle and one or more co-stimulatory domains (CSD). In another embodiment, the CAR is a bispecific CAR that is specific for two different antigens or epitopes. IAD activates intracellular signaling after ASTR specifically binds to the antigen of interest. For example, IAD can redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner, taking advantage of the antigen binding properties of antibodies. non-MHC-restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with endogenous T Cell Receptor (TCR) alpha and beta chains.

As used herein, the term "aggregate" of cells refers to a cluster of cells that adhere to each other.

As used herein, the term "constitutive T cell or NK cell promoter" refers to a promoter that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

As used herein, the term "inducible promoter" or "active promoter" refers to a promoter that, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell substantially only when a promoter-specific inducer is present in the cell. Inducible promoters have no basal transcriptional activity or have a lower level of basal transcriptional activity, but in the presence of an induction signal, transcriptional activity increases, sometimes dramatically.

As used herein, the term "insulator" refers to a cis-regulatory element that mediates intra-and inter-chromosomal interactions and can block interactions between enhancers and promoters. Typically, the spacers are 200 to 2000 base pairs in length and comprise an aggregation binding site for a sequence specific DNA binding protein.

As used herein, the term "microenvironment" means any portion or region of a tissue or body that has a constant or temporal, physical or chemical difference from other tissue or body regions. For example, a "tumor microenvironment" as used herein refers to an environment in which a tumor is present, which is a non-cellular region within the tumor and a region that is located just outside the tumor tissue, but is not associated with the intracellular compartment of the cancer cell itself. A tumor microenvironment may refer to any and all conditions of a tumor environment, including conditions that create a structural and/or functional environment for the exacerbation process to survive and/or amplify and/or spread. For example, a tumor microenvironment may include a change in conditions such as (but not limited to): pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronic acid, concentration of lactate, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidizing agents, reducing agents or cofactors, among other conditions as will be understood by those of skill in the art.

As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides of any length (ribonucleotides or deoxyribonucleotides). Thus, this term includes (but is not limited to): single, double or stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified non-natural or derivatized nucleotide bases.

As used herein, an "approved biologic" is a macromolecule that meets the requirements of a biologic provided by a government regulatory agency, such as, but not limited to, the United States Food and Drug Administration (USFDA), the European Medicines Agency (EMA), the chinese national medicines agency (NMHA) (the chinese FDA), or the japanese medicines and food safety agency (PFSB), and has obtained approval by such regulatory agency as a stand-alone biologic or as part of a combination product or process.

As used herein, the term "antibody" includes polyclonal and monoclonal antibodies, including intact antibodies as well as antibody fragments that remain specifically bound to an antigen. Antibody fragments may be (but are not limited to): fragment antigen binding fragment (Fab) fragment, fab 'fragment, F (ab') ₂ Fragments, fv fragments, fab '-SH fragments, (Fab') ₂ Fv fragments, fd fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), bivalent scFv, trivalent scFv, and single domain antibody fragments (e.g., sdabs, sdFv, nanobodies). The term includes genetically engineered and/or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, single chain antibodies, fully human antibodies, humanized antibodies, antigens including antibodies and non-antibody proteinsFusion proteins of specific targeting regions, heteroconjugate antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies), tandem di-scFv, and tandem tri-scFv. The term "antibody" is understood to include functional antibody fragments thereof, unless otherwise indicated. The term also includes whole or full length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, igM, igE, igA, and IgD.

As used herein, the term "antibody fragment" includes a portion of an intact antibody, e.g., the antigen binding or variable regions of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') ₂ Fv fragments; a bifunctional antibody; linear antibodies (Zapata et al, protein engineering (Protein Eng.))) 8 (10): 1057-1062 (1995); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments (referred to as "Fab" fragments, each with a single antigen binding site) and a residual "Fe" fragment (an indication reflecting the ability to crystallize readily). Pepsin treatment to produce F (ab') ₂ Fragments that have two antigen combining sites and are still capable of cross-linking antigens.

As used interchangeably herein, the term "single chain Fv", "scFv" or "sFv" antibody fragment includes V of an antibody _H Domain and V _L Domains, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises at V _H Domain and V _L Polypeptide linkers or spacers between the domains that enable sFv to form the desired structure for antigen binding. For reviews of sFvs, see monoclonal antibody pharmacology, stationary multivalent molecules (Pluckthun in The Pharmacology of Monoclonal Antibodies), volume 113, rosenburg and Moore editions, springer-Verlag, new York, pages 269-315 (1994).

As used herein, a "naturally occurring" VH domain and VL domain refers to VH and VL domains that have been isolated from a host without further molecular evolution to alter their affinity when produced in scFv format under specific conditions, as disclosed in U.S. patent 8709755B2 and application WO/2016/033331 A1.

As used herein, an "antibody mimetic" refers to an organic compound that specifically binds to a target sequence and has a structure that differs from a naturally occurring antibody. Antibody mimics may include proteins, nucleic acids, or small molecules, and the skilled artisan will understand when each type is relevant. The target sequence to which the antibodies of the present disclosure mimic specifically bind may be an antigen. Antibody mimetics can provide superior performance over antibodies, including but not limited to superior solubility, tissue penetration, stability to heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs. Antibody mimetics include, but are not limited to, affibodies (affils), affimers (affimers), affibodies (affiins), alpha bodies (alphabodies), alphamabs, anti-cargo proteins (anti-calins), peptide aptamers, armadillo repeat proteins, trimers, affimers (avimers) (also known as avimers), C-lectin domains, cysteine-binding miniproteins, cyclic peptides, cytotoxic T lymphocyte-associated protein-4, DARPin (designed ankyrin repeat protein (Designed Ankyrin Repeat Protein)), fibrinogen domains, fibronectin binding domains (FN 3 domains) (e.g., attachment proteins or monoclonal antibodies (monoclonal)), fynomers, kinking bacteria (knottins), kunitz domain peptides, nanofiltins, leucine-rich repeat domains, lipocalin domains, mAb 2 or Fcab ^TM Nanobodies, nanomembers, OBody, pronectin, single chain TCR, triangular tetrapeptide repeat domains (tetratricopeptide repeat domain), VHH or V-like domains.

As used herein, "complementarity determining regions" or "CDRs" refer to three hypervariable regions of four "framework" regions that interrupt the chain in each variable chain of an immunoglobulin and a T cell receptor. CDRs are mainly responsible for the specificity of binding. The CDRs of each immunoglobulin chain are called CDR1, CDR2 and CDR3, which are numbered sequentially starting from the N-terminus and are also typically identified by the chain in which the particular CDR is located. Thus, HCDR3 is located in the variable domain of the heavy chain of the antibody for which it was found, while LCDRl is CDR1 from the variable domain of the light chain of the antibody for which it was found. The sequences of the framework regions of the different light or heavy chains are relatively conserved in the species. The framework regions of antibodies, i.e., the combined framework regions that make up the light and heavy chains, are used to position and align CDRs in three-dimensional space. The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, such as Kabat, chothia, international ImmunoGenetics database (IMGT) and AbM (see, e.g., johnson and Wu, nucleic Acids Res.) (1 st. 2000; 28 (1): 214-218 and Johnson et al, nucleic Acids Res.) (29:205-206 (2001)), chothia & Lesk, (1987) journal of molecular biology (J. Mol. Biol.) (196,901-917), chothia et al (1989) Nature (Nature)) (342,877-883), chothia et al (1992) journal of molecular biology (J. Mol. Biol.) (227,799-817), al-Lazikam et al, (J. Mol. Biol.) (1997)) and (1997). Unless otherwise indicated, the CDRs herein are defined at vbase2.Org on the world Wide Web using "Fab Analysis" (Retter et al, nucleic Acids Res.) (33: D671-D674 and Mollova et al, BMC Systems Biol.) (S1, p 30).

As used herein, the term "idiotype" refers to a segment of an antibody or antibody mimetic that determines its antigen specificity, e.g., the structure of a variable region of an antibody, T cell receptor, or antibody mimetic that is a common feature between a set of antibodies, T cell receptors, or antibody mimetics, based on the antigen binding specificity and thus the structure of the variable region. The idiotype of an antibody typically includes variable regions, such as CDRs and framework regions. For antibodies, the idiotype is located in the Fab region. For antibodies formed with multiple chains, such as heavy and light chains, expression of the idiotype typically requires the involvement of variable regions of the heavy and light chains that form the antigen combining site. For antibodies formed with single chains, such as scFv, expression of the idiotype typically requires the participation of the variable region of one polypeptide that forms the antigen combining site. For antibody mimetics, the idiotype varies depending on the type of antibody mimetic, but includes the regions necessary to bind the cognate antigen.

As used herein, a "therapeutic antibody" or "therapeutic antibody mimetic" is an antibody or antibody mimetic that has been demonstrated to have therapeutic activity using in vivo assays, e.g., in humans.

As used herein, the term "recognition" refers to the ability of one molecule to bind to another molecule, such as the ability of a receptor to bind to its ligand or the ability of an antibody to bind to its target.

As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two agents, and is expressed as the dissociation constant (Kd). The affinity may be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold or more than the affinity of the antibody for the unrelated amino acid sequence. The affinity of the antibody for the protein of interest may be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomole (fM) or higher. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. With respect to antibodies and/or antigen binding fragments, the term "immunoreactive" and "preferential binding" are used interchangeably herein.

As used herein, the term "binding" refers to the direct association between two molecules due to, for example, covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic and/or hydrogen bonding interactions, including interactions such as salt and water bridges. Nonspecific binding refers to affinities of less than about 10 ^-7 Binding of M, e.g. with affinity less than 10 ^-6 M、10 ^-5 M、10 ^-4 M, etc.

As used herein, reference to "cell surface expression system (cell surface expression system)" or "cell surface presentation system (cell surface display system)" refers to the presentation or expression of a protein or portion thereof on the surface of a cell. Typically, cells are produced that express the relevant protein fused to a cell surface protein. For example, a protein is expressed as a fusion protein with a transmembrane domain.

As used herein, the term "element" includes polypeptides (including fusions of polypeptides, regions of polypeptides, and functional mutants or fragments thereof) and polynucleotides (including micrornas and shrnas, and functional mutants or fragments thereof).

As used herein, the term "region" is any segment of a polypeptide or polynucleotide.

As used herein, a "domain" is a region of a polypeptide or polynucleotide having functional and/or structural properties.

As used herein, the term "handle" or "handle domain" refers to a flexible polypeptide linking region that provides structural flexibility and is spaced apart from flanking polypeptide regions, and may consist of a natural or synthetic polypeptide. The handle may be derived from a hinge or hinge region of an immunoglobulin (e.g., igGl), which is generally defined as stretching from Glu216 to Pro230 (Burton (1985) molecular immunology (molecular. Immunol.), 22:161-206) of human IgGl. The hinge region of other IgG idiotypes can be aligned with the IgG1 sequence by allowing the first cysteine residue to form an inter-heavy chain disulfide bond (S-S) at the same position as the last cysteine residue. The handle may be naturally occurring or non-naturally occurring, including but not limited to an altered hinge region, as disclosed in U.S. patent No. 5,677,425. The handle may include a complete hinge region derived from antibodies of any class or subclass. The handle may also include regions derived from CD8, CD28 or other receptors that provide flexibility and similar function in spacing from the flanking regions.

As used herein, the term "isolated" means that the material is removed from its original environment (e.g., from the natural environment when it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector, and/or such polynucleotides or polypeptides may be part of a composition, and still be isolated, as such vector or composition is not part of its natural environment.

As used herein, a "polypeptide" is a single chain amino acid residue joined by a peptide bond. The polypeptide is neither folded into a fixed structure nor has any post-translational modifications. A "protein" is a polypeptide that is folded into a fixed structure. "polypeptide" and "protein" are used interchangeably herein.

As used herein, a polypeptide may be "purified" to remove impurity components of the natural environment of the polypeptide, e.g., materials that would interfere with diagnostic or therapeutic uses of the polypeptide, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The polypeptide may be purified to (1) greater than 90%, greater than 95% or greater than 98%, e.g., greater than 99% by weight, based on the weight of the antibody as determined by the Lawsry method, (2) to an extent sufficient to obtain at least 15N-terminal or internal amino acid sequence residues using a rotary cup sequencer, or (3) homogenized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining.

As used herein, the term "immune cells" generally includes white blood cells (leukocytes) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow. "immune cells" include, for example, lymphocytes (T cells, B cells, natural Killer (NK) cells), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

As used herein, "T cells" include all types of immune cells that express CD3, including helper T cells (CD 4 ⁺ Cells), cytotoxic T cells (CD 8) ⁺ Cells), regulatory T cells (tregs), and gamma-delta T cells. NKT cells that are cd3+, cd56+, and cd4+ or cd8+ are considered to be a type of T cell herein. Surface expression of CD3 can be transiently reduced or eliminated in T cells, as observed with some methods for modifying T cells disclosed herein. In the present disclosure, such modified cd4+ or cd8+ showers with transiently reduced/absent CD3 surface expressionThe babcells are still considered T cells. References herein to "CD" or clusters of differentiation markers (e.g., cd3+, cd4+, cd8+, cd56+) relate to surface expression of such polypeptides. It should be understood that surface expression is a continuum between positive and negative, and can be assessed by FACS analysis, wherein cells are determined to be positive and negative based on user cutoff as known in the art. Low and moderate expression of surface markers (e.g. CD3lo or CD3 int) as determined by FACS analysis is herein considered negative for surface markers (e.g. CD 3-).

As used herein, "NK cells" include lymphocytes that express CD56 on their surface (cd56+ lymphocytes). NKT cells that are cd3+, cd56+, and cd4+ or cd8+ are considered as types of NK cells herein.

As used herein, "cytotoxic cells" include CD8 ⁺ T cells, natural Killer (NK) cells, NK-T cells, gamma delta T cells (a CD4 ⁺ A subpopulation of cells) and neutrophils (which are cells capable of mediating a cytotoxic response).

As used herein, the term "stem cell" generally includes differentiated pluripotent or multipotent stem cells. "Stem cells" include, for example, embryonic stem cells (ES); mesenchymal Stem Cells (MSCs); induced differentiation pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSCs), bone marrow derived cells, etc.).

As used herein, the term "treatment" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (e.g., in a human), and includes: (a) Preventing the occurrence of a disease in a subject susceptible to the disease but not yet diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

As used herein interchangeably, the terms "individual," "subject," "host," and "patient" refer to a mammal, including, but not limited to, humans, rats (e.g., rats, mice), rabbits (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an agent or a combination of two agents sufficient to affect such treatment of a disease when administered to a mammal or other subject for treating the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, the age, weight, etc., of the subject being treated.

As used herein, the term "evolution" refers to the use of one or more mutation methods to produce different polynucleotides encoding different polypeptides that are themselves improved biomolecules and/or that contribute to the production of another improved biomolecule. "physiological" or "normal physiological" conditions are conditions such as (but not limited to) the following: pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronic acid, concentration of lactate, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of an oxidizing agent, reducing agent, or cofactor, as well as other conditions that would be considered normal for a subject at the site of administration or at a tissue or organ at the site of action.

As used herein, a "transduced cell" or a "stably transfected cell" is a cell that contains exogenous nucleic acid integrated into the genome of the cell. As used herein, a "genetically modified cell" is a cell that contains an exogenous nucleic acid, regardless of whether the exogenous nucleic acid is integrated into the genome of the cell, and regardless of the method used to introduce the exogenous nucleic acid into the cell. An exogenous nucleic acid within a cell that is not integrated into the genome of the cell may be referred to herein as "extrachromosomal". As used herein, a "modified cell" is a cell associated with a recombinant nucleic acid vector (also referred to herein as a "polynucleotide vector" or "gene vector"), which in an illustrative embodiment is a replication defective recombinant retroviral particle (also referred to herein as a "RIR retroviral particle" or "RIP") containing an exogenous nucleic acid, or a cell that has been modified by an exogenous nucleic acid gene. In general, in compositions and methods comprising replication defective recombinant retroviral particles, modified cells associate with replication defective recombinant retroviral particles through interactions between proteins on the surface of the cells and proteins on the surface of the replication defective recombinant retroviral particles (including pseudotyped elements and/or T cell activating elements). In compositions and methods involving transfection of nucleic acids in lipid-based reagents, such as lipid plasmid reagents, nucleic acid-containing lipid-based reagents (which are a type of recombinant nucleic acid vector) are associated with lipid bilayers of modified cells prior to fusion or internalization of the modified cells. Similarly, in compositions and methods involving transfection of chemical-based nucleic acids (e.g., polyethyleneimine (PEI) or calcium phosphate-based transfection), the nucleic acids are typically associated with positively charged transfection reagents to form recombinant nucleic acid vectors that are associated with negatively charged membranes of the modified cells prior to internalization of the complex by the modified cells. Other means or methods of stably transfecting or genetically modifying cells include electroporation, ballistic delivery, and microinjection. As used herein, a "polypeptide" may include a partial or complete protein molecule of a protein molecule, and any post-translational or other modifications.

Pseudotyped elements as used herein can include "binding polypeptides" that include one or more polypeptides (typically glycoproteins) that recognize and bind to a host cell of interest, and one or more "fusogenic polypeptides" that mediate fusion of a retrovirus with a cell membrane of a host cell of interest, thereby allowing the retroviral genome to enter the host cell of interest. As used herein, a "binding polypeptide" may also be referred to as a "T cell and/or NK cell binding polypeptide" or "target engagement element", and a "fusogenic polypeptide" may also be referred to as a "fusogenic element".

A "resting" lymphocyte (e.g., resting T cell) is a lymphocyte in the G0 phase of the cell cycle that does not express an activation marker (e.g., ki-67). Resting lymphocytes may include naive T cells that have never been contacted with a specific antigen, and memory T cells that have been altered by prior contact with the antigen. "resting" lymphocytes may also be referred to as "quiescent" lymphocytes.

As used herein, "lymphocyte depletion" refers to a method of reducing the number of lymphocytes in a subject (e.g., by administering a lymphocyte depletion agent). Partial body or systemic fractionated radiotherapy may also cause lymphocyte depletion. The lymphocyte depleting agent may be a chemical compound or composition capable of reducing the number of functional lymphocytes in a mammal when administered to said mammal. One example of such an agent is one or more chemotherapeutic agents. Such agents and dosages are known and may be selected by the treating physician depending on the subject being treated. Examples of lymphocyte depleting agents include, but are not limited to, fludarabine (fludarabine), cyclophosphamide, cladribine (cladribine), dinium Bai Sudi f tos (denileukin diftitox), alemtizumab, or combinations thereof.

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing a target RNA molecule. The RNA target may be mRNA, or it may be any other RNA that is susceptible to functional inhibition of RNAi. As used herein, an "inhibitory RNA molecule" refers to an RNA molecule that is present in a cell to produce RNAi and cause reduced expression of a transcript to which the inhibitory RNA molecule is targeted. An inhibitory RNA molecule as used herein has a 5 'stem and a 3' stem capable of forming an RNA duplex. The inhibitory RNA molecule may be, for example, miRNA (endogenous or artificial) or shRNA, a precursor of miRNA (i.e., pri-miRNA or Pre-miRNA) or shRNA, or dsRNA that is directly transcribed or introduced into a cell or subject as an isolated nucleic acid.

As used herein, "double-stranded RNA" or "dsRNA" or "RNA duplex" refers to an RNA molecule comprising two strands. A duplex molecule includes a molecule consisting of two RNA strands that hybridize to form a duplex RNA structure, or a single RNA strand that folds upon itself to form a duplex structure. Most but not necessarily all bases in the duplex region are base paired. The duplex region comprises a sequence complementary to the target RNA. The sequence complementary to the target RNA is an antisense sequence and is typically 18 to 29, 19 to 21, or 25 to 28 nucleotides long, or in some embodiments between 18, 19, 20, 21, 22, 23, 24, 25 as the low end and 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 as the high end, with a given range typically having a low end that is lower than the high end. Such structures typically include a 5 'stem, a loop, and a 3' stem joined by a loop adjacent to each stem (which is not part of a duplex). In certain embodiments, the loop comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the loop comprises 2 to 40, 3 to 21, or 19 to 21 nucleotides, or in some embodiments, between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 as the low end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 as the high end, wherein the given range generally has a low end that is lower than the high end.

The term "microrna flanking sequence" as used herein refers to a nucleotide sequence that includes microrna processing elements. The microRNA processing elements are minimal nucleic acid sequences that facilitate the production of mature microRNAs from precursor microRNAs. These elements are typically located within 40 nucleotide sequences flanking the stem-loop structure of the micrornas. In some cases, microrna processing elements are found within an extension of the nucleotide sequence flanking the microrna stem-loop structure between 5 and 4,000 nucleotides in length.

The term "linker" when used in reference to a multiplex inhibitory RNA molecule refers to a linking member that adds two inhibitory RNA molecules.

As used herein, "recombinant retrovirus" refers to a non-replicative or "replication defective" retrovirus unless it is specifically indicated as replicative retrovirus. The term "recombinant retrovirus" and "recombinant retroviral particle" are used interchangeably herein. Such retrovirus/retroviral particles may be any type of retroviral particle including, for example, gamma retrovirus and (in the illustrative embodiment) lentivirus. It is well known that such retroviral particles (e.g. lentiviral particles) are typically formed in packaging cells by transfecting the packaging cells with plasmids (which include packaging components such as Gag, pol and Rev) and envelope or pseudotyped plasmids (which encode pseudotyped elements) and transferred, genomic or retroviral (e.g. lentiviral) expression vectors (which are typically plasmids encoding genes or other relevant coding sequences thereon). Thus, retroviral (e.g., lentiviral) expression vectors include sequences that promote expression and packaging after transfection into a cell (e.g., 5'LTR and 3' LTR flanking, for example, a psi packaging element and heterologous coding sequence of interest). The term "lentivirus" and "lentiviral particle" are used interchangeably herein.

The "framework" of a miRNA consists of a loop sequence that separates the stems of the stem-loop structure in the miRNA, surrounding the "5 'microrna flanking sequences" and/or the "3' microrna flanking sequences" of the miRNA. In some examples, the "framework" is derived from a naturally occurring miRNA, such as miR-155. The term "5 'microrna flanking sequences" and "5' arms" are used interchangeably herein. The term "3 'microrna flanking sequences" and "3' arms" are used interchangeably herein.

As used herein, the term "miRNA precursor" refers to any length of RNA molecule that can be enzymatically processed into a miRNA, such as a primary RNA transcript, a pri-miRNA, or a pre-miRNA.

As used herein, the term "construct" refers to an isolated polypeptide or an isolated polynucleotide encoding a polypeptide. The polynucleotide construct may encode a polypeptide, such as a lymphoproliferative element. Those of skill in the art will understand that construct refers to an isolated polynucleotide or an isolated polypeptide, depending on the context.

As used herein, "MOI" refers to the rate of infection, where MOI is equal to the ratio of the number of viral particles to the number of cells used for infection. As a non-limiting example, FACS and reporter expression can be used to perform functional titration of the number of viral particles.

"peripheral blood mononuclear cells" (PBMCs) include peripheral blood cells with rounded nuclei and include lymphocytes (e.g., T cells, NK cells, and B cells) and monocytes. Some blood cell types that are not PBMCs include erythrocytes, platelets, and granulocytes (i.e., neutrophils, eosinophils, and basophils).

It is to be understood that the present disclosure and the aspects and embodiments provided herein are not limited to the particular examples disclosed, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of disclosing specific examples and embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

When a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other value or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically exclusive limit. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. When multiple low values and multiple high values are given for a range overlap, those skilled in the art will recognize that the selected range will include low values that are lower than the high values. All headings in this specification are for ease of reading and are not limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are listed.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a chimeric antigen receptor" includes a plurality of such chimeric antigen receptors and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As used herein, the term "or" is to be understood as inclusive unless specifically stated or otherwise apparent from the context. The term "and/or" as used herein in phrases such as "a and/or B" includes each of the following: a and B; a or B; a (alone); and B (alone). Similarly, the term "and/or" as used in phrases such as "A, B and/or C" includes each of the following: A. b and C; A. b or C; a or B; a or C; b or C; a and B; a and C; b and C; a (alone); b (alone); and C (alone). This logic extends to any number of items in the list associated with the term "and/or".

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations that are embodiments of the present invention are specifically included in the present invention and are disclosed herein as if each combination and each combination were individually and clearly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically contemplated herein and are disclosed herein as if each subcombination and each such subcombination was individually and clearly disclosed.

Detailed Description

The present disclosure overcomes the challenges of the prior art by providing improved methods and compositions for selectively killing or modulating the activity of modified cells, such as modified NK cells and, in the illustrative examples, modified T cells, using anti-idiotype polypeptides expressed on the cell surface. For example, provided herein are anti-idiotype polypeptides that are improved safety switches for cell therapies. One challenge with prior art safety switches as etags is that they include EGFR family member domains and are therefore structurally limited to EGFR-only domains. Furthermore, they may produce undesirable side effects when delivered to a subject, as they are structurally related to growth factors (e.g., EGFR) found in the subject. The anti-idiotype polypeptide safety switches provided herein are more robust and flexible safety switches than etags in design and development. The anti-idiotype polypeptide safety switch can be designed to be recognized by virtually any antibody, including any clinical antibody such as a therapeutic antibody. The example section herein discloses exemplary methods for developing anti-idiotype antibodies using phage display assays. In addition, the anti-idiotype polypeptides provided herein include designs of antibodies that can be used to modulate cellular activity (including but not limited to proliferation and apoptosis) and do not require driving cytotoxicity.

More specifically, some methods and compositions herein provide a versatile and effective solution for controlling cytokine release syndrome in a subject having modified cells that express a Chimeric Antigen Receptor (CAR) or a recombinant T Cell Receptor (TCR). Thus, the method provides an important step for improving the safety and effectiveness of cell therapy methods. Illustrative compositions for selectively killing modified cells, such as modified NK cells and modified T cells in the illustrative examples, are easier to develop and safer than existing compositions and methods. Aspects of the anti-idiotype polypeptides, methods of using the same, and polynucleotides encoding the same provided herein provide novel methods and compositions for modulating the activity of modified cells. In addition, compositions are provided that have a number of uses, including their use in these improved methods, including cell preparation compositions. Some of these compositions include modified and in the illustrative embodiments genetically modified lymphocytes that include safety switches for use with cell therapies to affect reduction or elimination of infused cells upon occurrence of a dangerous adverse event. Some of these compositions have improved proliferation and survival quality, including when cultured in vitro, e.g., in the absence of growth factors. Such modified and in the illustrative embodiments genetically modified lymphocytes will have utility, for example, in the following: as a research tool to better understand the mechanisms that control and stop cytokine release syndrome and factors that affect T cell proliferation and survival; and commercial production, such as the production of certain factors (e.g., growth factors and immunomodulators) that may be collected and tested or used in commercial products. And such modified and genetically modified lymphocytes have utility in the treatment of cancer and other diseases.

Anti-idiotype polypeptide

Anti-idiotype polypeptides and polynucleotides encoding these polypeptides are provided herein (disclosed in detail in other sections herein), which have many utility in life sciences and medicine. In illustrative embodiments, such polypeptides are particularly useful in modified cells, such as in cells and gene therapies. Anti-idiotype polypeptides expressed on the cell surface can recognize target antibodies or target antibody mimics that are in contact with these cells. These antibodies and antibody mimics may be used, for example, to label cells expressing an anti-idiotype polypeptide for immune system killing, to modulate a characteristic (such as, for example, a proliferative or apoptotic state) or activity of a cell, to label a cell, to provide a target for enrichment and/or purification, to enrich a cell, or to aggregate a cell. Those of skill in the art will understand how to use anti-idiotype polypeptides in these and other methods in view of this disclosure. Accordingly, provided herein are methods of providing any of the above uses by expressing any of the polynucleotides disclosed herein, including nucleic acids encoding the anti-idiotype polypeptides disclosed herein.

FIG. 3 shows a non-limiting exemplary embodiment of an anti-idiotype polypeptide herein. As shown in fig. 3A, in an illustrative aspect, the anti-idiotype polypeptides herein include an extracellular recognition domain (sometimes referred to as an anti-idiotype extracellular recognition domain, an anti-Id ERD, or an anti-Id ECD, and represented as an "anti-idiotype" in fig. 3A-F and 3H) and generally include a Membrane Association Domain (MAD), which in an illustrative embodiment is separated from the anti-Id ECD by a handle. In an illustrative embodiment, the anti-Id ERD comprises a recognition domain of an anti-idiotype antibody or an anti-idiotype antibody mimetic. The recognition domain of the anti-idiotype polypeptide recognizes the idiotype of the target antibody or the idiotype of the target antibody mimetic. In an illustrative embodiment, the anti-idiotype extracellular recognition domain comprises an idiotype-binding variable region of an anti-idiotype antibody or an anti-idiotype antibody mimetic. In an illustrative embodiment, the handle separates the MAD and the anti-idiotype extracellular recognition domain.

In some embodiments, the extracellular recognition domain recognizes the idiotype of any antibody or antibody mimetic known in the art. In certain illustrative embodiments, the extracellular recognition domain recognizes an idiotype of a clinical antibody or a clinical antibody mimetic. In some illustrative embodiments, such clinical antibodies are regulatory bodies (e.g., FDA approved biologies in the united states). In some embodiments, binding of the anti-idiotype polypeptide to the target antibody does not block or prevent binding between the target antibody and its cognate antigen. In illustrative embodiments, binding of the anti-idiotype polypeptide to the target antibody blocks or prevents binding between the target antibody and its cognate antigen.

Typically, the anti-idiotype polypeptide comprises a membrane association domain (sometimes referred to herein as MAD). The membrane associating domain of the anti-idiotype polypeptide attaches, tethers or anchors the recognition domain from the anti-idiotype antibody or antibody mimetic to the cell membrane. In some embodiments, the membrane associating domain comprises one or more of a transmembrane domain and a GPI anchor, as further disclosed elsewhere herein. In some embodiments, the transmembrane domain may be a heterologous transmembrane domain or an endogenous transmembrane domain, any of which may be a transmembrane domain of an antibody.

As shown in fig. 3B-G, in some embodiments, the anti-idiotype polypeptides provided herein further comprise one or more intracellular domains (sometimes referred to herein as ICDs). Furthermore, in certain illustrative embodiments, MADs comprising such anti-idiotype polypeptides of ICD are transmembrane domains ("TMs" in FIGS. 3B-G). One or more intracellular domains may activate or inhibit pro-apoptotic or anti-apoptotic pathways and/or pro-survival or anti-survival pathways, and in certain embodiments modulate other cellular processes/pathways. Details regarding these various embodiments and other embodiments in which the anti-idiotype polypeptides herein comprise ICDs are provided throughout this specification. Figures 3B-G provide some non-limiting examples. In some embodiments, ICDs function structurally to ensure that anti-id ERDs are stably expressed on and remain bound to the cell membrane. In other embodiments, ICDs may have functional properties that are modulated by binding to dimerization or multimerization that is induced by the binding of anti-id ERD to its target antibody or mimetic.

In some embodiments (fig. 3C), the anti-id ERD (the "anti-idiotype" in fig. 3C) serves as the inducible extracellular dimerization domain of LE herein. In such embodiments, dimerization of LE by binding of the target antibody to anti-id ERD may activate signaling domains in ICD, driving proliferation and/or cell survival. As shown, the ICD of the LE may include P3 and optionally P4 domains, as disclosed herein with respect to LE ICDs. In some embodiments, the intracellular domain may activate one or more of the Jak/Stat pathway, TRAF pathway, PI3K pathway, or PLC pathway. The disclosure relating to the mechanisms used to activate these pathways is provided herein in "lymphoproliferative sections". Illustrative examples of these embodiments are inducible chimeric lymphoproliferative elements, which are inducible when the target antibody binds to an extracellular domain that recognizes the idiotype of the target antibody.

In some embodiments (fig. 3D), the anti-id ERD (the "anti-idiotype" in fig. 3D) acts as the ASTR for the CAR or TCR. Thus, the CAR or TCR comprises an anti-id ERD, a handle that attaches the ERD to the transmembrane domain, and an ICD of the CAR or TCR. Thus, binding of the target antibody to the anti-id ERD can activate the signaling domain in ICD of CAR or TCR.

In some embodiments, the intracellular domains are pro-apoptotic and may include one or more intracellular signaling domains from a caspase protein and/or one or more intracellular signaling domains from a member of the tumor necrosis factor receptor superfamily. Thus, such embodiments are specific examples of safety switches provided herein. As shown in fig. 3E, such embodiments may include an anti-idiotype polypeptide, wherein the ICD includes a death domain. Such ICDs may include all ICDs from TNF receptor superfamily members or, in certain illustrative embodiments, a portion of an ICD from TNF receptor superfamily members that includes a death domain such as FAS. Illustrative examples of such anti-idiotype polypeptides include constitutive dimerization domains in the extracellular domain, which may be all or part of the stalk or transmembrane domain, thereby providing such anti-idiotype polypeptides with the ability to form higher order multimers, such as tetramers. Furthermore, the ICD of any of the embodiments shown in fig. 3E may include death domains, or other functional domains from promoter cysteases such as, for example, cysteases 2, 8, 9, and 10.

In some embodiments (fig. 3F), the anti-id ERD (the "anti-idiotype" of fig. 3D) acts as one of the ASTRs of the bispecific CAR or TCR, with a second AST that normally binds to an antigen expressed by a cancer cell being present. Such embodiments may be referred to herein as anti-idiotype bispecific CARs. In such embodiments, the CAR or TCR comprises an anti-id ERD, a second AST, an anti-id ERD, and 2 ^nd AST is attached to the handle of the transmembrane domain, and ICD of CAR or TCR. Thus, binding of target antibody to anti-id ERD or 2 ^nd Binding of AST to its antigen may activate the signaling domain in the ICD of the CAR or TCR.

Fig. 3G illustrates another embodiment of an anti-idiotype polypeptide comprising an ICD herein. In such embodiments, a cleavage site, typically activated by dimerization, is added between or as part of the transmembrane domain in the anti-idiotype polypeptide and the ICD. Thus, in some embodiments, these anti-idiotype polypeptides include a transmembrane domain that includes an amino acid sequence that acts as a substrate cleavage site upon dimerization by certain proteases. In certain embodiments, such cleavage sites may be substrate cleavage sites for gamma-secretase complexes, as discussed in more detail herein. The intracellular domains used in such embodiments may encode a variety of intracellular polypeptides, including certain caspases, including promoter caspases, e.g., caspases 2, 8, 9, and 10. In other embodiments, the ICD is a transcription factor. In such embodiments, the transcription factor is sequestered outside the nucleus until the anti-id ERD binds to its target antibody that induces dimerization and cleavage, releasing the transcription factor so that the transcription factor can enter the nucleus to be active in regulating gene expression.

Anti-idiotype polynucleotide

Provided herein in one aspect are polynucleotides comprising a nucleic acid encoding an anti-idiotype polypeptide, which may be referred to herein as an anti-idiotype polynucleotide. FIG. 1 provides a non-limiting exemplary polynucleotide that is part of a lentiviral vector, in this non-limiting example a third generation lentiviral expression vector comprising a nucleic acid encoding an anti-idiotype polypeptide. The encoded anti-idiotype polypeptides include an anti-idiotype extracellular recognition domain ("anti-idiotype"), a handle, and a Membrane Association Domain (MAD). The non-limiting polynucleotide vector of FIG. 1 includes a promoter ("promoter") positioned to drive expression of nucleic acids encoding different functional polypeptides, including anti-idiotype polypeptides. In addition, the polynucleotide vector of fig. 1 includes the various functional domains shown, including various viral functional domains, and encodes an optionally inhibitory RNA molecule within a promoter.

FIG. 2 provides a non-limiting example of some of the polynucleotides provided herein, including nucleic acids encoding one or more anti-idiotype polypeptides, which in themselves represent separate aspects of the disclosure. As shown in fig. 2A, in some embodiments, the nucleic acid encoding the anti-idiotype polypeptide encodes a Membrane Association Domain (MAD) and an anti-idiotype extracellular recognition domain (referred to as an "anti-idiotype" in fig. 2). In an illustrative embodiment, the nucleic acid encoding the handle separates the nucleic acid encoding the MAD from the nucleic acid encoding the anti-idiotype extracellular recognition domain and is in frame with the nucleic acid encoding the MAD and the nucleic acid encoding the anti-idiotype extracellular recognition domain. In some embodiments, provided herein are polynucleotides encoding additional functional groups expressed from the same promoter (i.e., on the same transcriptional unit) or different promoters (i.e., different transcriptional units). For example, a polynucleotide comprising a nucleic acid encoding an anti-idiotype extracellular recognition domain may comprise a nucleic acid encoding an engineered signaling polypeptide, cytokine, and/or inhibitory RNA in addition to the anti-idiotype extracellular recognition domain. Accordingly, provided herein in one aspect is a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter, wherein the one or more transcriptional units comprise:

a) Polynucleotide sequences encoding one or more inhibitory RNA molecules, a first engineered signaling polypeptide and/or cytokine, and

b) A polynucleotide sequence encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain that recognizes the idiotype of a target antibody or target antibody mimetic.

The polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide may be DNA or RNA. In some illustrative embodiments, they are mRNA. Such embodiments may include embodiments in which the anti-idiotype antibody is directed against an antibody that forms the astm of the CAR. Thus, mRNA encoding an anti-idiotype antibody can be delivered directly to a subject, and when taken up and expressed by cells of the subject, such cells can form artificial antigen presenting cells that drive proliferation of CAR-T cells administered to the subject that express a CAR having an ASTR that is a target antibody recognized by the anti-idiotype polypeptide.

Methods for preparing synthetic mRNA are well known in the art. In addition, such polynucleotides may be polynucleotide vectors, such as expression vectors. Further details regarding polynucleotides and polynucleotide vectors such as RIP (including lentiviral particles) are provided throughout this disclosure, including the claims. These further details include details regarding polynucleotides comprising nucleic acids encoding anti-idiotype polypeptides. Some non-limiting notable chapters include a recombinant retroviral particle chapter, a nucleic acid chapter, and an exemplary embodiment chapter.

The nucleic acid encoding the anti-idiotype polypeptide may be upstream or downstream (i.e., 5 'or 3') of those encoding other functional groups. Thus, in such embodiments, the anti-idiotype polynucleotide is expressed as a polypeptide separate from other functional polypeptides.

In certain illustrative embodiments, the polynucleotides herein encode anti-idiotype polypeptides and are suitable, structured, and/or effective for expression in T cells and/or NK cells, and thus are suitable, structured, and/or effective for T cell and/or NK cell therapies. Examples of such polynucleotides typically include promoters that are active in T cells and/or NK cells, driving expression of the anti-idiotype extracellular recognition domain and the membrane association domain, and thus the anti-idiotype extracellular recognition domain and the membrane association domain, on the same transcriptional unit, the expression of which is driven by the promoter. Thus, in some embodiments, the anti-idiotype polypeptide is expressed as part of a single polynucleotide that also encodes a Chimeric Antigen Receptor (CAR), an engineered T Cell Receptor (TCR) (fig. 2B), a lymphoproliferative element (fig. 2C), or a cytokine (fig. 2D). Furthermore, in some embodiments, the anti-idiotype polypeptide is expressed as part of a single polynucleotide encoding the CAR or TCR, the anti-idiotype polypeptide, and the lymphoproliferative element (fig. 2E), or the cytokine (fig. 2F), or both the lymphoproliferative element and the cytokine (fig. 2G). Such polynucleotide embodiments and the anti-idiotype polypeptides encoded thereby, particularly embodiments wherein both the anti-idiotype polypeptides and CAR or TCR are encoded, are particularly suitable, adapted, and/or effective when present and expressed in T cells and/or NK cells for safety switches as provided herein, including as part of CAR-T therapy, TIL therapy, and CAR-NK therapy, as well as other safety switch methods provided herein.

In some embodiments, the polynucleotide encoding the anti-idiotype polypeptide is separated from the polynucleotide encoding the CAR, TCR, cytokine, and/or polynucleotide encoding the proliferative element by an Internal Ribosome Entry Site (IRES) or ribosome jump sequence and/or cleavage signal (as shown, for example, in fig. 2B-2G). The IRES or ribosome jump and/or cleavage signal can be any IRES or ribosome jump sequence and/or cleavage signal known in the art. The ribosome-hopping sequence can be, for example, T2A with the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 83). Other examples of cleavage signals and/or ribosome hopping sequences include FMDV 2A (F2A); equine type rhinitis virus 2A (E2A for short); porcine teschovirus (porcine teschovirus) -1 2A (P2A); and the Flat moth (Thoseasaigna) virus 2A (T2A).

FIG. 4 provides other examples of anti-idiotype polynucleotides provided herein. For example, fig. 4A shows a polynucleotide construct comprising a nucleic acid encoding a bispecific CAR (i.e., a CAR with anti-id ERD as one astm), followed by a nucleic acid T2A element, followed by a nucleic acid encoding LE. Figure 4B shows a polynucleotide construct comprising a nucleic acid encoding a CAR or TCR followed by a nucleic acid T2A element followed by a nucleic acid encoding an anti-idiotype polypeptide comprising an anti-id ERD, a handle, a transmembrane domain, and an intracellular domain. FIG. 4C shows a polynucleotide construct comprising a nucleic acid encoding an anti-idiotype polypeptide comprising an anti-id ERD, a handle, a transmembrane domain, and an intracellular domain, followed by a nucleic acid T2A element, followed by a nucleic acid encoding LE. FIG. 4D shows a polynucleotide construct comprising a nucleic acid encoding an anti-idiotype polypeptide comprising an anti-id ERD, a handle, a transmembrane domain, and an intracellular domain, followed by a nucleic acid T2A element, followed by a nucleic acid encoding a cytokine. Fig. 4E shows a polynucleotide construct identical to fig. 4B, but with the nucleic acid encoding the ICD of the anti-idiotype polypeptide followed by the nucleic acid T2A element, followed by the nucleic acid encoding LE. Fig. 4F shows a polynucleotide construct identical to fig. 4B, but with the nucleic acid encoding LE followed by a nucleic acid T2A element followed by a nucleic acid encoding a cytokine. Fig. 4G shows a polynucleotide construct identical to fig. 4E, but with the nucleic acid encoding the ICD of the anti-idiotype polypeptide followed by the nucleic acid T2A element, followed by the nucleic acid encoding the cytokine.

Some non-limiting specific exemplary constructs are as follows:

f1-3-247 encodes CD19 CAR and a polypeptide lymphoproliferative element consisting of, from amino to carboxy terminus: kozak-type sequence GCCGCCACCAT/UG (SEQ ID NO: 331) with T at the "T/U" residue and with an optional last G, CD8 signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 72) (wherein the sequence ATGG from the Kozak-type sequence also encodes the first four nucleotides of the CD8 signal peptide), FLAG-TAG (DYKDDDK; SEQ ID NO: 74), linker (GSTSGS; SEQ ID NO: 349), anti-CD 19scFv, CD8 handle and transmembrane region and intracellular domain from CD3z followed by T2A and a lymphoproliferative element comprising E006-T016-S186-S050 portion encoding an extracellular domain comprising a variant of the leucine zipper motif and eTAG, the transmembrane domain of CSF2RA, the intracellular domain of MPL and the intracellular domain of CD40, wherein each portion of the lymphoproliferative element is linked by a GGS linker.

F1-3-P100 encodes a CD19 CAR and an anti-idiotype polypeptide having P3 and P4 domains of LE, said anti-idiotype polypeptide consisting of, from amino to carboxy terminus: kozak-type sequence GCCGCCACCAT/UG (SEQ ID NO: 331) with T at the "T/U" residue and optionally the last G, CD8 signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 72) wherein the sequence ATGG from the Kozak-type sequence also encodes the first four nucleotides of the CD8 signal peptide, FLAG-TAG (DYKDDDK; SEQ ID NO: 74), linker (GSTSGS; SEQ ID NO: 349), anti-CD 19scFv, CD8 handle and transmembrane region and intracellular domain from CD3z followed by T2A, anti-cetuximab scFv of the table shown in FIG. 8, handle, transmembrane and ICD (STMICD 2, SEQ ID NO: 677), and lymphoproliferative elements S121-S105 portion encoding the intracellular domain of IL7Rα and the intracellular domain of IL2Rβ to which the GGS linker is linked.

FIG. 5A shows a schematic representation of an illustrative bicistronic lentiviral genome vector with divergent transcription units. Under transcriptional control of the NFAT-responsive minimal IL-2 promoter (6 x NFAT), a first transcription unit comprising a lymphoproliferative element that is also an anti-idiotype polynucleotide and a subsequent polyadenylation sequence (PolyA) is encoded in the opposite direction. Optionally, a spacer element (Ins) separates the first transcription unit and the second transcription unit. The second transcriptional unit encodes a CAR (CAR) under the transcriptional control of a constitutive promoter (promoter) and in the forward direction. Triangles shown in dashed lines represent 3 possible positions, in any one or more of which one or more mirnas may optionally be inserted into the vector. The triangles shown in dotted lines represent 1 possible positions of exons within a promoter (e.g., EF 1-a), where one or more inhibitory RNAs may optionally be inserted into the vector. "SA" and "SD" correspond to splice donor and splice acceptor sites.

FIG. 5B shows a schematic representation of an illustrative bicistronic lentiviral genome vector with divergent transcription units. Under the transcriptional control of the NFAT-responsive minimal IL-2 promoter (6 x NFAT), the first transcription encoding the lymphoproliferative element and subsequent polyadenylation sequence (PolyA) is encoded in the opposite direction. Optionally, a spacer element (Ins) separates the first transcription unit and the second transcription unit. The second transcriptional unit encodes a CAR (CAR) under the transcriptional control of a constitutive promoter (promoter), followed by a T2A element and an anti-idiotype extracellular recognition domain, a handle and a MAD, and in the forward direction. Triangles shown in dashed lines represent 3 possible positions, in any one or more of which one or more mirnas may optionally be inserted into the vector. The triangles shown in dotted lines represent 1 possible positions of exons within a promoter (e.g., EF 1-a), where one or more inhibitory RNAs may optionally be inserted into the vector. "SA" and "SD" correspond to splice donor and splice acceptor sites. The ECD of the lymphoproliferative element in this design optionally comprises an eTAG or a second anti-idiotype extracellular recognition domain. F1-6-744 encodes a bicistronic lentiviral genome vector having different transcription units. The first transcription unit encodes the lymphoproliferative element E016-T016-S186-S050 under the control of the NFAT responsive minimal IL-2 promoter, which is encoded entirely in the reverse direction. The second transcriptional unit encodes a HER2 CAR consisting of an anti-HER 2scFv, a CD8 stalk and transmembrane region, a CD137 intracellular domain and an intracellular activation domain from CD3z followed by T2A and eTag. The first transcription unit and the second transcription unit are separated in the forward direction by a 250cHS4 spacer (SEQ ID NO: 358).

Cells expressing anti-idiotype polypeptides

In some aspects, provided herein are modified cells, and in illustrative embodiments, genetically modified cells are provided, the cells comprising any one of the anti-idiotype polynucleotides provided herein. Further, provided herein are modified cells or, in illustrative embodiments, genetically modified cells that express any one of the anti-idiotype polypeptides provided herein. In some embodiments, the modified or genetically modified cells may further express an inhibitory RNA molecule disclosed herein and/or other polypeptides, such as engineered signaling polypeptides. In an illustrative embodiment, the modified or genetically modified cell can express an anti-idiotype polypeptide and a lymphoproliferative element CAR and/or a recombinant TCR. In illustrative embodiments, the modified or genetically modified cells can express anti-idiotype polypeptides and cytokines. In some embodiments, the cytokine is in a secreted form. In some embodiments, the cytokine is membrane-associated. In some embodiments, the cell may be an immortalized cell.

In some embodiments, the cells expressing the anti-idiotype polypeptide are lymphocytes, e.g., TIL, lymphocytes other than B cells, and in illustrative embodiments are T cells and/or NK cells. In some embodiments, the cell expresses a chimeric TCR, or the cell is a CAR-T cell and/or a CAR-NK cell, or a tumor infiltrating lymphocyte. In some embodiments of any of the aspects herein, the T or NK cell is a NKT cell. NKT cells are a subset of T cells that express CD3 and typically co-express the αβ T cell receptor and also express a variety of molecular markers commonly associated with NK cells (e.g., NK1.1 or CD 56). In some embodiments, the cell is a primary cell. In some embodiments, the cell is a human cell. In some embodiments, the cells generally do not produce antibodies. The skilled artisan will appreciate that reference to lymphocytes that do not express antibodies in the context of modified cells comprising or expressing an anti-idiotype polypeptide herein refers to the natural ability of the cells, and that such modified cells comprise and in illustrative embodiments express an anti-idiotype polypeptide.

In some embodiments, the modified cells having a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide are present in a cell suspension within a commercial containerCommercial containers such as those suitable for cell therapies such as cell cryopreservation infusion bags. The number of cells in suspension in a commercial container may be sufficient to provide 1x10 ⁵ Individual cells to 1x10 ⁹ Individual cells, 1x10 ⁶ Individual cells to 1x10 ⁹ Individual cells, or 1x10 ⁶ Individual cells to 5x10 ⁸ Individual cells, e.g., CAR positive living T cells and/or NK cells per kg body weight of the subject to which the cells are to be delivered. Thus, in some embodiments, the commercial container may comprise the above-described range x50-150kg or 50-100kg. In some embodiments, the commercial container comprises 1x10 ⁷ Up to 1x10 ¹¹ 1x10 ⁸ Up to 1x10 ¹¹ Or 1x10 ⁸ Up to 5x10 ¹⁰ Individual cells, modified cells, e.g., CAR positive living T cells and/or NK cells, and/or in an illustrative embodiment, cells positive for an anti-idiotype extracellular recognition domain. Thus, the anti-idiotype polypeptides herein can be used to confirm that modified cells expressing the CAR also express the anti-idiotype polypeptide, e.g., as a release standard, to help ensure that a safety switch is present on such cells. Further details regarding modified cells herein in commercial containers can be found herein as non-limiting examples in the "kits and commercial products" section.

In one aspect, provided herein are modified or genetically modified T cells or NK cells prepared using a method according to any one of the methods provided herein, wherein the cells are modified to contain a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide. In some embodiments, the T cell or NK cell is further modified to express the first engineered signaling polypeptide. In an illustrative embodiment, the first engineered signaling polypeptide may be an LE, TCR, or CAR, which includes an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the T cell or NK cell may further comprise a second engineered signaling polypeptide that may be a CAR, TCR, or lymphoproliferative element. In some embodiments, the lymphoproliferative element may be a chimeric lymphoproliferative element. In some embodiments, the T cell or NK cell may further comprise a pseudotyped element on the surface. In some embodiments, the T cell or NK cell may further comprise an activating element on the surface. The CAR, lymphoproliferative element, pseudotyped element, and activating element of a genetically modified T cell or NK cell can comprise any of the aspects, embodiments, or sub-embodiments disclosed herein. In illustrative embodiments, the activating element may be an anti-CD 3 antibody, such as an anti-CD 3 scFvFc or an anti-CD 3 antibody mimetic.

In some aspects, provided herein are aspects comprising genetically modified and/or transduced T cells or NK cells comprising a polynucleotide encoding an anti-idiotype polypeptide and a self-driven CAR. Details regarding aspects of such genetically modified and/or transduced T cells or NK cells containing such polynucleotides, and compositions and methods including self-driven CARs, are disclosed in greater detail herein.

In some embodiments, provided herein are genetically modified lymphocytes, in illustrative embodiments TIL, T cells, and/or NK cells, or the self-driven CAR aspects provided herein, that relate to aspects for transducing T cells and/or NK cells in blood or a component thereof, the lymphocytes comprising transcriptional units encoding one, two, or more (e.g., 1-10, 2-10, 4-10, 1-6, 2-6, 3-6, 4-6, 1-4, 2-4, 3-4) inhibitory RNA molecules. In some embodiments, such inhibitory RNA molecules are lymphoproliferative elements and thus may be included in any aspect or embodiment disclosed herein as lymphoproliferative elements, so long as they induce proliferation of T cells and/or NK cells or otherwise meet the assays of lymphoproliferative elements provided herein. In some embodiments, an inhibitory RNA molecule directed against any target identified in the inhibitory RNA molecule section herein.

In some embodiments of aspects of immediately above T cells or NK cells comprising one or more (e.g., two or more) inhibitory RNA molecules and CAR or nucleic acid encoding the same, the astm of the CAR is MRB astm and/or the astm of the CAR binds to an antigen associated with a tumor. Furthermore, in some embodiments of the above aspects, the first nucleic acid sequence is operably linked to a riboswitch, e.g., capable of binding a nucleoside analog, and in illustrative embodiments an antiviral drug, e.g., acyclovir (acyclovir).

In the methods and compositions disclosed herein, expression of the engineered signaling polypeptide is regulated by a control element, and in some embodiments, the control element is a polynucleotide comprising a riboswitch. In certain embodiments, the riboswitch is capable of binding to a nucleoside analog and, when the nucleoside analog is present, expresses one or both of the engineered signaling polypeptides.

The cells of the present disclosure, e.g., lymphocytes, such as primary cells and/or non-naturally occurring antibody producing lymphocytes, and in illustrative embodiments T cells and/or NK cells, may include more than one type of anti-idiotype polypeptide on their surface, i.e., anti-idiotype polypeptides having different extracellular recognition domains. Thus, in some embodiments, a cell may include a first anti-idiotype polypeptide on its surface that recognizes a first target antibody or antibody mimetic and initiates a first reaction upon binding, and a second anti-idiotype polypeptide on its surface that recognizes a second target antibody or antibody mimetic and initiates a second reaction upon binding. For example, the addition of a first target antibody or antibody mimetic can activate a pro-survival signal of the first anti-idiotype polypeptide expressed in a cell through the intracellular domain of the first anti-idiotype polypeptide, and the addition of a second target antibody or antibody mimetic can induce ADCC through antibody or antibody mimetic effector function. Thus, in some embodiments, the polynucleotide encodes one, two, three, or four or more anti-idiotype polypeptides. In some embodiments, the cell contains such polynucleotides and expresses one, two, three, or four or more encoded anti-idiotype polypeptides. The skilled artisan will understand how to combine different anti-idiotype polypeptides to obtain different desired responses.

Safety switch

Safety switches for cell therapies have been developed to affect the reduction or elimination of infused cells in the event of adverse events. Any of the replication defective recombinant retroviral particles provided herein can comprise a nucleic acid encoding a safety switch as part of or separate from a nucleic acid encoding any of the engineered signaling polypeptides provided herein. Thus, any of the engineered signaling polypeptides provided herein (e.g., engineered signaling polypeptides in modified, genetically modified, and/or transduced lymphocytes to be introduced or reintroduced into a subject) can include a safety switch. For example, any of the engineered T cells disclosed herein can include a safety switch.

Safety switching techniques can be broadly divided into three categories according to their mechanism of action; antibody or antibody mimetic mediated cytotoxicity, pro-apoptotic signaling and metabolism (gene-directed enzyme prodrug therapy, GDEPT). The previously disclosed safety switch includes a cell surface molecule that is a truncated tyrosine kinase receptor. In some of these examples, the truncated tyrosine kinase receptor is a member of the Epidermal Growth Factor Receptor (EGFR) family (e.g., erbB1 (HER 1), erbB2, erbB3, and ErbB 4), for example as disclosed in us patent 8,802,374 or WO 2018226897. Thus, some of these existing safety switches are polypeptides recognized by antibodies that recognize the extracellular domain of EGFR members. For example, SEQ ID NO. 82 is an exemplary polypeptide that is bound by an antibody that recognizes the extracellular domain of an EGFR member and recognized under appropriate conditions. Such truncated EGFR polypeptides are sometimes referred to herein as etags. In an illustrative embodiment, eTag is recognized by a commercially available monoclonal antibody (e.g., matuzumab), nixib (necitumumab), panitumumab (panitumumab) and in an illustrative embodiment cetuximab (cetuximab), e.g., by

Mediated Antibody Dependent Cellular Cytotoxicity (ADCC) pathways, etags have been demonstrated to have cell killing potential. The inventors of the present disclosure have successfully expressed eTag in PBMCs using lentiviral vectors, and have found that expression of eTag in vitro by PBMCs exposed to cetuximab provides an effective elimination mechanism for PBMCs.eTag may be used in some embodiments of the disclosure, but in such embodiments, typically an anti-idiotype extracellular domain is also present.

In some embodiments, the extracellular recognition domain (i.e., cell tag) is itself an antibody that binds to a predetermined binding partner antibody (e.g., target antibody) as disclosed herein, including functional antibody fragments. In illustrative embodiments, the cell tag antibodies are specific for the target antibody and, for example, do not bind to the antibody constant region alone, or in some embodiments do not bind to the antibody constant region at all, or in illustrative embodiments do not bind to the antibody constant region unless they interact with the target antibody (Ab 1) to bind to the cell tag (extracellular recognition domain) (Ab 2). In illustrative embodiments, the cell-tagged antibody (i.e., an extracellular recognition domain comprising the variable region of the antibody) is an anti-idiotype antibody or antibody mimetic. In some embodiments, the anti-idiotype antibody (Ab 2) recognizes an epitope on a predetermined binding partner antibody (i.e., target antibody) (Ab 1) that is different from the antigen binding site on Ab 1. In an illustrative embodiment, ab2 binds to the variable region of Ab 1. In other illustrative embodiments, ab2 binds to the antigen binding site of Ab1, and in illustrative embodiments competes with Ab1 for binding to the antigen binding site of Ab 1. In certain embodiments, ab2 may be from any animal, including humans and mice, or humanized or chimeric antibodies or antibody derivatives, including, for example, antibody fragments (Fab, fab ', F (Ab') 2, scFv, diabodies, bispecific antibodies, and antibody fusion proteins Ab2 associates with a membrane through a membrane association domain.

In some embodiments, the safety switch also functions as a marker (flag) to tag or label a polynucleotide, polypeptide, or engineered cell. Such safety switches can be detected using standard laboratory techniques including PCR, southern blotting, RT-PCR, northern blotting, western blotting, histology and flow cytometry. For example, at least one of the present inventors used detection of eTAG by flow cytometry as an in vivo tracking marker for T cell implantation in mice. In other embodiments, the engineered cells are enriched using cell tags, using antibodies or ligands optionally bound to a solid substrate such as a column or bead. For example, others have shown that the application of biotinylated cetuximab in combination with anti-biotin microbeads to immunomagnetic selection successfully enriches T cells that have been transduced with a construct containing eTAG from as low as 2% of the population lentivirus to greater than 90% purity without observable toxicity to the cell preparation.

In some embodiments provided herein, the anti-idiotype polypeptide is a safety switch (also referred to herein as a safety switch polypeptide or an anti-idiotype polypeptide safety switch) comprising a recognition domain of an anti-idiotype antibody or an anti-idiotype antibody mimetic and a membrane association domain. Such safety switch polypeptides can be designed more efficiently and in more optional sequences and designs than prior art safety switches. In one aspect, such safety switch polypeptides are designed such that the extracellular recognition domain recognizes an idiotype of an antibody or antibody mimetic capable of inducing cytotoxicity.

Thus, in one aspect, the safety switch is based on antibody-mediated cytotoxicity when the antibody or antibody mimetic binds to an anti-idiotype polypeptide expressed on the cell surface, and more specifically to an extracellular recognition domain of the anti-idiotype polypeptide (also referred to herein as a cell tag or more specifically an anti-idiotype cell tag). In some embodiments, the antibody or antibody mimetic binds to a cell tag and induces Complement Dependent Cytotoxicity (CDC) and/or antibody dependent cell-mediated cytotoxicity (ADCC). In some embodiments, binding of the antibody or antibody mimetic to the anti-idiotype polypeptide induces, promotes and/or activates one or more of ADCC, CDC, antibody-mediated complement activation, antibody-dependent cellular phagocytosis, and antibody-dependent disease enhancement. Details concerning other antibody and antibody mimetic functions, including the corresponding Fc domains for eliciting such responses, are discussed herein in "antibody and antibody mimetic effector functions". The anti-idiotype polypeptide may be immunogenic to further stimulate the immune system. Thus, in some embodiments, the cell tag is immunogenic. In other embodiments, the cell tag polypeptide is non-immunogenic. In another aspect, the safety switch polypeptide is designed such that the anti-idiotype polypeptide comprises an intracellular domain having one or more cell death-inducing signals, and the polypeptide is capable of inducing a cell death signal when the anti-idiotype polypeptide binds to a target antibody or antibody mimetic comprising an idiotype recognized by the anti-idiotype polypeptide. Cell death induction signals may be induced based on dimerization-induced apoptosis signaling. In some embodiments, the safety switch is based on a dimerization-induced apoptosis signal. In some embodiments, such safety switches comprise an extracellular dimerization domain comprising a recognition domain of an anti-idiotype antibody or antibody mimetic linked in-frame to a membrane association domain and an intracellular domain comprising a component of an apoptotic pathway. Thus, dimerization mediated by the binding of antibodies or antibody mimics to anti-idiotype polypeptides results in apoptosis. In some embodiments, the safety switch comprises an Inducible FAS (iFAS) consisting of one or more inducible dimerization domains (i.e., anti-idiotype polypeptides) fused to the cytoplasmic tail of the FAS receptor and localized to the membrane by a membrane association domain. As discussed in the "intracellular domain" section herein, in some embodiments, the safety switch includes one or more domains from a caspase, such as caspase-1 or caspase-9.

As discussed in the "anti-idiotype fusion polypeptide" section herein, the anti-idiotype polypeptides, including the safety switches discussed in this section, can be expressed as fusions with other polypeptides disclosed herein, including lymphoproliferative elements, CARs, and/or recombinant TCRs. In other embodiments, the anti-idiotype polypeptide itself is expressed as a polypeptide. In any of these embodiments, the anti-idiotype polypeptide may include any domain disclosed herein to be included in a lymphoproliferative element, CAR, and/or TCR, such as an extracellular domain, a stalk, a transmembrane domain, an intracellular activation domain, a regulatory domain, a linker, or an intracellular domain.

Anti-idiotype method

The anti-idiotype compositions provided herein have many uses, including in vitro and in vivo methods for, e.g., cell tags, and in illustrative embodiments methods for delivering polynucleotides or polynucleotide vectors (e.g., RIP) and/or modified cells to a subject. For example, such methods for delivering polynucleotides, polynucleotide vectors, and/or modified cells to a subject can utilize signaling modulated by binding of a target antibody to an anti-idiotype extracellular recognition domain of an anti-idiotype polypeptide provided herein. Such methods may be methods of inducing proliferation or death of a target cell using a polynucleotide or polynucleotide vector, wherein the target cell is a modified cell that is delivered, or a cell that is present in a subject that is modified in vivo in the subject by transduction, transfection, electroporation, or other gene delivery means, wherein in some embodiments the polynucleotide or polynucleotide vector encoding the anti-idiotype polynucleotide is delivered directly. In illustrative embodiments, target cell proliferation or death is induced by binding of a target antibody to an anti-idiotype extracellular recognition domain expressed on a target cell.

Thus, in one aspect, provided herein is a method for administering a modified cell and/or a polynucleotide, such as a polynucleotide vector, comprising a nucleic acid encoding an anti-idiotype polypeptide to a mammalian subject. The method of this aspect comprises delivering a polynucleotide, such as a polynucleotide vector and/or modified cell, to a mammalian subject. The polynucleotide or polynucleotide vector may be any polynucleotide vector provided herein comprising a nucleic acid encoding an anti-idiotype polypeptide provided herein. In certain embodiments, such anti-idiotype polypeptides include a membrane associating domain, which in illustrative embodiments is separated from an anti-idiotype extracellular recognition domain on the anti-idiotype polypeptide by a handle. In some embodiments, the anti-idiotype polypeptide includes an intracellular domain, in some embodiments, which is an intracellular signaling domain. The modified cell may be any modified cell provided herein comprising and in an illustrative embodiment expressing any polynucleotide provided herein comprising a nucleic acid encoding an anti-idiotype polypeptide.

In an illustrative embodiment, the modified cell is a genetically modified cell. In some non-limiting embodiments, such modified cells or genetically modified cells are lymphocytes or lymphocytes other than B cells, and in some embodiments are T cells and/or NK cells, e.g., CAR-T cells and/or CAR-NK cells. In certain embodiments, the polynucleotide is RNA or DNA, and in illustrative embodiments is mRNA, e.g., synthetic anti-idiotype mRNA as disclosed herein. In an illustrative embodiment, the polynucleotide vector is a replication defective retroviral particle (RIP), such as a recombinant lentiviral particle.

Systems and methods for processing cells removed from a subject, e.g., methods for isolating and modifying blood cells such as PBMCs, may include conventional closed cell processing systems and methods, or "newer" methods and systems as disclosed in more detail herein. Thus, provided herein in certain aspects, in addition to, or as part of, or in combination with, methods of administering modified cells (typically prior to the delivery step of such methods for administration), is a method of transducing, modifying, and/or genetically modifying Peripheral Blood Mononuclear Cells (PBMCs) or lymphocytes (typically T cells and/or NK cells, and in certain illustrative embodiments resting T cells and/or resting NK cells) in a reaction mixture, the method utilizing a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide in illustrative embodiments. Such methods may include a contacting step comprising contacting the lymphocyte with a polynucleotide vector comprising a nucleic acid encoding an anti-idiotype polypeptide, such as, but not limited to, a replication defective recombinant retroviral particle (RIP) in a reaction mixture. Such reaction mixtures themselves represent separate aspects provided herein. In some embodiments, the reaction mixture comprises blood or a component thereof and/or an anticoagulant. The reaction mixture in the illustrative embodiments comprises lymphocytes and identical copies of a polynucleotide vector, e.g., replication defective recombinant retroviral particles, comprising a nucleic acid encoding an anti-idiotype polypeptide provided herein. In addition, the reaction mixture may include a T cell activating element. In certain embodiments, the reaction mixture includes one or more additional blood components set forth below that are present in the illustrative embodiments, as the reaction mixture comprises at least 10% whole blood. In an illustrative embodiment, the RIP comprises a T cell binding polypeptide and a fusogenic polypeptide on its surface, which in an illustrative embodiment is a pseudotyped element. In such methods, contacting (and incubating under contact conditions) facilitates association of lymphocytes with RIP, wherein the RIP gene modifies and/or transduces the lymphocytes. In certain illustrative embodiments, particularly directed to "newer" methods for processing T cells, this may be performed in a shorter time, as discussed herein, the RIP may further include T cell activating elements on its surface.

In some embodiments, the method further comprises instructing a medical professional, patient, or caregiver that if a life threatening adverse event occurs, the target antibody should be administered to the subject, and/or that the target antibody or antibody mimetic should be delivered to the subject in response to the life threatening adverse event. Such adverse events include grade 3 and/or grade 4 adverse events, including grade 3 and/or grade 4 CRS or ICANS (Lee et al, "ASCT consensus classification of immune effector cell-related cytokine release syndrome and neurotoxicity"; blood and bone marrow transplantation biology (Biology of Blood and Marrow Transplantation); 25:6235-638 (2019)). In an illustrative embodiment, the target antibody or antibody mimetic comprises an idiotype recognized by an anti-idiotype extracellular recognition domain. The target antibody or antibody mimetic can be any of the target antibodies and antibody mimetics disclosed herein, including, but not limited to, approved biological target antibodies and antibody mimetics. In some embodiments, the target antibody or antibody mimetic is delivered in an amount sufficient to selectively kill at least 1%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the genetically modified T cells and/or NK cells in the subject.

Methods for delivering modified cells, polynucleotides, polynucleotide vectors, and antibodies or antibody mimics to a subject are known in the art. In some embodiments of any aspect herein, the delivery of the modified or genetically modified cell, polynucleotide, or polynucleotide vector (e.g., RIP) or target antibody or antibody mimetic is performed by intravenous administration/infusion, intraperitoneal administration/injection, subcutaneous administration/injection, or intramuscular administration/injection. In some embodiments, the modified cells (e.g., lymphocytes) introduced into the subject are autologous cells, and in other embodiments, they are allogeneic cells. In embodiments utilizing allogeneic cells, the cells (e.g., lymphocytes) are typically from different humans, and the cells (e.g., lymphocytes) from the subject are not modified. In some embodiments, blood is not collected from the subject to harvest cells during the method.

The present disclosure provides various therapeutic methods involving delivery of engineered T cell receptors or CARs. The engineered T cell receptors or CARs of the present disclosure can mediate cytotoxicity against target cells when present in modified T lymphocytes or NK cells or in polynucleotide vectors delivered directly to a subject. Such methods generally involve administering a modified lymphocyte, or a substantially purified or purified polynucleotide vector (e.g., a RIR) as provided herein to a subject. The engineered T cell receptor or CAR binds to an antigen present on the target cell, thereby mediating killing of the target cell by T lymphocytes or NK cells genetically modified to produce the engineered T cell receptor or CAR. To increase the effectiveness and/or safety of such methods, in certain illustrative embodiments, modified T cells and/or NK cells, polynucleotides, or polynucleotide vectors delivered into a subject are further modified such that they are capable of expressing or expressing an anti-idiotype polypeptide and CAR, TCR, cytokine, and/or lymphoproliferative element as provided herein. In some embodiments, the modified cells express an anti-idiotype polypeptide and one or more of a CAR, TCR, cytokine, and/or lymphoproliferative element from the same polynucleotide, and sometimes on the same polypeptide, as disclosed herein. In an illustrative embodiment, the modified cells are lymphocytes, in an illustrative embodiment T cells and/or NK cells. In certain illustrative embodiments, such methods herein comprise delivering a polynucleotide and polynucleotide vector provided herein, including a promoter active in lymphocytes, in illustrative embodiments in T cells and/or NK cells, and encoding an anti-idiotype polypeptide, directly into a subject.

Such methods involving delivery of modified T cells and/or NK cells, or polynucleotides or polynucleotide vectors having promoters active in T cells and/or NK cells, are particularly suitable, adapted, and/or effective in methods wherein the anti-idiotype polypeptide modulates T cells and/or NK cell proliferation (e.g., may be used as a lymphoproliferative element) and/or induces cell killing (e.g., may be used as a safety switch). Methods of using anti-idiotype polypeptides herein to promote selective killing of modified cells, e.g., modified CAR-T, CAR-NK or TIL cells, may be referred to herein as safety switching methods. In an illustrative embodiment, such safety switching methods provided herein utilize any polynucleotide including a nucleic acid encoding an anti-idiotype polynucleotide and corresponding encoded and expressed anti-idiotype polypeptides in the illustrative embodiments. In some embodiments, such safety switching methods involve delivery of modified CAR-T cells, modified CAR-NK cells, or modified TIL cells to a subject, and may involve delivery of a target clinical antibody. Antibody delivery may be directed and administered to induce proliferation of modified cells in a subject, and/or if a subject develops a clinical symptom indicating that the delivered modified cells or cells derived thereof are causing adverse events in the subject, such as adverse events of grade 1 or grade 2, and in some embodiments adverse events of grade 3 and grade 4, or otherwise putting the subject at risk of medical complications or even death. Thus, in some aspects, provided herein are methods, systems, and kits wherein the same anti-idiotype polypeptides or polynucleotides generally used in these examples include an intracellular proliferation-inducing signaling domain that can be used to induce proliferation or killing, depending on whether the antibody contacted with the modified cell expressing the anti-idiotype polypeptide has a structure that induces cell killing or only induces dimerization of the anti-idiotype polypeptide. Typically, especially in the safety switching method, the delivery of the target clinical antibody will be performed on a later date, i.e. days, weeks or months after the first or last delivery of the modified cells to the subject.

In some embodiments, methods for administering modified cells, such as modified T cells and/or NK cells, can include receiving information about and/or testing for adverse events in a subject, as well as information indicating to a medical professional, patient, and/or caregiver to administer a target antibody if the patient experiences certain adverse events, and in some embodiments, such target antibodies. For example, the indication may specify that the target antibody is to be administered if the patient experiences certain grade 3 or grade 4 adverse events. Such adverse events may include, but are not limited to, tumor lysis syndrome, cytokine Release Syndrome (CRS), macrophage activation syndrome, and/or neurotoxicity. In some embodiments herein, the levels of Interferon (IFN) -gamma, IL-2, soluble IL-2Ralpha, IL-6, soluble IL-6R, and granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, IL-8, IL-10, IL-12, tumor Necrosis Factor (TNF) -alpha, IFN-alpha, monocyte Chemotactic Protein (MCP) -1, macrophage Inflammatory Protein (MIP) 1-alpha may be detected and measured in illustrative embodiments to identify CRS. To detect neurotoxicity, some embodiments herein include detecting and/or receiving reports of reduced level of consciousness, confusion, epilepsy, and cerebral edema.

A method of treating a disease or disorder in a subject having a disease or disorder can include contacting a polynucleotide vector such as an expression vector (including a nucleic acid sequence encoding an anti-idiotype polypeptide provided herein) with peripheral blood cells obtained from the subject to produce modified T cells and/or NK cells, such as modified cytotoxic T cells and/or NK cells, prior to administering the modified T cells and/or NK cells to the subject.

In some embodiments, the methods provided herein, such as methods of delivering modified cells to a subject (which may be adoptive cell therapies), methods for generating a durable population of cells (persistent population), methods for delivering formulations, and the like, are particularly useful for treating cancer, as non-limiting examples. Such cancer may be any type of cancer. For example, such methods can be used to treat a subject suffering from or suffering from a tumor associated with: ovarian cancer, soft tissue sarcoma, peripheral T-cell carcinoma, colorectal cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T-cell lymphoma, non-hodgkin's lymphoma, urothelial cancer, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung cancer, hodgkin's lymphoma, renal cell carcinoma, mesothelioma, metastatic uveal melanoma, renal cancer, blood cancer, HER 2-expressing cancer, non-melanoma skin cancer, liposarcoma, hepatocellular carcinoma, small lymphocytic lymphoma, prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, thyroid medullary carcinoma, triple negative breast cancer, neuroendocrine prostate cancer, bladder cancer, paraganglioma, medulloblastoma, superficial basal cell carcinoma, head and neck squamous cell carcinoma, hematological malignancy, melanoma B-cell lymphoma, recurrent/refractory acute myelogenous leukemia, angiosarcoma, osteosarcoma, refractory cervical cancer, cholangiocarcinoma, osteosarcoma, biliary tract carcinoma, castration-resistant prostate cancer, gastroesophageal adenocarcinoma, rhabdomyosarcoma, carcinoma, non-muscle invasive bladder carcinoma, uveal melanoma, small cell lung cancer, cervical cancer, primary open angle glaucoma, follicular lymphoma, synovial sarcoma, liver cancer, carcinomatosis, pial brain tumor, T-cell lymphoma, small cell lung cancer, mantle cell lymphoma, B-cell malignancy, endometrial carcinoma, mucous-like/round cell liposarcoma, metastatic merck cell carcinoma, neuroblastoma, chronic lymphocytic leukemia, tenosynovial giant cell carcinoma, sarcoma, acute myelogenous leukemia, skin carcinoma, eustachian sarcoma, recurrent/refractory Wen sarcoma, bone cancer, glioma, salivary gland cancer, gastric cancer, benign tumor, low grade malignant serous ovarian cancer, metastatic breast cancer, multiple myeloma, diffuse large B-cell lymphoma, recurrent/refractory lymphoma, metastatic colorectal cancer, advanced malignant tumor, acute lymphoblastic leukemia, solid tumors expressing mesothelin.

In some embodiments, the methods herein can be used to treat tumors that express any one or more of the tumor-associated antigens and/or tumor-specific antigens provided herein, and the engineered T cell receptor and CAR can be designed to recognize such targets. As non-limiting examples, such tumor-associated or tumor-specific antigens include blood tumor antigens provided elsewhere in this specification, and in some non-limiting examples, include the following antigens, most or all of which are considered to be associated with solid tumors: AXL, CD44v6, CAIX, CEA, CD133, c-Met, EGFR, EGFRvIII, epcam, ephA2, GD2, GPC3, GUCY2C, HER1, HER2, ICAM-1, IL13rα2, IL11rα, kras G12D, L1CAM, MAGE, MET, mesothelin, MUC1, MUC16ecto, NKG2D, NY-ESO-1, PSCA, ROR-2, WT-1.

In some embodiments, any of the methods provided herein involving the step of administering may be combined with administration of another cancer therapy, which in certain embodiments may be, for example, a subcutaneously delivered cancer vaccine. In other embodiments, and optionally in further combination with cancer vaccine administration, such methods provided herein comprising administering genetically modified T cells and/or NK cells to a subject, particularly where the subject has, suffers from, or is suspected of having cancer, may further comprise delivering an effective dose of an immune checkpoint inhibitor to the subject. Such checkpoint inhibitor delivery may occur before, after, or simultaneously with administration of the genetically modified T cells and/or NK cells. Immune checkpoint inhibitors are known and a variety of compounds are approved and in clinical development. Checkpoint molecules (many of which are targets of checkpoint inhibitor compounds) include, but are not limited to, anti-PD 1 antibodies.

In some embodiments, the administration is for treating cancer in a subject, and wherein the tumor of the subject regresses within 60 days, 45 days, 30 days, or 14 days after the administration. In some embodiments, the tumor is a hematological cancer, such as DLBCL, which in an illustrative example expresses any of the hematological cancer antigens provided herein. In other embodiments, the tumor is a solid tumor that expresses a solid tumor antigen, which in certain illustrative embodiments is a HER2 positive solid tumor, such as, but not limited to, breast cancer. In some embodiments, the administration is for treating cancer in a subject, and wherein the subject experiences stable disease, at least partial response, or complete response within 90 days, 75 days, 60 days, 45 days, 30 days, or 14 days after the administration, in illustrative embodiments by RECIST1.1 criteria. In some embodiments, the tumor is reduced by at least 10%, 20%, 25%, 30%, 50% or more. In some embodiments, a partial response occurs when the sum of tumor lesions is reduced by 30% or more and is confirmed at least 4 weeks after a previous scan without the appearance of new lesions and/or the short axis of any pathological lymph nodes is reduced to less than 10 mm. In some embodiments, a complete response occurs when all target and non-target lesions disappear. In some embodiments, the administration is for treating cancer in a subject, and wherein the subject experiences at least a partial response or experiences a complete response within 60 days, 45 days, 30 days, or 14 days after the administration. In some embodiments, the subject is a human afflicted with cancer. In some embodiments, the cell preparation is administered 2, 3, 4, 5, 6 or more times, or in illustrative embodiments, only once to the subject prior to the stable disease, or in illustrative embodiments, a partial or complete response is achieved. In some embodiments, the second formulation is administered to the subject at a second, third, fourth, etc., time point between 1 day and 1 month, 2 months, 3 months, 6 months, or 12 months after administration of the first cell formulation, wherein the second formulation may be the same as the first formulation, or may comprise any of the formulations provided herein.

In any aspect provided herein that includes delivery of modified lymphocytes (e.g., modified T cells and/or NK cells), the method can be performed on a mammalian subject that has undergone a lymphocyte depletion process, as known in the art. However, in illustrative embodiments, administration of modified T cells and/or NK cells or RER retroviral particles (RIP) is performed in a method that does not require lymphatic depletion of the subject for successful implantation in the subject and/or for successful reduction of tumor volume in the subject, or on a mammalian (e.g., human) subject that did not experience lymphatic depletion prior to the first days, weeks, or months, or even prior to such administration (e.g., subcutaneous administration). In certain embodiments, administration is performed on a mammalian (e.g., human) subject that does not suffer from low white blood cell count, lymphopenia (lymphopenia), or lymphopenia (lymphocytopenia). In certain embodiments, subcutaneous administration is performed on subjects having lymphocyte counts within the normal range (i.e., 1,000 and 4,800 lymphocytes in 1 microliter (μl) of blood). In certain embodiments, the subcutaneous administration is performed on a subject having 1,000 lymphocytes/μl blood to 5,000 lymphocytes/μl blood, greater than 300 lymphocytes/μl blood, greater than 500 lymphocytes/μl blood, greater than 1,000 lymphocytes/μl blood, greater than 1,500 lymphocytes/μl blood, or greater than 2,000 lymphocytes/μl blood. In certain embodiments, subcutaneous administration is performed on a lymphorich mammalian (e.g., human) subject.

Further details regarding the steps and systems for performing the anti-idiotype methods may be found throughout the present disclosure, including as non-limiting examples in the section entitled "steps and reaction mixtures for the methods herein".

Target antibodies and antibody mimics

The anti-idiotype polypeptides of the present disclosure bind to the idiotype of the target antibody and/or target antibody mimetic. In some embodiments, the target antibody may include one or more domains derived from or derived from a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a chicken antibody, a sheep antibody, a bovine antibody, a llama antibody, a chimeric antibody, or in illustrative embodiments, a human antibody. In some embodiments, different target antibodies or target antibody mimics may be used to bind the same anti-idiotype polypeptide to elicit different responses, e.g., a first target antibody is used to promote cell proliferation and/or survival and a second target antibody mimic is used to promote cell death.

In some embodiments, the target antibody or antibody mimetic is a therapeutic antibody or therapeutic antibody mimetic, respectively. In some embodiments, the target antibody or antibody mimetic may be a clinical antibody or a clinical antibody mimetic, respectively. In some embodiments, the clinical antibody or clinical antibody mimetic is the subject of an FDA approved new drug research application (IND) or equivalent approved regulatory application for human initial clinical testing in another country or jurisdiction. In some embodiments, the target antibody or target antibody mimetic according to the new drug research application or equivalent application is a stand alone product that has not been tested for other active therapeutic agents or components as part of IND. In some illustrative embodiments, the clinical antibody or antibody mimetic is a regulatory agency (e.g., the U.S. FDA) approved biologic. In some embodiments, the target antibody may be one or more of cetuximab (cetuximab), momordica-CD 3 (murominab-CD 3), efalizumab (murominab-CD 3), toximab-i 131 (tositumomab-i 131), nebukumab (banabaumab), edelomab (edereceptacle-mab), katuuzumab (cathetuzumab), daclizumab (daclizumab), olanexiab (olaparimab), aciumab (abciximab), rituximab (rituximab), basiliximab (palizumab), minilizumab (palivizumab), infliximab (infuzumab), trazumab (trastuzumab), adalimumab (adalimumab), ubbeanab (62), oxuzumab (atuzumab), oxybizumab (guanab), oxybizumab (atuzumab (62), oxybizumab (guanab), oxybizumab (atuzumab (guanab), oxybizumab (guanitumomab), oxybizumab (atuzumab) and (14, oxybizumab (tuzumab) and (tulizumab) and (tuzumab) and (tutimuzumab) and may be used in the methods of treating the following the symptoms, the therapeutic system comprises an oxuzumab (oxyuzumab), a siltuzumab (siltuzumab), a ramucirumab (ramucirumab), a vedolizumab (vedolizumab), a nivolumab (nivolumab), a pemuzumab (pembruzumab), a bokutuzumab (blituzumab), an alemtuzumab (allotuzumab), an everkutuzumab (everkutuzumab), an idazodiac bead antibody (idarutuzumab), a bezizumab (neguzumab), a detuzumab (dintuzumab), a stituzumab (stituzumab), a mezuumab (mepolizumab), an alemtuzumab (alizuumaab), a dacuzumab (dacuzumab), a Ai Luozhu monoclonal antibody (eotuzumab), an eizumab (allouzumab), an eizuab (oxytuzumab), a eizumab (stituzumab), a eizukutuzumab (stituzumab), an antibody (stituzumab), a-35, a-antibody (stituzutuzumab), a-antibody (stituzumab) and a-antibody (stituzutuzumab), a-antibody (stituzutuzumab) and a-antibody), a-drum-antibody (stituzutuzutuzumab) and a-antibody (stituzukutuzumab) and a-antibody (stitujauab) and a-under (stitujauzumab) and a) have the therapeutic activity and been determined by the therapeutic system Lenalizumab (landeleumab-flyo), mo Geli group mab (mogamulizumab) (mogamulizumab-kpkc), tildrakizumab (tildrakizumab-asmn), remainderuzumab (freezumab) (freemam), eculizumab (ravulizumab) (ravulizumab-cfm), eculizumab (ravulizumab) (ravulizumab-cvz), cimapru Li Shan antibody (cemiplimab-rwlc), ibamzumab (ibalizumab-uiyk), epalizumab (moxetumomab pasudotox) (moxetumomab pasudotox-tdfk), carboxizumab (plazizumab) (placizumab-nzsg), panuzumab (moxetumomab pasudotox), daclizumab (placizumab-dppuzumab) Risankizumab (rispanlizumab) (rispankizumab-rzaa), vistin-poisatozumab (polatuzumab vedotin) (polatuzumab vedotin-piiq), luo Moshan-anti (polatuzumab vedotin) (romisozumab-aqqg), blosailizumab (brouclizumab-dbll), rispanlizumab (crizanlizumab) (crizanlizumab-tmca), enrolment mab (enfortumab vedotin) (enfortumab vedotin-ejfv), desitrozumab ([ fam-trastuzumab deruxtecan) (fam-trastuzumab deruxtecan-nxki), tetuzumab (tetuzumab) (tetuzumab-trbw), ai Punai-bead mab (epotinizumab-jjjmmr), 4-sibutrab (isuzumab) (isuzumab-393), and (facituzumab govitecan-466-hziatuzumab), infliximab (sacituzumab govitecan) (inflabilizumab-cdon), fluvastatin (tafanstaimab) (tafanstaimab-cxix), bei Lan Taimazethapyr (belantamab mafodotin) (belantamab mafodotin-blmf), saririzumab (satraplizumab-mwge), ati Wei Shankang (ato) flunomab, maltifanab (mattivinab), malti Wei Shankang (maftivisab), odesiimab-ebgn, naxitamab-gqgk, margetuximab-cmkb, asuwimbzykl, everavilamab (evaginaceumab), dortimamab (dorlimimab) (dorlimimab-gxyy), trastuimab (loncastuximab tesirine) (loncastuximab tesirine-lpyrol), epothilamab (amivanmamma) (amitraumaw), amanmab (aman), aman ab (adavanambambab), amab (52), rufimbritumab (52), rufimbritumomab (52), rufimbritude (52), rufimbritumomab (52), rufimbritude (52), rufimbrituab (52), rufimbritumomab (52), rufimbrituab (52), and favalituab (favalimab-35, and favalituab (favalamab-zoma-zoprandib), binding of telbizumab and raffmab to its target antibody or antibody mimetic prevents and/or blocks binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic in illustrative embodiments. In some embodiments, when the anti-idiotype polypeptide binds to a target antibody or antibody mimetic, the extracellular recognition domain of the anti-idiotype polypeptide is capable of, suitable for, and/or configured to prevent and/or block binding of the target antibody or antibody mimetic to a cognate antigen of the target antibody or antibody mimetic. In an illustrative embodiment, the target antibody is cetuximab. In further illustrative embodiments, binding of the anti-idiotype polypeptide to cetuximab prevents and/or blocks binding of cetuximab to Epidermal Growth Factor Receptor (EGFR). In some embodiments, when the anti-idiotype polypeptide binds to cetuximab, the extracellular recognition domain of the anti-idiotype polypeptide is capable of, suitable for, and/or configured to prevent and/or block binding of cetuximab to EGFR.

In some embodiments, the clinical antibody or clinical antibody mimetic has been shown to have an acceptable safety (i.e., adverse event) profile in one or more clinical trials. In some embodiments, the clinical antibody or clinical antibody mimetic has passed a human clinical safety test in an independent clinical trial of the clinical antibody or clinical antibody mimetic. In some embodiments, independent regulatory approval applications for clinical antibodies or clinical antibody mimics have been filed to the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), chinese National Medicines Administration (NMAN) (chinese FDA), or japanese medicine and food safety administration (PFSB). In some embodiments, approval applications (e.g., biological product approval applications (BLAs)) for clinical antibodies or clinical antibody mimics have been filed by the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), chinese national medicines administration (NPA) (chinese FDA), or japan medicine and food safety administration (PFSB). In some embodiments, the clinical antibody or clinical antibody mimetic is an approved biological antibody or antibody mimetic approved by the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), chinese National Medicines Administration (NMAN) (chinese FDA), or japanese medicine and food safety administration (PFSB).

In some embodiments, the target antibody or antibody mimetic may be a bispecific antibody. In some embodiments, the target antibody or antibody mimetic may be a multispecific antibody. Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen. In certain embodiments, a bispecific antibody or antibody mimetic can bind to two different epitopes of a target antigen. In some embodiments, one binding specificity is for one target antigen that is not an antibody or antibody mimetic, and the other binding specificity is for a second target antigen that is an antibody or antibody mimetic.

In some embodiments, the target antibody or antibody mimetic recognized by the anti-idiotype polypeptide comprises an Fc domain from IgM, igD, igG, igA or IgE. In some embodiments, the antibody or antibody mimetic recognized by the anti-idiotype polypeptide comprises an Fc domain from IgM, igD, igG1, igG2, igG3, igG4, igA1, igA2, or IgE. In some embodiments, target antibodies or antibody mimics having IgM Fc domains may be used to drive higher order multimerization.

In some embodiments, the target antibody or antibody mimetic comprises a glycosylated residue. In some embodiments, the target antibody or antibody mimetic comprises an a-Gal epitope. In some embodiments, the target antibody or antibody mimetic comprises one or more glycoforms. In some embodiments, glycoforms or antibody mimics include target antibodies having alpha-1, 3-Gal residues. In some embodiments, the glycoform comprises a target antibody having an N-glycolylneuraminic acid residue. In some embodiments, the glycoform comprises a target antibody having a mannooligosaccharide.

Wherein the cell line from which the antibody or antibody mimetic is produced can affect glycosylation of residues in the polypeptide. In some embodiments, the target antibody or antibody mimetic may be produced in a CHO cell line, BHK cell line, NS0 cell line, SP2/0 cell line, YB2/0 cell line, or HEK293 cell line or derivatives thereof (e.g., HT-1080 cell line, huh-7 cell line, or per.c6 cell line). In illustrative embodiments, the target antibody or antibody mimetic may be produced in a CHO cell line. In other illustrative embodiments, the target antibody or antibody mimetic may be produced in an NS0 cell line. In other illustrative embodiments, the target antibody or antibody mimetic may be produced in an SP2/0 cell line.

In some embodiments, the anti-idiotype polypeptide recognizes a target antibody mimetic. In some embodiments, the target antibody mimetic can be an affinity, an affinity mer, an affinity block, an alpha mer, an alphamab, an anti-carrier, a peptide aptamer, an armadillo repeat protein, a trimer, an affinity multimer (also known as an avidity multimer), a C-type lectin domain, a cysteine-binding microgrotein, a cyclic peptide, a cytotoxic T-lymphocyte-associated protein-4, DARPin (designed ankyrin repeat protein), a fibrinogen domain, a fibronectin binding domain (FN 3 domain) (e.g., an attachment protein or monoclonal antibody), fynomer, a kink bacterium, a Kunitz domain peptide, nanofitin, a leucine-rich repeat domain, a lipocalin domain, mAb 2, or Fcab ^TM Nanobodies, nanomembers, OBody, pronectin, single chain TCRs, triangular tetrapeptide repeat domains, VHH or V-like domains. The antibody mimetic may include an Fc domain to control the effect of binding of the anti-idiotype antibody mimetic to the target antibody mimetic, as discussed in the section "antibody and antibody mimetic effector functions".

Antibody variants

In any of the embodiments disclosed herein, the amino acids in the polypeptide sequence of the extracellular recognition domains of the anti-idiotype polypeptides or antibodies and antibody mimetics that bind to the anti-idiotype polypeptides can contain substitutions or variants. Anti-idiotype polypeptides, antibodies, and antibody mimetics obtained by performing substitutions or variations in the amino acid sequence can be referred to as anti-idiotype polypeptide variants, antibody variants, and antibody mimetic variants, respectively. In some embodiments, the antibodies and antibody mimetics that bind to the anti-idiotype polypeptide can be antibody variants and antibody mimetic variants, respectively. Anti-idiotype polypeptide variants, antibody variants, and antibody mimetic variants can be prepared by introducing appropriate changes to the nucleotide sequence of a nucleic acid encoding an antibody or antibody mimetic. Alternatively, antibody variants and antibody mimetic variants may be prepared by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues within the amino acid sequence of an anti-idiotype polypeptide, antibody or antibody mimetic. Any combination of deletions, insertions, and substitutions may be made to produce an anti-idiotype polypeptide variant, antibody variant, or antibody mimetic variant, provided that the anti-idiotype polypeptide variant, antibody variant, or antibody mimetic variant has the desired characteristics, e.g., idiotype or antigen binding. Without being limited by theory, variations may be introduced into an antibody in order to improve the binding affinity and/or other biological properties of the antibody. In some embodiments, the variants include one or more amino acid substitutions.

In some embodiments, an alanine (Ala) residue may be substituted with valine (Val), leucine (Leu), or isoleucine (Ile). In some embodiments, the arginine (Arg) residue may be replaced with lysine (Lys), glutamine (gin), or asparagine (Asn). In some embodiments, the asparagine (Asn) residue may be substituted with glutamine (gin), histidine (His), aspartic acid (Asp), lysine (Lys), or arginine (Arg). In some embodiments, an aspartic acid (Asp) residue may be substituted with glutamic acid (Glu) or asparagine (Asn). In some embodiments, the cysteine (Cys) residue may be substituted with serine (Ser) or alanine (Ala). In some embodiments, the glutamine (Gln) residue can be substituted with asparagine (Asn) or glutamic acid (Glu). In some embodiments, the glutamic acid (Glu) residue may be substituted with aspartic acid (Asp) or glutamine (Gln). In some embodiments, glycine (Gly) residues may be substituted with alanine (Ala). In some embodiments, the histidine (His) residue may be substituted with asparagine (Asn), glutamine (Gln), lysine (Lys), or arginine (Arg). In some embodiments, the isoleucine (Ile) residue may be replaced with leucine (Leu), valine (Val), methionine (Met), alanine (Ala), phenylalanine (Phe), or norleucine. In some embodiments, the leucine (Leu) residue may be substituted with norleucine, isoleucine (Ile), valine (Val), methionine (Met), alanine (Ala), or phenylalanine (Phe). In some embodiments, the lysine (Lys) residue may be substituted with arginine (Arg), glutamine (gin), or asparagine (Asn). In some embodiments, the methionine (Met) residue may be substituted with leucine (Leu), phenylalanine (Phe), or isoleucine (Ile). In some embodiments, the phenylalanine (Phe) residue may be substituted with tryptophan (Trp), leucine (Leu), valine (Val), isoleucine (Ile), alanine (Ala), or tyrosine (Tyr). In some embodiments, the proline residue may be substituted with alanine (Ala). In some embodiments, serine (Ser) residues may be substituted with threonine (Thr). In some embodiments, the threonine (Thr) residue may be replaced with valine (Val) or serine (Ser). In some embodiments, tryptophan (Trp) may be substituted with tyrosine (Tyr) or phenylalanine (Phe). In some embodiments, the tyrosine (Tyr) residue may be substituted with tryptophan (Trp), phenylalanine (Phe), threonine (Thr), or serine (Ser). In some embodiments, the valine (Val) residue may be substituted with isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), alanine (Ala), or norleucine.

As discussed elsewhere herein, the target antibody or antibody mimetic may include an Fc region. Without being limited by theory, glycosylation of the Fc region of a target antibody or antibody mimetic can affect antibody effector function. In some embodiments, an antibody or antibody mimetic comprising an Fc region can be altered by altering the carbohydrate attached to the Fc region. Natural antibodies produced by mammalian cells typically include branched-chain double-antennary oligosaccharides, which are typically attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of a double-antennary oligosaccharide structure. In some embodiments, modification of oligosaccharides in an antibody or antibody mimetic of the invention can be performed to produce an antibody or antibody mimetic variant with certain improved properties. In some embodiments, the antibody or antibody mimetic variant comprises a carbohydrate structure that lacks fucose attached directly or indirectly to the Fc region. In some embodiments, the amount of fucose in an antibody or antibody mimetic variant can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO2008/077546, the amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g. complexes, mixtures and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, asn297 may also be located within about 3 amino acids upstream or downstream of position 297, i.e., position 294 to position 300, due to minor sequence changes in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US 2003/0157108; US 2004/0093621.

In some embodiments, the antibody or antibody mimetic variant may comprise a bishemi-glycan wherein a bisantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). The bisected GlcNAc structure is a β1, 4-linked GlcNAc attached to the β mannose residues of the core, representing a particular type of N-glycosylation modification. The transfer of the beta-linked core mannose (Man) residue at position 4 by beta 1, 4-mannosyl-glycoprotein 4-beta-N-acetylglucosamine transferase (GlcNAc-III) to complex or mixed N-glycans is considered a bisecting structure that is not generally considered an antenna because it is not further extended by an appropriate enzyme. Such antibodies and antibody mimetic variants may have reduced fucosylation and/or improved ADCC antibody effector function. In some embodiments, the antibody or antibody mimetic variant can include at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibodies and antibody mimetic variants can have improved antibody-mediated complement function. In some embodiments, the Fc region of an antibody or antibody mimetic may include modifications, e.g., substitutions, in one or more amino acids, to produce an Fc region variant. In some embodiments, the Fc region variant may include a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4 Fc region). In some embodiments, the antibody or antibody mimetic variant comprises an Fc region with one or more amino acid substitutions that may have improved ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region of SEQ ID No. 698 (the heavy chain of an E27 anti-IgE antibody). In some embodiments, the antibody or antibody mimetic variant comprises a modification in the Fc region that results in a change (i.e., an improvement or decrease) in CC1q binding and/or Complement Dependent Cytotoxicity (CDC). ). In some embodiments, the antibodies or antibody mimics disclosed herein may be derivatized with non-protein moieties known in the art. In some embodiments, derivatized non-protein moieties suitable for antibodies or antibody mimics include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol) and/or polyvinyl alcohol, and combinations thereof. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if attached to more than one polymer they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular characteristics or functions of the antibody or antibody mimetic to be improved, and whether the antibody or antibody mimetic derivative is to be used in therapy under defined conditions. The antibody or antibody mimetic polymer conjugate may be prepared using any suitable technique for derivatizing antibodies with polymers. In some embodiments, the antibodies and antibody mimetic polymer conjugates disclosed herein include species in which the polymer is covalently attached to one or more specific sites on the parent antibody, i.e., the polymer attachment is targeted to a specific region or one or more specific amino acid residues in the parent antibody or antibody mimetic. Site-specific conjugation of the polymer may be performed by attachment to cysteine residues in the parent antibody or antibody mimetic. In such embodiments, the coupling chemistry may, for example, utilize free thiol groups of cysteine residues in disulfide bridges that are not in the parent antibody. The polymer may be activated with any functional group capable of specifically reacting with the free thiol or thiol group on the parent antibody, such as maleimide, thiol, triflate, tesylate, aziridine, exilane and 5-pyridyl functional groups. The polymer may be coupled to the parent antibody using any scheme suitable for the chemistry of the coupling system chosen. In some embodiments, one or more cysteine residues naturally present in the parent antibody or antibody mimetic are used as attachment sites for polymer conjugation. In another embodiment, one or more cysteine residues are engineered into one or more selected sites in the parent antibody or antibody mimetic to provide one or more specific attachment sites for the polymer. In some embodiments, an antibody fragment such as a Fab may be derivatized to form an antibody fragment-polymer conjugate, and the polymer attached to one or more cysteine residues in the light or heavy chain of the fragment that typically forms an interchain disulfide bond connecting the light and heavy chains.

The skilled artisan will appreciate that any of the embodiments disclosed herein that include antibodies and antibody mimics may alternatively include antibody variants and antibody mimetic variants, respectively, disclosed in this section.

Antibodies and antibody mimetic effector functions

The domains of the antibodies and/or antibody mimics bound by the anti-idiotype polypeptide may control or modulate the downstream effect that the anti-idiotype polypeptide has upon binding to its target antibody or target antibody mimetic. The Fc domain of an antibody may alter the downstream effects of antibody binding to an antigen. Thus, the illustrative methods provided herein include contacting a cell expressing an anti-idiotype polypeptide with a target antibody comprising an idiotype, or delivering the antibody to a subject to effect such contact, which methods can utilize an antibody whose Fc domain is selected to provide a specific function. Without being limited by theory, the Fc domain of an antibody may also affect the function of the antibody to bind to an anti-idiotype polypeptide, also referred to herein as antibody effector function. In some embodiments, the target antibody or antibody mimetic comprises an Fc domain that is capable of cross-linking with an Fc receptor on an effector cell to elicit antibody-dependent cellular cytotoxicity (ADCC) resulting in cell death of the bound target antibody or antibody mimetic. In some embodiments, the effector cells may be one or more of NK cells, monocytes, macrophages, and granulocytes. Fc domain variants include five isoforms IgM, igD, igG, igA and IgE, each of which has unique structural features that affect antibody function. IgG isotypes are further divided into four subclasses, namely IgG1, igG2, igG3 and IgG4 and IgA isotypes are further divided into two subclasses, namely IgA1 and IgA2. In some embodiments, the antibody recognized by the anti-idiotype polypeptide comprises an Fc domain from IgM, igD, igG, igA or IgE. In some embodiments, the antibody recognized by the anti-idiotype polypeptide comprises an Fc domain from IgM, igD, igG1, igG2, igG3, igG4, igA1, igA2, or IgE. In some embodiments, the antibody recognized by the anti-idiotype polypeptide comprises an Fc domain from IgG1, igG2, igG3, or IgG 4. In some embodiments, the antibody recognized by the anti-idiotype polypeptide comprises an Fc domain from IgA1 or IgA2. In some embodiments, the Fc domain of an antibody recognized by an anti-idiotype polypeptide is or is derived from the Fc domain of an antibody of any animal. In some embodiments, the Fc domain of an antibody recognized by an anti-idiotype polypeptide is or is derived from a rat, mouse, or human Fc domain in the illustrative embodiments, and retains at least 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the Fc domain of an antibody recognized by an anti-idiotype polypeptide may be chimeric IgG1, human IgG2, human IgG4, human IgM, humanized IgG1, humanized IgG2/4, humanized IgG4, mouse IgG1, or mouse IgG2a.

In some embodiments, the anti-idiotype polypeptide induces, promotes or activates antibody effector function when the antibody or antibody mimetic binds to the anti-idiotype extracellular recognition domain of the anti-idiotype polypeptide expressed on the cell surface. In some embodiments, binding of the antibody or antibody mimetic to the anti-idiotype polypeptide induces, promotes or activates one or more of antibody-mediated complement activation, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis, antibody-dependent disease enhancement, and opsonization. In some embodiments, the antibody or antibody mimetic is an anti-idiotype multi-domain antibodyPeptide binding does not induce, promote or activate ADCC. In some embodiments, the antibody comprises an Fc domain derived from IgM, igG, or IgA, and the binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates one or more of macrophage opsonophagocytosis, active oxygen burst or release of cytokines, and antimicrobial peptides. In some embodiments, the antibody comprises an Fc domain derived from IgM, igG, or IgA, and the binding of the anti-idiotype polypeptide to the antibody induces, promotes, or activates one or more of antigen uptake, DC maturation, and antigen presentation. In some embodiments, the antibody comprises an Fc domain derived from IgM, igG, or IgA, and the binding of the anti-idiotype polypeptide to the antibody induces, facilitates, or activates antigen capture on follicular dendritic cells for presentation to B cells. In some embodiments, the antibody comprises an Fc domain derived from IgD, igG or IgE, and binding of the anti-idiotype polypeptide to the antibody induces, promotes or activates degranulation of granulocytes and releases vasoactive mediators, chemokines and T _H One or more of the type 2 cytokines. In some embodiments, the antibody comprises an Fc domain derived from IgM or IgG, and the binding of the anti-idiotype polypeptide to the antibody induces, promotes or activates complement-mediated chemokine production, cytotoxicity, and opsonophagocytosis. In some embodiments, the antibody comprises an Fc domain derived from IgM or IgG, and the binding of the anti-idiotype polypeptide to the antibody induces, promotes or activates one or more of neutrophil activation, opsonophagocytosis, reactive oxygen species burst, and induces neutrophil extracellular traps. In some embodiments, the antibody comprises an Fc domain derived from IgG, and binding of the anti-idiotype polypeptide to the antibody induces, promotes or activates one or more of NK cell degranulation and cytotoxicity.

Anti-idiotype composition

In one aspect, provided herein are anti-idiotype polypeptides comprising an extracellular recognition domain and a membrane associated domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody or target antibody mimetic.

In addition, provided herein in another aspect are polynucleotides encoding anti-idiotype polypeptides. For example, in one aspect, provided herein is a polynucleotide comprising one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter, wherein the one or more transcriptional units comprise:

a) Polynucleotide sequences encoding one or more inhibitory RNA molecules and/or a first engineered signaling polypeptide, and

b) A polynucleotide sequence encoding an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane associated domain, wherein the extracellular recognition domain comprises a domain that recognizes the idiotype of a target antibody or antibody mimetic.

In some embodiments, the anti-idiotype polypeptide further includes one or more of a handle domain, an intracellular domain, and/or a linker. The anti-idiotype polypeptide may also be part of a fusion polypeptide, as disclosed in more detail below. In such embodiments, the membrane associating domain may be part of a polypeptide fused to an anti-idiotype polypeptide.

In some embodiments, the anti-idiotype polypeptide may be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 amino acids in length. In some embodiments, the anti-idiotype polypeptide may be 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 25 to 1000, 25 to 900, 25 to 800, 25 to 700, 25 to 600, 25 to 500, 25 to 400, 25 to 300, 25 to 200, 25 to 100, 25 to 50, 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, or 100 to 200 amino acids in length.

Anti-idiotype extracellular recognition domain

The anti-idiotype extracellular recognition domain of an anti-idiotype polypeptide is typically an anti-idiotype antibody or an anti-idiotype antibody mimetic, in whole or more typically in part. The extracellular recognition domain of the anti-idiotype polypeptide can be, has a structure for, is designed for, is selected for, is effective for and/or is suitable for recognizing, binds to or otherwise interacts with an idiotype of a target antibody or an idiotype of a target antibody mimetic. Thus, the recognition domain of an anti-idiotype antibody or antibody mimetic and the target antibody or antibody mimetic having the idiotype bound thereto are members of a specific binding pair.

In some embodiments, the extracellular recognition domain of the anti-idiotype polypeptide recognizes the idiotype of any antibody or antibody mimetic known in the art, a non-limiting example of which may be a clinical antibody or clinical antibody mimetic, as discussed in further detail herein. In some embodiments, the extracellular recognition domain of an anti-idiotype polypeptide herein comprises an idiotype binding variable region of an anti-idiotype antibody or an idiotype binding region of an anti-idiotype antibody mimetic. Such variable regions may include frameworks derived from human framework regions. Such anti-idiotype antibodies or antibody mimics may be any of the antibody fragments or any type of antibody mimics provided herein. As non-limiting examples, the extracellular recognition domain may be an antibody, such as a full length antibody, a single chain antibody, a Fab fragment, a Fab 'fragment, (Fab') 2 fragment, an Fv fragment, an scFv, a bivalent single chain antibody, a bifunctional antibody, a chimeric antibody, or a disbid. In some embodiments, the extracellular recognition domain is a single chain Fv (scFv). In some embodiments, the heavy chain is located N-terminal to the light chain in the engineered signaling polypeptide. In other embodiments, the light chain is located N-terminal to the heavy chain in the engineered signaling polypeptide. In any of the disclosed embodiments, the heavy and light chains can be separated by a linker, as discussed in more detail herein. In any of the disclosed embodiments, the heavy or light chain can be N-terminal to the engineered signaling polypeptide and typically is C-terminal to another domain (e.g., a signal sequence or signal peptide).

In some embodiments, the extracellular recognition domain may include one or more domains from or derived from a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a chicken antibody, a sheep antibody, a bovine antibody, or a llama antibody, or in illustrative embodiments, a human antibody. Other antibody-based recognition domains (cAb VHH (camelid antibody variable domain) and humanized versions, igNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domain) are suitable for use as extracellular recognition domains for the anti-idiotype polypeptides provided herein and methods of use thereof. In some cases, the T Cell Receptor (TCR) -based recognition domain may be an extracellular recognition domain. In some embodiments, the extracellular recognition domain may be bispecific or include a domain from a bispecific antibody. In some embodiments, the extracellular recognition domain may be multispecific or include a domain from a multispecific antibody. Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antibody or antibody mimetic and the other is for another target antibody or antibody mimetic. In certain embodiments, the bispecific antibody can bind to two different epitopes of the target antibody or antibody mimetic. In some embodiments, the extracellular recognition domain can include a recognition domain of an anti-idiotype antibody or antibody mimetic, and further include an ASTR of any of the CARs disclosed herein.

In some embodiments, the extracellular recognition domain can be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids in length. In some embodiments, the extracellular recognition domain may be 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 10 to 30, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 100, 20 to 50, 20 to 30, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 125 to 275, 150 to 275, or 150 to 250 amino acids in length.

Methods for preparing the anti-idiotype extracellular recognition domains provided herein can utilize known methods for preparing anti-idiotype antibodies and antibody mimics, applying strategies such as those provided in the examples herein for the exemplary phage display strategy. Such methods may include preparing an antibody or antibody mimetic library using almost any method known in the art for creating such libraries, and screening the library for idiotypes that bind to the target antibody. Libraries may be constructed by altering the residues of an existing anti-idiotype antibody or may involve a general antibody or antibody mimetic library that does not begin with a selected previous anti-idiotype antibody. Once the library is designed and constructed, the translated antibodies or antibody mimetic protein scaffolds are screened for isolation of mutants with the desired properties. The most commonly used display systems include phage display, ribosome display, mRNA display, yeast display and bacterial cell surface display (Lipovsek and Pluckthun, & J immunology methods) 2004Jul;290 (1-2): 51-67). The skilled artisan will understand how to use this and similar systems to identify suitable anti-idiotype antibodies and antibody mimics, typically using all or a portion of the target antibody as bat, which may be present on a solid support such as a bead during screening. Screening can be performed in several rounds, where conditions can be varied to improve isolated library members for one or more characteristics, including affinity for an idiotype. Other antibodies or antibody mimics having similar constant regions but different idiotypes to the target antibody can be used to subtract out any library member that does not specifically bind to the target antibody. Examples of screening, identifying, and isolating anti-idiotype antibodies using phage display are provided in the examples herein.

In some embodiments, the binding of the anti-id ERD of the anti-idiotype polypeptide to its target antibody or antibody mimetic prevents and/or blocks the binding of the target antibody or antibody mimetic to the cognate antigen of the target antibody or antibody mimetic. In some embodiments, when the anti-idiotype polypeptide binds to a target antibody or antibody mimetic, the extracellular recognition domain of the anti-idiotype polypeptide is capable of, suitable for, and/or configured to prevent and/or block binding of the target antibody or antibody mimetic to a cognate antigen of the target antibody or antibody mimetic. In an illustrative embodiment, the target antibody is cetuximab and the binding of the extracellular recognition domain of the anti-idiotype polypeptide to cetuximab prevents and/or blocks binding of cetuximab to the Epidermal Growth Factor Receptor (EGFR). In some embodiments, when the anti-idiotype polypeptide binds to cetuximab, the extracellular recognition domain of the anti-idiotype polypeptide is capable of, suitable for, and/or configured to prevent and/or block binding of cetuximab to EGFR. In some embodiments, binding of the anti-idiotype polypeptide to the target antibody does not block or prevent binding between the target antibody and its cognate antigen. In illustrative embodiments, binding of the anti-idiotype polypeptide to the target antibody blocks or prevents binding between the target antibody and its cognate antigen. In some embodiments, the extracellular recognition domain recognizes an antigen binding site of a target antibody or antibody mimetic. In some embodiments, the extracellular recognition domain recognizes the variable region, but does not recognize the antigen binding site of the target antibody or antibody mimetic. In some embodiments, the extracellular recognition domain recognizes a portion of a variable region that is not part of the antigen binding site of the target antibody or antibody mimetic. In some embodiments, wherein the extracellular recognition domain recognizes an antigen binding site of the target antibody or antibody mimetic, binding of the extracellular recognition domain to the target antibody or antibody mimetic prevents and/or blocks binding of the target antibody or antibody mimetic to a cognate antigen of the target antibody or antibody mimetic. The antigen binding site may include residues important for the target antibody or antibody mimetic to recognize its cognate antigen.

In some embodiments, the anti-id ERD recognizes (e.g., is capable of binding) cetuximab and comprises any of the sequences provided in this paragraph. In some embodiments, anti-ID ERD may be encoded by a polynucleotide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the polynucleotides in one or more of SEQ ID NOs 376-436. In any of the embodiments disclosed herein that include an anti-ID ERD, the anti-ID ERD can include a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in one or more of SEQ ID NOs 437-497. In some embodiments, the extracellular recognition domain can include a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in one or more of SEQ ID NOs 498-599. In some embodiments, the extracellular recognition domain may comprise a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in one or more of SEQ ID NOs 600-672.

In some embodiments, the anti-id ERD recognizes (e.g., is capable of binding) cetuximab and comprises any of the sequences provided in this paragraph. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the extracellular recognition domain, respectively, can comprise any one of the following combinations: SEQ ID NOs 498, 528 and 560; 499. 529 and 561; 500. 530 and 562; 501. 531 and 563; 502. 532 and 564; 503. 533 and 565; 504. 534 and 566; 505. 535 and 567; 506. 536 and 568; 507. 537 and 569; 508. 538 and 570; 509. 539 and 571; 510. 540 and 572; 511. 541 and 573; 511. 542 and 574; 501. 543 and 575; 512. 544 and 576; 513. 545 and 577; 501. 543 and 578; 514. 546 and 579; 513. 545 and 580; 515. 547 and 581; 516. 548 and 582; 517. 549 and 583; 517. 549, 584; 518. 550 and 585; 519. 551 and 586; 520. 552 and 587; 521. 553 and 588; 522. 554 and 589; 523. 555 and 590; 513. 545 and 591; 517. 549 and 592; 524. 556 and 593; 513. 545 and 594; 511. 542 and 595; 525. 557 and 596; 526. 558 and 597; 527. 559 and 598; or 503, 533 and 599, or a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in any of the foregoing SEQ ID NOs.

In some embodiments, the anti-id ERD recognizes (e.g., is capable of binding) cetuximab and comprises any of the sequences provided in this paragraph. In some embodiments, LCDR1, LCDR2, and LCDR3 of the extracellular recognition domain, respectively, can include any one of the following combinations: SEQ ID NOs 600, 625 and 634; 601. 625 and 635; 600. 625 and 636; 602. 625 and 637; 603. 626 and 638; 603. 626 and 639; 604. 630 and 640; 605. 628 and 641; 606. 625 and 642; 603. 626 and 643; 607. 632 and 644; 608. 625 and 645; 609. 625 and 646; 610. 630 and 647; 604. 630 and 648; 611. 625 and 649; 606. 625 and 650; 612. 625 and 649; 607. 632 and 651; 600. 625 and 652; 600. 625 and 653; 600. 625 and 654; 613. 632 and 655; 614. 625 and 656; 604. 630 and 657; 615. 630 and 658; 616. 625 and 659; 606. 625 and 660; 617. 625 and 661; 604. 630 and 662; 618. 632 and 663; 604. 630 and 664; 619. 630 and 665; 620. 630 and 666; 621. 631 and 667; 606. 627 and 642; 622. 629 and 668; 623. 629 and 669; 600. 625 and 670; 600. 625 and 671; 600. 625 and 672; or 624, 633 and 659, or a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in any of the foregoing SEQ ID NOs.

In some embodiments, the anti-id ERD recognizes (e.g., is capable of binding) cetuximab and comprises any of the sequences provided in this paragraph. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the extracellular recognition domain, respectively, can comprise any one of the following combinations: SEQ ID NOs 498, 528, 560, 600, 625 and 634; 499. 529, 561, 601, 625 and 635; 500. 530, 562, 600, 625, and 636; 501. 531, 563, 602, 625, and 637; 502. 532, 564, 603, 626 and 638; 503. 533, 565, 600, 625, and 634; 504. 534, 566, 603, 626 and 639; 504. 534, 566, 604, 630, and 640; 505. 535, 567, 605, 628 and 641; 506. 536, 568, 606, 625 and 642; 507. 537, 569, 603, 626 and 643; 508. 538, 570, 607, 632 and 644; 509. 539, 571, 608, 625, and 645; 510. 540, 572, 609, 625 and 646; 511. 541, 573, 610, 630, and 647; 511. 542, 574, 604, 630 and 648; 501. 543, 575, 611, 625, and 649; 501. 543, 575, 606, 625, and 650; 501. 543, 575, 612, 625, and 649; 512. 544, 576, 607, 632 and 651; 513. 545, 577, 600, 625 and 652; 501. 543, 578, 600, 625, and 653; 514. 546, 579, 600, 625, and 654; 513. 545, 580, 613, 632 and 655; 513. 545, 580, 606, 625 and 650; 515. 547, 581, 614, 625 and 656; 516. 548, 582, 600, 625, and 634; 517. 549, 583, 604, 630, and 657; 517. 549, 584, 615, 630, and 658; 518. 550, 585, 616, 625, and 659; 519. 551, 586, 606, 625, and 660; 519. 551, 586, 617, 625 and 661; 520. 552, 587, 604, 630 and 662; 521. 553, 588, 618, 632, 663; 522. 554, 589, 604, 630 and 664; 523. 555, 590, 619, 630 and 665; 513. 545, 591, 620, 630 and 666; 517. 549, 592, 621, 631, and 667; 524. 556, 593, 606, 627 and 642; 513. 545, 594, 622, 629 and 668; 511. 542, 595, 600, 625 and 634; 525. 557, 596, 623, 629 and 669; 500. 530, 562, 600, 625, and 670; 526. 558, 597, 600, 625, and 671; 504. 534, 566, 600, 625, and 672; 527. 559, 598, 600, 625, and 634; 503. 533, 599, 600, 625, and 634; or 518, 550, 585, 624, 633 and 659, or a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in any of the foregoing SEQ ID NOs.

In some embodiments, the extracellular recognition domain may be derived or derived from an anti-idiotype antibody mimetic. In some embodiments, the anti-idiotype antibody mimetic can be an affinity, an affinity mer, an affinity block, an alpha body, an alphamab, an anti-carrier protein, a peptide aptamer, an armadillo repeat protein, a trimer, an affinity multimer (also known as an avidity multimer), a C-type lectin domain, a cysteine-binding microgrotein, a cyclic peptide, a cytotoxic T lymphocyte-associated protein-4, DARPin (engineered ankyrin repeat protein), a fibrinogen domain, a fibronectin binding domain (FN 3 domain) (e.g., an attachment protein or monoclonal antibody), fynomer, a kink bacteria, a Kunitz structureDomain peptides, nanofitins, leucine rich repeat domains, lipocalin domains, mAb 2 or Fcab ^TM Nanobodies, nanomembers, OBody, pronectin, single chain TCRs, triangular tetrapeptide repeat domains, VHH or V-like domains. Without being limited by theory, antibody mimetics can provide superior properties over antibodies, including but not limited to superior solubility, tissue penetration, stability against heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs.

In some embodiments, the extracellular recognition domain of the anti-idiotype polypeptide comprises an affinity mer capable of binding to a target antibody. The average size of the affinity mer may be in the range of 12-14 kDa. Without being limited by theory, affinity polymers capable of binding to a particular target are designed by selecting an appropriate scaffold suitable for incorporation of the recognition domain. Scaffolds are three-dimensional protein structures that are suitable for mutation and insertion with sufficient flexibility in their primary structure, the introduced modifications not compromising their secondary structure and overall stability. The scaffold is typically a small, thermostable single domain protein without any disulfide bonds or glycosylation. The affinity polymers may be made from two scaffolds, an adhron scaffold and a human stefin a scaffold. The stefin a scaffold was engineered from human stefin a protein, whereas the adhron scaffold was originally synthesized based on cystatin sequences. After scaffold selection, libraries were designed by employing computer methods and constructed via molecular biology protocols. Once candidates for mutagenesis are identified, mutant constructs and libraries of antibody mimics can be generated at the DNA level by employing site-directed or random mutagenesis strategies. Once the library is designed and constructed, the translated protein scaffold is screened for isolation of mutants with the desired properties. The most commonly used display systems include phage display, ribosome display, mRNA display, yeast display and bacterial cell surface display. The skilled artisan will understand how to use this and similar systems to identify appropriate anti-idiotype affinity polymers.

In some embodiments, the extracellular recognition domain of the anti-idiotype polypeptide comprises DARPin capable of binding to a target antibody. DRPins are made from closely packed repeats of 33 amino acid residues. Each repeat sequence forms a structural unit consisting of a beta turn followed by two antiparallel alpha helices. DARPins are small proteins with molecular weights in the range of 14-18kDa, which are extremely thermostable and resistant to proteases and denaturants. DARPins typically have a scaffold that is a constant region and have variable sites that do not alter the conformation of the protein by amino acid substitutions. The process of designing scaffolds involves designing libraries of protein variants by random site-specific mutagenesis; and selecting molecules using techniques such as yeast display, phage display, and ribosome display. DRPins can recognize targets with high specificity and affinity that exceed that of antibodies. The skilled artisan will understand how to use this and similar systems to identify the appropriate anti-idiotype DARPins.

In some embodiments, the extracellular recognition domain of the anti-idiotype polypeptide comprises a nanobody capable of binding to a target antibody. Nanobodies are antigen-binding fragments, which are about 12-15kDa in size. Without being limited by theory, nanobodies include the antigen binding ability of the original heavy chain antibody that evolves to be fully functional without the light chain. Nanobodies consist of three antigen Complementarity Determining Regions (CDRs) and four Framework Regions (FR). CDRs are the binding regions of the nanobody to the antigen or target antibody. To generate nanobodies, libraries are prepared from samples including camels. Phage display can be used to screen and enrich nanobody-phage with specific binding capacity from nanobody libraries. Functional verification and validation of nanobody expression may also be performed to select the best candidate. The skilled artisan will understand how to use this and similar systems to identify appropriate anti-idiotype nanobodies.

Handle domain

In some embodiments, the anti-idiotype polypeptide comprises a stem located at a portion of the anti-idiotype polypeptide that is outside the cell and between the extracellular recognition domain and the membrane association domain. In some embodiments, the handle has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type CD8 handle region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFA (SEQ ID NO: 2)), at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type CD28 handle region (FCKIEVMYPPPYLDNEKSNGTII HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3)), or at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type immunoglobulin heavy chain handle region. In anti-idiotype polypeptides, the handle employed allows extracellular recognition domains and typically the entire anti-idiotype polypeptide to maintain increased binding to the target antibody or antibody mimetic. In some embodiments, the handle domain may be a heavy chain Fc domain.

The handle region may be about 4 amino acids to about 50 amino acids in length, such as about 4aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 40aa, or about 40aa to about 50aa.

In some embodiments, the stem of the anti-idiotype polypeptide comprises at least one cysteine. For example, in some embodiments, the handle may include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 4). If present, the cysteine in the stalk of the first anti-idiotype polypeptide may be capable of forming a disulfide bond with the stalk in the second anti-idiotype polypeptide.

The handle may comprise an immunoglobulin hinge region amino acid sequence known in the art; see, e.g., tan et al (1990) Proc. Natl. Acad. Sci. USA, 87:162; and Huck et al (1986) nucleic acid research (nucleic acids Res.) 14:1779. As non-limiting examples, an immunoglobulin hinge region may comprise a domain having at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO: 5); CPPC (SEQ ID NO: 4); CPEPKSCDTPPPCPR (SEQ ID NO: 6) (see, e.g., glaser et al (2005), "J.Biol.chem.)," 280:41494); ELKTPLGDTTHT (SEQ ID NO: 7); KSCDKTHTCP (SEQ ID NO: 8); KCCVDCP (SEQ ID NO: 9); KYGGPPCP (SEQ ID NO: 10); EPKSCDKTHTCPPCP (SEQ ID NO: 11) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 12) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 13) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 14) (human IgG4 hinge), etc. The handle may include a hinge region having the amino acid sequence of a human IgG1, igG2, igG3 or IgG4 hinge region. The handle may comprise one or more amino acid substitutions and/or insertions and/or deletions compared to the wild-type (naturally occurring) hinge region. For example, his229 of a human IgG1 hinge may be substituted with Tyr such that the handle includes the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 15) (see, e.g., yan et al (2012) J.Biol.chem.) (287:5891). The handle may comprise an amino acid sequence derived from human CD 8; for example, the handle may comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16), or a variant thereof.

In some embodiments, the stem domain of an anti-idiotype polypeptide may include any of the dimerization moieties disclosed herein. In any aspect or embodiment wherein the stalk domain of the anti-idiotype polypeptide comprises a dimerization motif, the dimerization motif may be selected from the group consisting of: leucine zipper motif-containing polypeptides, CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271 and CD324, and mutants and/or active fragments thereof that retain dimerization capacity.

Membrane association domain

The membrane associating domain of the anti-idiotype polypeptide attaches the extracellular recognition domain of the anti-idiotype polypeptide to the cell membrane. In some embodiments, the membrane association is a transmembrane domain. In some embodiments, the transmembrane domain is a heterologous transmembrane domain. In other embodiments, the transmembrane domain is an endogenous transmembrane domain. In some embodiments, the transmembrane domain is from or derived from an antibody. In illustrative embodiments, the transmembrane domain is derived or derived from IgD. In some embodiments (including those in which the transmembrane domain is derived or derived from IgD), the polynucleotide, vector, or cell may further comprise nucleic acids encoding IgA and IgB. CA (CA)

In some embodiments, the transmembrane domain may include a transmembrane domain from or derived from: BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD, CD5, CD7, CD8A, CD8B, CD, CD11A, CD11A, CD11A, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49A, CD49A, CD64, CD79A, CD79A, CD80, CD84, CD86, CD96 (tactile), CD100 (SEMA 4D), CD103, C134 CD137, CD154, CD160 (BY 55), CD162 (SELPLG), CD226 (DNAM 1), CD229 (Ly 9), CD247, CRLF2, CRTAM, CSF2RA, CSF2RB, CSF 3A, CD 1A, CD 2A, CD2, GHR, HVEM (LIGHTR), IA4, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA IL 4A, CD RA, IL 6A, CD ST, IL7RA Ins PPCL, IL 9A, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD RA, IL31RA, ITGA1, ITGA4, ITGA6, A, CD1, ITGB2, ITGB7, KIRDS2, LEPR, LFA-1 (CD 11a, CD 18), A, CD (KLRF 1), OSMR, PAG/A, CD1, SLAM (SLAMF 1, CD150, IPO-3), AMF4 (CD 244, 2B 4), SLF 6 (NTB-A, CD 108), SLAMF7, SLAMF8 (SLSF 8, TNSF 2, TNSF 6, or a mutant thereof. In some embodiments, the transmembrane domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in one or more of: CD 8. Alpha. TM (SEQ ID NO: 17); CD 8. Beta. TM (SEQ ID NO: 18); CD4 handle (SEQ ID NO: 19); CD3Z TM (SEQ ID NO: 20); CD28 TM (SEQ ID NO: 21); CD134 (OX 40) TM: (SEQ ID NO: 22); CD7 TM (SEQ ID NO: 23); CD8 handle and TM (SEQ ID NO: 24); CD28 handle and TM (SEQ ID NO: 25); the hPDGFRB hinge and TM of SEQ ID NO. 676 (this sequence also includes an 8aa intracellular domain); CD28 hinge and TM of SEQ ID NO. 677 (this sequence also includes the 9aa intracellular domain); the CD80 hinge and TM domain of SEQ ID NO. 678 (this sequence also includes the intracellular domain); or CD28 hinge and TM of SEQ ID NO:679 (the sequence also includes an intracellular domain).

In some embodiments, the transmembrane domain of an anti-idiotype polypeptide may include any of the dimerization moieties disclosed herein. In any aspect or embodiment wherein the transmembrane domain of the anti-idiotype polypeptide comprises a dimerization motif, the dimerization motif may be selected from the group consisting of: leucine zipper motif-containing polypeptides, CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271 and CD324, and mutants and/or active fragments thereof that retain dimerization capacity.

In some embodiments, the membrane associated domain is Glycosyl Phosphatidylinositol (GPI). In some embodiments, the anti-idiotype polypeptide is fused to a lymphoproliferative element, CAR, and/or recombinant TCR, and the membrane associating domain can be a transmembrane domain of the lymphoproliferative element, CAR, and/or recombinant TCR.

Intracellular domains

The intracellular domains of the anti-idiotype polypeptides, if present, are typically all or more typically a portion of one or more of the intracellular domains known in the art or disclosed herein. The intracellular domain of the anti-idiotype polypeptide may perform a variety of functions including, for example, a non-signaling function that anchors the anti-idiotype polypeptide, and a signaling function that activates proliferation, survival and/or cell death signaling or modulates transcriptional activity, for example. Thus, in some embodiments, the anti-idiotype polypeptide includes an intracellular domain. In some embodiments, for example, when one or more of the anti-idiotype polypeptide domains is part of a fusion polypeptide, the intracellular domain of the fusion polypeptide can be the anti-idiotype polypeptide intracellular domain. In other embodiments, the intracellular domain of the fusion polypeptide can be the intracellular domain of a polypeptide fused to an anti-idiotype polypeptide, such as the intracellular domain of a lymphoproliferative element, CAR, and/or recombinant TCR. The intracellular domains that can be used include any of the lymphoproliferative elements, CARs, and recombinant TCRs disclosed elsewhere herein. Thus, in some embodiments, the intracellular domains of an anti-idiotype polypeptide may include one or more, two or more, or three or more intracellular domains from other polypeptides, such as any of the intracellular domains of the lymphoproliferative elements, CARs, and/or recombinant TCRs disclosed herein. In some embodiments, the intracellular domain may include any one or more of the intracellular activation domains, regulatory domains, or intracellular signaling domains disclosed elsewhere herein. In some embodiments, the intracellular domain of the anti-idiotype polypeptide may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, or 200 amino acids in length. In some embodiments of the present invention, in some embodiments, the intracellular domain of the anti-idiotype polypeptide may be 1 to 1000, 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 1 to 25, 5 to 1000, 5 to 900, 5 to 800, 5 to 700, 5 to 600, 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 5 to 25, 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 10 to 25, 25 to 1000, 25 to 900, 25 to 800, 25 to 700, 25 to 600 25 to 500, 25 to 400, 25 to 300, 25 to 200, 25 to 100, 25 to 50, 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 1000, 200 to 900, 200 to 800, 200 to 700, 200 to 600, 200 to 500, 200 to 400, 200 to 300, 300 to 1000, 300 to 900, 300 to 800, 300 to 700, 300 to 600, 300 to 500, or 300 to 400 amino acids. In some embodiments, the intracellular domain is an anchor for the anti-idiotype polypeptide. In some embodiments, the intracellular domain has no signaling activity.

In some embodiments, signaling through the intracellular domain of the anti-idiotype polypeptide is inducible, and the signaling can be activated by the addition of a target antibody or antibody mimetic. Such anti-idiotype polypeptides may be referred to herein as activity modulators, wherein upon addition of a target antibody or antibody mimetic, two or more anti-idiotype polypeptides may dimerize by binding to the same molecule of the target antibody or antibody mimetic. This dimerization of the anti-idiotype polypeptides dimerizes the intracellular domains of both anti-idiotype polypeptides, resulting in an activation-based dimerization if the appropriate intracellular domain is used. In some embodiments, the intracellular domain of the activity-modulating anti-idiotype polypeptide can be activated by dimerization, and the only dimerizing moiety on the anti-idiotype polypeptide is the extracellular recognition domain. In other embodiments, the intracellular domain of the activity-modulating anti-idiotype polypeptide can be activated by dimerization, and the anti-idiotype polypeptide includes a separate inducible or constitutive dimerization moiety on the anti-idiotype polypeptide in addition to the extracellular recognition domain. In some embodiments, the intracellular domain of the activity-modulating anti-idiotype polypeptide can be activated by multimerization (e.g., trimerization) of at least three domains, and the only dimerizing moiety on the anti-idiotype polypeptide is the extracellular recognition domain. In other embodiments, the intracellular domains of the activity-modulating anti-idiotype polypeptide can be activated by multimerization (e.g., trimerization) of at least three domains, and the anti-idiotype polypeptide includes a separate inducible or constitutive dimerization moiety on the anti-idiotype polypeptide in addition to the extracellular recognition domain. In an illustrative embodiment, the intracellular domains of the active-modulating anti-idiotype polypeptide activated by multimerization (e.g., trimerization) of at least three domains include separate constitutive dimerization moieties on the anti-idiotype polypeptide in addition to the extracellular recognition domain. In some embodiments, multimerization (e.g., dimerization, trimerization, etc.) of the anti-idiotype polypeptide activates proliferation and/or survival signaling. Such embodiments include, for example, fusion polypeptides in which an extracellular recognition domain is attached to a lymphoproliferative element, as well as anti-idiotype polypeptides having an intracellular domain from a lymphoproliferative element (e.g., an intracellular signaling domain from a cytokine receptor). In some embodiments, multimerization of the anti-idiotype polypeptide activates cell death signaling. Such embodiments include, for example, anti-idiotype polypeptides having the intracellular domains of some of the apoptosis-inducing polypeptides disclosed herein. In some embodiments, the target antibody or antibody mimetic recognized by the anti-idiotype polypeptide comprises an Fc domain from IgM or IgA, and binding of the target induces multimerization (e.g., trimerization and higher order multimerization). In such embodiments, the target antibody or antibody mimetic can activate an intracellular domain that requires higher order multimerization (e.g., trimerization). In some embodiments, the anti-idiotype polypeptide recognizes nebulomab including an Fc domain from IgM, and binding of the anti-idiotype polypeptide to nebulomab induces multimerization and activation of the intracellular domain of the anti-idiotype polypeptide.

In some embodiments, the intracellular domains can include one or more, two or more, three or more, or all of the domains, motifs and/or mutations of any of the intracellular domains disclosed herein. In some embodiments, the intracellular domains may include one or more, two or more, three or more, or all domains, motifs and/or mutations of any intracellular domain known to induce proliferation and/or survival of T cells and/or NK cells. In some embodiments, the intracellular domain may activate a Jak pathway, a Stat pathway, a Jak/Stat pathway, a TRAF pathway, a PI3K pathway, and/or a PLC pathway. Suitable intracellular domains for activating various pathways are disclosed herein as "lymphoproliferative elements" and may be used as the intracellular domain of an anti-idiotype polypeptide. In some embodiments, the intracellular domains of the anti-idiotype polypeptide may include all or part of one or more intracellular signaling domains from one or more cytokine receptors. In further embodiments, the one or more cytokine receptors may be selected from the group consisting of CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL RA, IL2RB, IL2RG, IL3RA, IL4R, IL RA, IL6R, IL ST, IL7R, IL RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL13R, IL RA1, IL13RA2, IL15R, IL RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23R, IL R, IL RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TGF beta R, TGF beta-bait, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18.

In some embodiments, the intracellular domain of the anti-idiotype polypeptide comprises one or more apoptotic domains known to induce cell death in T cells and/or NK cells. Such intracellular domains are also referred to herein as intracellular apoptosis domains, and include domains from apoptosis-inducing polypeptides, referred to herein as apoptotic polypeptides and functional fragments thereof. The intracellular apoptosis domain of the anti-idiotype polypeptide may include one or more Caspase Activation and Recruitment Domains (CARDs), death Domains (DDs), death Effector Domains (DED), thermal protein domains (PYD), and/or caspase proteolytic domains. Typically, such apoptotic polypeptides are capable of inducing an apoptotic signal upon dimerization. The skilled artisan will understand how to identify and incorporate these intracellular apoptosis-inducing domains (and other apoptosis-inducing domains) into the anti-idiotype polypeptides of the present disclosure. In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide may include one or more CARD, DD, DED, PYD and/or caspase proteolytic domains. In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide may include one or more means for performing the function of a caspase, such as CARD, DD, DED, PYD and/or caspase proteolytic domains of caspase 2, 8, 9 or 10. In some embodiments, the intracellular apoptosis domain may include one or more CARDs from Apaf-1, DARK, CED-4, CED-3, dronc, CARMA1, bcl-10, nod1, nod2, RIP2, ICEBERG, RIG-I, MDA5, MAV5, ASC, NALP1, caspase 2, caspase 5, and/or caspase 9, and/or functional fragments thereof. In some embodiments, the intracellular apoptosis domain may include one or more DDs from TNF-R1, fas, p75, TRADD, FADD, RIP, myD88, IRAK, pelle, tube, PIDD, RAIDD, and/or MALT1 and/or functional fragments thereof. In some embodiments, the intracellular apoptosis domain may include one or more DED from FADD, caspase 8, caspase 10, c-FLIP, v-FLIP, MC159, PEA-15, ddd, and/or ddd 2 and/or functional fragments thereof. In some embodiments, the intracellular apoptosis domain may include one or more PYDs from ASC, ASC2, NALP1, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, and/or NALP12, and/or functional fragments thereof. In some embodiments, the intracellular apoptosis domain may include one or more domains from caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, FAS (CD 95 or Apo-1 antigen), TNF-R1, death receptor 3 (DR 3), death receptor 4 (DR 4 or TRAIL receptor-I), death receptor 5 (DR 5 or TRAIL receptor-II), FADD, APAF1, CRADD/RAIDD, ASC, bcl-2 family members, bax, bak, RIPK3 and/or RIPK1-RHIM, and/or functional fragments thereof. Without being limited by theory, bax and Bak are pro-apoptotic Bcl-2 family members that can cause mitochondrial depolarization (or mislocalization of anti-apoptotic family members such as Bcl-xL or Bcl-2). Without being limited by theory, the RIPK3 and RIPK1-RHIM domains may trigger a related form of pro-inflammatory cell death, known as necrotic apoptosis, due to MLKL-mediated membrane cleavage. In illustrative embodiments, the intracellular apoptosis domain comprises one or more of a caspase 2 polypeptide, a caspase 8 polypeptide, a caspase 9 polypeptide, and/or a caspase 10 polypeptide. Caspase 2, caspase 8, caspase 9, and caspase 10 are also referred to herein as promoter caspases. In some embodiments, the intracellular domains of the anti-idiotype polypeptide include a fusion polypeptide of one or more domains of a promoter caspase and one or more domains of an effector caspase. In some embodiments, the promoter caspases may be caspase 2, caspase 8, caspase 9, and/or caspase 10. In some embodiments, the effector caspases may be caspase 3, caspase 6, and/or caspase 7. In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide may include at least one domain from one or more of caspase 2, caspase 8, caspase 9, or caspase 10, or functional fragments thereof. In some embodiments, the functional fragment is an intracellular domain from a caspase polypeptide lacking one or more domains. In some embodiments, the one or more domains lacking in the functional fragment may be CARD, DD, DED, PYD and/or a caspase proteolytic domain. In some embodiments, the functional fragment is a caspase 2 polypeptide lacking CARD. In some embodiments, the functional fragment is a caspase 8 polypeptide lacking DED.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a caspase 2 polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID NOs 680. In some embodiments, the intracellular apoptosis domain includes all of the domains of the protein of SEQ ID NO. 680. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the polypeptide of SEQ ID NO 680. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the polypeptide of SEQ ID NO 680. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or a stretch of at least 10, 15, 20, or all of the CARD of caspase 2 (amino acids 32 to 118 of SEQ ID NO: 680) and/or the CASc domain of caspase 2 (amino acids 192 to 447 of SEQ ID NO: 680). In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in SEQ ID NO:680, wherein the polypeptide sequence does not comprise a CARD (amino acids 32 to 118 of SEQ ID NO: 680) and/or a stretch of at least 10, 15, 20 or all amino acids in the CASc domain of caspase 2 (amino acids 192 to 447 of SEQ ID NO: 680). In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acids 327 to 452 of SEQ ID NO:680 or to a stretch of at least 10, 15, 20, or all of amino acids 359 to 452 of SEQ ID NO: 680.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a caspase 8 polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID No. 681. In some embodiments, the intracellular apoptosis domain comprises all of the domains of the protein of SEQ ID NO: 681. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 681. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the DED (amino acids 3 to 84 of SEQ ID NO: 681) of caspase 8, DD (amino acids 134 to 212 of SEQ ID NO: 681) of caspase 8, and/or a segment of the CASc domain of amino acids 242 to 494 of caspase 8 of SEQ ID NO: 681. In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in SEQ ID No. 681, wherein the polypeptide sequence does not comprise a DED of caspase 8 (amino acids 3 to 84 of SEQ ID No. 681), a DD of caspase 8 (amino acids 134 to 212 of SEQ ID No. 681) and/or a stretch of at least 10, 15, 20 or all amino acids in the CASc domain of amino acids 242 to 494 of caspase 8 of SEQ ID No. 681. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acids 384 to 496 of SEQ ID No. 681 or to a stretch of at least 10, 15, 20, or all amino acids 411 to 496 of SEQ ID No. 681.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a caspase 9 polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 682. In some embodiments, the intracellular apoptosis domain comprises all of the domains of the protein of SEQ ID NO 682. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO 682. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or a stretch of at least 10, 15, 20 or all of the CARD (amino acids 17 to 90 of SEQ ID NO: 682) of caspase 9 and/or the CASc domain of caspase 9 (amino acids 152 to 414 of SEQ ID NO: 682). In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID No. 682, wherein the polypeptide sequence does not comprise a CARD of caspase 9 (amino acids 17 to 90 of SEQ ID No. 682) and/or a stretch of at least 10, 15, 20 or all amino acids of the CASc domain of caspase 9 (amino acids 152 to 414 of SEQ ID No. 682). In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acids 294 to 416 of SEQ ID NO:682 or to a stretch of at least 10, 15, 20 or all amino acids 336 to 416 of SEQ ID NO: 682.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a caspase 10 polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO: 683. In some embodiments, the intracellular apoptosis domain includes all of the domains of the protein of SEQ ID NO: 683. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 683. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or a stretch of at least 15, 20 or all of the DD (amino acids 18 to 99 of SEQ ID NO: 683) of caspase 10, the DED (amino acids 112 to 190 of SEQ ID NO: 683) of caspase 10, and/or the CASc domain of caspase 10 (amino acids 233 to 474 of SEQ ID NO: 683). In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in SEQ ID NO:683, wherein the polypeptide sequence does not comprise a stretch of at least 10, 15, 20 or all amino acids in DD of caspase 10 (amino acids 18 to 99 of SEQ ID NO: 683), DED of caspase 10 (amino acids 112 to 190 of SEQ ID NO: 683) and/or CASc domain of caspase 10 (amino acids 233 to 474 of SEQ ID NO: 683). In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acids 365 to 478 of SEQ ID NO:683 or a stretch of at least 10, 15, 20 or all amino acids 388 to 478 of SEQ ID NO: 683.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide may include one or more means for activating a promoter caspase or an effector caspase or both. In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide may include one or more means for binding to the death domain. In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide may include one or more means for performing the function of caspase 2, 3, 9, or 10 or the death domain of FAS or TNF receptor ICD. In some illustrative embodiments, the anti-idiotype polypeptides further comprise a dimerization moiety, wherein the dimerization moiety is constitutively dimerized. In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide intracellular domain comprises one or more domains from a FAS (CD 95 or Apo-I antigen) polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID nos. 684. In some embodiments, the intracellular apoptosis domain comprises all of the domains of the protein of SEQ ID NO: 684. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 684. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD (amino acids 257 to 341 of SEQ ID NO: 684) of the FAS polypeptide. In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID NO:684, wherein the polypeptide sequence does not comprise a stretch of at least 10, 15, 20 or all amino acids in DD (amino acids 257 to 341 of SEQ ID NO: 684) of a FAS polypeptide.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a TNF-R1 (tumor necrosis factor receptor superfamily member 1) polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID NO: 685. In some embodiments, the intracellular apoptosis domain comprises all of the domains of the protein of SEQ ID NO: 685. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 685. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the DD (amino acids 358 to 438 of SEQ ID NO: 685) of the TNF-R1 polypeptide. In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID NO:685, wherein the polypeptide sequence does not comprise a stretch of at least 10, 15, 20 or all amino acids in DD (amino acids 358 to 438 of SEQ ID NO: 685) of a TNF-R1 polypeptide.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a DR-3 (tumor necrosis factor receptor superfamily member 25) polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID nos. 686. In some embodiments, the intracellular apoptosis domain includes all of the domains of the protein of SEQ ID NO: 686. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 686. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the DD (amino acids 32 to 145 of SEQ ID NO: 686) of the DR-3 polypeptide. In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID NO:686, wherein said polypeptide sequence does not comprise a stretch of at least 10, 15, 20 or all amino acids in DD (amino acids 32 to 145 of SEQ ID NO: 686) of a DR-3 polypeptide.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a DR-4 (tumor necrosis factor receptor superfamily member 10A; TRAIL receptor-I) polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID nos. 687. In some embodiments, the intracellular apoptosis domain includes all of the domains of the protein of SEQ ID NO: 687. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 687. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the DD (amino acids 367 to 454 of SEQ ID NO: 687) of the DR-4 polypeptide. In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID NO:687, wherein said polypeptide sequence does not comprise a stretch of at least 10, 15, 20 or all amino acids in DD (amino acids 367 to 454 of SEQ ID NO: 687) of a DR-4 polypeptide.

In some embodiments, the intracellular apoptosis domain of the anti-idiotype polypeptide comprises one or more domains from a DR-5 (tumor necrosis factor receptor superfamily member 10B; TRAIL receptor-II) polypeptide. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of SEQ ID nos. 688. In some embodiments, the intracellular apoptosis domain comprises all of the domains of the protein of SEQ ID NO: 688. In some embodiments, the intracellular apoptosis domain comprises one or more domains of the protein of SEQ ID NO: 688. In some embodiments, the intracellular apoptosis domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the DD (amino acids 341 to 428 of SEQ ID NO: 688) of the DR-5 polypeptide. In some embodiments, the intracellular apoptotic domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID NO:688, wherein said polypeptide sequence does not comprise a stretch of at least 10, 15, 20 or all amino acids in the DD (amino acids 341 to 428 of SEQ ID NO: 688) of a DR-5 polypeptide.

In some embodiments, the anti-idiotype polypeptide comprises a cleavable signal. In some embodiments, the cleavable signal may comprise a proteolytic cleavage site that binds to an induced Notch receptor. In some embodiments, the cleavable signal may comprise a gamma secretase recognition sequence.

In some embodiments, the anti-idiotype polypeptide comprises a Notch receptor comprising an extracellular domain having a recognition site for a target antibody or target antibody mimetic, a transmembrane domain having a binding-induced proteolytic cleavage site, and one or more intracellular domains. In some embodiments, the intracellular domain may include one or more transcription factors. Without being limited by theory, the conjugation of the extracellular domain of the Novch receptor to its ligand (which may be a target antibody or antibody mimetic) results in intramembrane proteolysis (sequential proteolysis of depolymerizing elements and metalloprotease (ADAM) metalloproteases and gamma secretase complexes) by dimerization of the receptor. The induced cleavage of the receptor releases the intracellular fragment of Notch. In some embodiments, the Notch receptor required for dimerization may not be present in the anti-idiotype polypeptide. In further embodiments, the anti-idiotype polypeptide may include an extracellular recognition domain that may provide dimerization upon addition of an antibody or antibody mimetic. Notch intracellular domains are transcriptional regulators that release from the membrane and translocate into the nucleus after dimerization to activate target genes that play a critical role in cell-cell signaling during development. In some embodiments, the intracellular domain of the anti-idiotype polypeptide may comprise a Notch intracellular domain. Such intracellular domains on anti-idiotype polypeptides that modulate transcription are referred to herein as intracellular transcription domains, and can increase or decrease expression of a target gene that binds to an intracellular transcription domain. In some embodiments, the intracellular domain is a transcriptional activator and increases expression. In some embodiments, the intracellular domain is a transcriptional repressor and reduces expression. In some embodiments, the intracellular domain of the anti-idiotype polypeptide may be an artificial transcription factor. In some embodiments, the artificial transcription factor may include domains from zinc finger nucleases, gal4 and/or tetR, or any other transcription factor known in the art. In some embodiments, the intracellular domain is a site-specific nuclease. The site-specific nuclease may be one or more of a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas9 system. In some embodiments, the intracellular domain comprises one or more domains of a caspase 9 polypeptide. In some embodiments, the intracellular domain is a recombinase. In some embodiments, the intracellular domain is an inhibitory immunoreceptor. In some embodiments, the intracellular domain is an activated immune receptor. In some embodiments, release of the intracellular domain modulates cell proliferation. In some embodiments, release of the intracellular domain modulates apoptosis in the cell. In some embodiments, release of the intracellular domain induces cell death by a mechanism other than apoptosis. In some embodiments, release of the intracellular domain modulates gene expression in the cell by transcriptional regulation, chromatin regulation, translation, trafficking, or post-translational processing. In some embodiments, release of the intracellular domain modulates differentiation of the cell. In some embodiments, release of the intracellular domain modulates migration of the cell. In some embodiments, release of the intracellular domain modulates expression and secretion of the molecule from the cell. In some embodiments, release of the intracellular domain modulates adhesion of the cell to a second cell or to an extracellular matrix. In some embodiments, release of the intracellular domain de novo induces expression of the gene product in the cell. In some embodiments, release of the intracellular domain de novo induces expression of a gene product in the cell, wherein the gene product is a transcriptional activator, transcriptional repressor, chimeric antigen receptor, second chimeric Notch receptor polypeptide, translation regulator, cytokine, hormone, chemokine, or antibody. Some examples of such Notch receptors and intracellular domains that release upon binding of a target antibody or antibody mimetic to a Notch receptor provided in US10590182B2 are incorporated herein.

In some embodiments, the anti-idiotype polypeptide may include a cleavable signal, which may be one or more gamma secretase substrate sequences that are recognizable by a gamma secretase for cleavage. The gamma secretase substrate sequence (also referred to as a gamma secretase recognition sequence) is a sequence derived from the gamma secretase substrate due to cleavage of the substrate. In some embodiments, one or more gamma secretase substrate sequences may be derived from one or more polypeptides, such as ERBB4, INSR, IGF1R, CSF1R, VEGFR, VEGFR2, VEGFR3, FGFR4, PTK7, TRKA, TRKB, MUSK, MET, AXL, MER, TYRO3, TIE1, EPHA2, EPHA4, EPHA5, EPHA7, EPHB2, EPHB3, EPHB4, EPHB6, RYK, alcadein alpha, alcadein beta, alcadein gamma, APLP1, APLP2, APP, CD44, E-Cadherin, epCAM, GHR, IL-1R2, neurogulin-1, notch-2, notch-3, notch-4, P75 NTR, podoplanin, PTK7, and syncan-3.

In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence of an Alcadein alpha polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO. 689. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence of an Alcadein beta polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 690. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence of a beta Alcadein gamma polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 691. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence for an APLP1 polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in SEQ ID NO 692. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence for an APLP2 polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 693. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence for a Notch-1 polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 694. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence for a Notch-2 polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 695. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence for a Notch-3 polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 696. In some embodiments, the anti-idiotype polypeptide may include a gamma secretase substrate sequence for a Notch-4 polypeptide. In some embodiments, an anti-idiotype polypeptide comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 697.

Connector

In some embodiments, the anti-idiotype polypeptide may include a linker between any two adjacent domains. For example, the linker can be between the transmembrane domain and the first intracellular domain. As another example, the intracellular recognition domain of the anti-idiotype polypeptide may be an antibody, and the linker may be between the heavy and light chains. As another example, a linker can be between the extracellular recognition domain and the transmembrane domain, and another linker can be between the transmembrane domain and the intracellular domain. As another example, the linker can be between the first intracellular domain and the second intracellular domain. As another example, the linker can be between the extracellular recognition domain and the intracellular domain.

The linker peptide may have any of a variety of amino acid sequences. Proteins may be linked by spacer peptides which are generally flexible, but other chemical bonds are not excluded. The linker may be a peptide between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be generated by coupling the proteins using synthetic oligonucleotides encoding the linkers. Peptide linkers with a degree of flexibility may be used. The linker peptide may have virtually any amino acid sequence, provided that a suitable linker will have a sequence that results in a generally flexible peptide. In general, the use of small amino acids such as glycine and alanine is useful in the production of flexible peptides. The creation of such sequences is conventional to those skilled in the art.

Suitable linkers may be readily selected and may be any of a variety of suitable lengths, such as 1 amino acid (e.g., gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G) _n Glycine-serine polymers (including, for example (GS) _n 、(GSGGS) _n 、(GGS) _n 、(GGGS) _n And (GGGGS) _n Where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore can act as neutral chains between components. Glycine polymers are of particular interest because glycine has significantly more phi-psi space than even alanine and is less restricted than residues with longer side chains (see Scheraga, review of computational chemistry (rev. Computational chem.)) 11173-142 (1992). Exemplary flexible connectors include but are not limited to Not limited to GGGGSGGGGS (SEQ ID NO: 674), GGGGSGGGGSGGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 372), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 675), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), GGSSRSS (SEQ ID NO: 673), GGGGSGGGSGGGGS (SEQ ID NO: 65), GGSG (SEQ ID NO: 66), GGSGG (SEQ ID NO: 67), GSGSGSG (SEQ ID NO: 68), GSGGG (SEQ ID NO: 69), GGGSG (SEQ ID NO: 70), GSSSG (SEQ ID NO: 71), and the like. Those skilled in the art will recognize that the design of a peptide that binds to any of the elements described above may include a linker that is wholly or partially flexible, such that the linker may include a flexible linker and one or more portions that impart less flexibility to the structure.

Anti-idiotype fusion polypeptide

The anti-idiotype polypeptide may act as a single polypeptide or may act as a fusion with other polypeptides. The fusion polypeptide may comprise only the extracellular recognition domain of one or more anti-idiotype polypeptides, or the fusion polypeptide may further comprise one or more other domains of an anti-idiotype polypeptide. Based on the presence of extracellular recognition domains from the anti-idiotype polypeptides, the fusion polypeptides may have different activities based on the presence or absence of the target antibody or antibody mimetic. For example, a constitutively active lymphoproliferative element may comprise an extracellular recognition domain from an anti-idiotype polypeptide that recognizes a target antibody. In the absence of target antibodies, the constitutively active lymphoproliferative elements may promote proliferation and/or survival of cells expressing the fusion polypeptide. Binding of the target antibody to the extracellular recognition domain in the presence of the target antibody, e.g., a target antibody that induces ADCC, can result in cell death of the cell expressing the fusion polypeptide with the constitutive lymphoproliferative element. In some embodiments, the extracellular recognition domain of the fusion polypeptide can perform any function of the anti-idiotype polypeptides disclosed herein, including, for example, acting as a safety switch and acting as an activity modulator. In such fusion polypeptides, the membrane associated domain of the anti-idiotype polypeptide may be provided by the polypeptide fused to the anti-idiotype polypeptide.

In some embodiments, the anti-idiotype polypeptide or the anti-idiotype extracellular recognition domain of the anti-idiotype polypeptide is expressed as part of a fusion polypeptide. In some embodiments, the fusion polypeptide may be an anti-idiotype polypeptide or a fusion between the extracellular recognition domain of an anti-idiotype polypeptide and a first engineered signaling polypeptide, which is disclosed in more detail elsewhere herein. Thus, in some embodiments, the anti-idiotype polypeptide or the extracellular recognition domain of the anti-idiotype polypeptide is fused to the lymphoproliferative element, CAR, and/or recombinant TCR. In some embodiments, the anti-idiotype polypeptide or the extracellular recognition domain of the anti-idiotype polypeptide is fused to a cytokine. In some embodiments, the fusion polypeptide may further comprise one or more other domains of the anti-idiotype polypeptide.

In some embodiments, the fusion polypeptide comprising the anti-idiotype polypeptide can further comprise a CAR. In some embodiments, the fusion polypeptide may comprise a recombinant TCR. In embodiments in which the anti-idiotype polypeptide is fused to a CAR or recombinant TCR, the extracellular recognition domain of the anti-idiotype polypeptide may be expressed as part of the antigen-specific binding region of the CAR (as in the ASTR discussed elsewhere herein) or the antigen-binding site of the recombinant TCR, or the extracellular recognition domain may be expressed as part of a separate domain on the fusion polypeptide. For example, the CAR and extracellular recognition domain from the anti-idiotype polypeptide may each be part of a bispecific antibody that is attached to other domains of the fusion polypeptide.

In some embodiments, an anti-idiotype polypeptide, such as a safety switch or an activity modulator, is expressed fused to a lymphoproliferative element to form a fusion polypeptide. Thus, in some embodiments, the fusion polypeptide may include a lymphoproliferative element. Such constructs offer the advantage of taking up less genomic space on the RNA genome than the polypeptide alone, particularly in combination with other "space saving" elements provided herein. The lymphoproliferative element of the fusion polypeptide may be any of the lymphoproliferative elements disclosed elsewhere herein. In some embodiments, the lymphoproliferative elements of the fusion polypeptide may include one or more or all of the domains, motifs and/or mutations of the intracellular signaling domains disclosed herein or otherwise known to induce proliferation and/or survival of T cells and/or NK cells. In some embodiments, the lymphoproliferative elements of the fusion polypeptide are constitutively active. In some embodiments, the lymphoproliferative element of the fusion polypeptide is an inducible lymphoproliferative element. In some such embodiments, binding of the target antibody or antibody mimetic to the extracellular recognition domain on the fusion polypeptide dimerizes the two intracellular signaling domains of the inductive lymphoproliferative element to drive proliferation of cells such as T cells and/or NK cells. In some embodiments, the inducible lymphoproliferative element of the fusion polypeptide activates proliferation and/or survival signaling after binding of the anti-idiotype extracellular recognition domain to the target antibody or antibody mimetic, as also discussed in the "activity modulator" section herein. In some embodiments, a fusion polypeptide comprising an induced lymphoproliferative element may comprise a dimerization domain in addition to an anti-idiotype extracellular recognition domain. In illustrative embodiments, fusion polypeptides comprising an induced lymphoproliferative element do not include a dimerization domain in addition to an anti-idiotype extracellular recognition domain.

In one illustrative embodiment, eTag is expressed as a fusion polypeptide fused to the 5' end of the c-Jun domain (SEQ ID NO: 104), the transmembrane domain from CSF2RA (SEQ ID NO: 129), the first intracellular domain from MPL (SEQ ID NO: 283), and the second intracellular domain from CD40 (SEQ ID NO: 208). When expressed as a polypeptide that is not fused to a CAR or lymphoproliferative element, the cell tag may be associated with the cell membrane by its native membrane attachment sequence or by a heterologous membrane attachment sequence such as a GPI-anchor or transmembrane sequence. In an illustrative embodiment, the cell tag is expressed on T cells and/or NK cells, but not on replication defective recombinant retroviral particles. In some embodiments, the polynucleotides, polypeptides, and cells comprise 2 or more safety switches.

Cell preparation and method of administration

In some embodiments (e.g., embodiments in which the sample does not undergo PBMC isolation or granulocyte depletion procedure), at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the neutrophils, basophils, and/or eosinophils present in the blood sample subjected to the methods for modification herein are present in the cell preparation, including at the time of the optional delivery (i.e., administration) step. In some embodiments (e.g., those embodiments in which the sample is not subjected to a B cell depletion procedure), at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the B cells present in the blood sample subjected to the methods for modification herein are present in the cell preparation, including at the time of the optional delivery step. In some embodiments (e.g., those wherein the sample does not undergo a monocyte depletion procedure), at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the monocytes present in the blood sample subjected to the methods for modification herein are present in the cell preparation, including at the time of the optional delivery step.

In some embodiments, and in illustrative embodiments in which the cell preparation is administered subcutaneously or intramuscularly, the cell preparation including the modified lymphocytes is less voluminous than conventional CAR-T methods (which are typically infusion-delivery methods), and may be less than or less than about 1ml, about 2ml, about 3ml, about 4ml, about 5ml, about 10ml, about 15ml, about 20ml, or about 25ml.

In certain embodiments, the short contact time results in many modified lymphocytes in the cell preparations herein having binding polypeptides, fusogenic polypeptides on their surface, and in some embodiments, T cell activating elements originating on the surface of the retroviral particle, by association with the recombinant retroviral particle or fusion with the plasma membrane by the retroviral envelope, including at the optional step of delivery. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the modified lymphocytes in the cell preparation comprise pseudotyped elements and/or T cell activating elements, e.g., T cell activating antibodies. In some embodiments, the pseudotyped element and/or the T cell activating element can be bound to the surface of the modified lymphocyte by, for example, T cell receptor CD28, OX40, 4-1BB, ICOS, CD, CD53, CD63, CD81, CD82, and/or the pseudotyped element and/or the T cell activating element can be present in the plasma membrane of the modified lymphocyte.

Provided herein are cell preparations that include, for example, T cells and/or NK cells. In illustrative embodiments, such formulations are provided by the methods provided herein. Any of the cell preparations provided herein can comprise a self-driven CAR-T cell. In one aspect, provided herein is a cell formulation comprising a population of self-driven CAR-T cells, e.g., modified, genetically modified, transcribed, transfected, and/or stably integrated self-driven CAR-T cells, in a delivery solution.

Since the time of contacting the lymphocyte with the recombinant nucleic acid vector is advantageously short, and in some illustrative embodiments provided herein such post-contact modified lymphocyte is ex vivo, in these embodiments, some or all of the T cells and NK cells have not expressed the recombinant nucleic acid or have not integrated the recombinant nucleic acid into the genome of the cell, and in embodiments including these, some retroviral particles may be associated with, but may not have fused with, the target cell membrane prior to use or inclusion in any of the methods or compositions provided herein, including but not limited to being introduced or reintroduced into the subject, or prior to use in preparing a cell preparation. Thus, provided herein are various cell preparation aspects and embodiments that may, for example, result from the illustrative methods provided herein, such as, in the illustrative embodiments, a point-of-care method involving subcutaneous administration. Such cell preparations, including but not limited to those described below and in the illustrative examples section herein, may be present when the cells are contacted with the recombinant retroviral vector and optionally after cell collection after rinsing, and in illustrative examples, may be present up to and including subcutaneous administration to a subject.

In some embodiments, provided herein are cell preparations comprising T cells and/or NK cells, wherein less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, or 5% of the cells in the cell preparations are T cells and/or NK cells. In some embodiments, there is provided a cell preparation comprising lymphocytes, NK cells, and/or T cells, wherein at least 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the lymphocytes, NK cells, and/or T cells in the illustrative embodiments are modified cells, e.g., cells modified with a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide provided herein. Such polynucleotides may optionally encode a CAR, TCR, inhibitory RNA, or LE as provided herein. In some embodiments, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the lymphocytes that are the low end of the range are modified, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the lymphocytes that are the high end of the range are modified, e.g., 5% to 95%, 10% to 90%, 25% to 75%, and 25% to 95%. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified lymphocytes within the cell preparation are not genetically modified, transduced or stably transfected. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% and 70% of the modified lymphocytes at the low end of the range are not genetically modified, transduced or stably transfected, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% or all of the modified lymphocytes at the high end of the range are not genetically modified, transduced or stably transfected, e.g., 5% to 95%, 10% to 90%, 25% to 75% and 25% to 95%. In some embodiments, the polynucleotides of the genetically modified lymphocytes may be extrachromosomal or integrated into the genome of these cell preparations, which are formed after contact and incubation, and upon optional administration. In some embodiments of these cell preparations, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the genetically modified lymphocytes have extrachromosomal polynucleotides. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% and 70% of the modified or genetically modified lymphocytes at the low end of the range have extrachromosomal polynucleotides, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% or all of the modified or genetically modified lymphocytes at the high end of the range have extrachromosomal polynucleotides, e.g., 5% to 95%, 10% to 90%, 25% to 75% and 25% to 95%. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the modified or genetically modified lymphocytes are not transduced or stably transfected in these cell preparations, e.g., as a result of the methods provided herein for genetically modifying T cells and/or NK cells. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the modified or genetically modified lymphocytes at the low end of the range are not transduced, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the modified or genetically modified lymphocytes at the high end of the range are not transduced or are not stably transduced, e.g., 5% to 95%, 10% to 90%, 25% to 75%, and 25% to 95%.

Without being limited by theory, unlike traditional CAR-T cell processing methods in which cells are cultured ex vivo for days or weeks and many cells divide, in the illustrative methods provided herein in which T cells and/or NK cells are contacted with a retroviral particle to modify T cells and/or NK cells within hours of delivery, some or most of the reverse transcriptase and integrase present within the retroviral particle moves into T cells and/or NK cells after it fuses with the retroviral particle, will remain in the modified T cells and/or NK cells upon delivery.

The volume of the cell preparation or other solution administered varies depending on the route of administration, as described elsewhere herein. Cell preparations for subcutaneous or intramuscular injection typically have a smaller volume than cell preparations delivered by infusion. In some embodiments, the volume of the cell preparation or other solution (including suspensions of modified and in illustrative embodiments genetically modified lymphocytes) does not exceed 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml, 25ml, 30ml, 35ml, 40ml, 45ml, or 50ml. In some embodiments, the volume of the cell preparation or other solution comprising a suspension of modified lymphocytes may be 0.20ml, 0.25ml, 0.5ml, 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml or 25ml as the low end of the range to 0.5ml, 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml, 25ml, 30ml, 35ml, 40ml, 45ml or 50ml, 30ml, 35ml, 40ml, 45ml, 50ml, 75ml, 100ml, 125ml, 250ml, 500ml or 1000ml as the high end of the range. Thus, as non-limiting examples, the volume may be 0.2ml to 10ml, 0.5 to 2ml, 1ml to 250ml, 1ml to 100ml, 10ml to 100ml, or 1ml to 10ml. In certain illustrative embodiments, less than 10ml, 1ml to 25ml, and in illustrative embodiments 1ml to 3ml, 1ml to 5ml, or 1ml to 10ml of the cell preparation comprising the modified lymphocytes in a delivery solution is administered subcutaneously or intramuscularly. In an illustrative embodiment, the volume of the solution containing the modified lymphocytes may be at 0.20ml, 0.25ml, 0.5ml, 1ml, 2ml, 3ml, 4ml and 5ml as the low end of the range and 0.5ml, 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml as the high end of the range, Between 20ml, 25ml, 30ml, 35ml, 40ml, 45ml and 50 ml. In one exemplary embodiment, the method is performed by subcutaneously administering 1ml of 7.0X10 ⁷ T cell delivery formulation at 1.0X10 per ml ⁶ Individual T cells/kg 70kg subjects were dosed. In some embodiments, when the volume of the solution is at least 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml, or 25ml, the solution may comprise hyaluronidase. In embodiments herein in which lymphocytes are filtered, particularly after they are modified, and/or in particular in which transduction is performed on top of a filter, the delivery solution may be used to re-suspend and/or elute cells from the filter in volumes that may be those described above. Thus, in some embodiments, the delivery solution provided herein is an eluting solution.

In some embodiments, the modified and in the illustrative embodiments genetically modified lymphocytes are introduced or reintroduced into the subject by intradermal, intratumoral or intramuscular administration and in the illustrative embodiments subcutaneous administration using a cell preparation present in a subcutaneous delivery device (e.g., a sterile syringe suitable for subcutaneous delivery of a solution). In some embodiments, a subcutaneous delivery device is used that holds a solution (e.g., a cell preparation herein) and has an open or openable end, which in the illustrative embodiment is the open end of a needle, for subcutaneous administration of the solution (e.g., cell preparation) from a liquid-holding portion of the device. Such subcutaneous delivery devices are effective for subcutaneous delivery and in the illustrative embodiments are suitable for subcutaneous delivery, or are effective for subcutaneous injection, or are suitable for subcutaneous injection. Non-limiting examples of subcutaneous delivery devices suitable for subcutaneous delivery of a solution include subcutaneous catheters, such as indwelling subcutaneous catheters, for example, as follows

(Becton Dickinson) and unnecessary closed indwelling subcutaneous catheter systems, e.g. with wings, e.g. as +.>

(Becton Dickinson). In some embodiments, the delivery deviceThe device may comprise a pump, such as an infusion pump or peristaltic pump. In some embodiments, the cell preparation is in fluid connection with any of the needles disclosed herein (e.g., needles compatible with, effective for, suitable for, or effective for subcutaneous delivery).

In some embodiments, the delivery solution, composition or cell preparation in the kit comprises one or more cytokines (e.g., IL-2, IL-7, IL-15 or IL-21, IL-21) and/or cytokine receptor agonists (e.g., IL-15 agonists). In some embodiments, the cytokine does not bind to a cytokine receptor contained in the delivery solution, kit, or cell preparation; and/or does not bind to a cytokine receptor encoded by the polynucleotide in the delivery solution, cell preparation, or kit. In some embodiments, the cytokine may be a modified cytokine that selectively activates a complex that drives proliferation without being limited by theory. In an illustrative embodiment, the modified cytokine is a modified IL-2, such as a fusion protein having a circularly permuted extracellular domain of IL-2 and IL-2Rα (see, e.g., lopes et al, J.cancer immunotherapy (J Immunather Cancer) month 4 2020; 8 (1): e 000673). In some embodiments, the cytokine, modified cytokine or cytokine receptor agonist may also be administered in one administration or in a separate administration from the cell preparation, either before, simultaneously with, or after administration including the delivery solution or cell preparation. In some embodiments, two or more separate administrations may be in ascending doses. In some embodiments, two or more administrations may be at the same dose. In some embodiments, two or more administrations may include the same or different cytokines, modified cytokines, and/or cytokine receptor agonists. In some embodiments, the separate administrations may be a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 administrations. In some embodiments, separate administrations occur over several consecutive days.

In an illustrative embodiment, cells in a first mixture of cells (e.g., cells obtained from a subject) are modified with a recombinant nucleic acid vector encoding a target antigen, which may be referred to herein as an "artificial antigen presenting cell" or "aAPC," and cells in an isolated second mixture of cells from the same subject are modified to express an antigen-binding CAR. In some embodiments, the modified cell in which the vector encoding the target antigen is modified is a T cell, which may be referred to herein as a "T-APC". As non-limiting examples, such modified T-APCs can include B cells, dendritic cells, and macrophages, and in illustrative embodiments, dendritic cells and macrophages, for example, wherein the corresponding CAR-T target is a B cell cancer target, and can be produced using the methods provided herein, wherein the reaction mixture for modification (e.g., transduction) includes a T cell binding polypeptide, such as a polypeptide directed against CD 3. In further illustrative embodiments, the cell mixture is whole blood, isolated TNC, isolated PBMC. For example, the first cell mixture can be modified with a recombinant nucleic acid vector encoding a fusion protein of an extracellular domain of Her2 and a transmembrane domain of PDGF, and the second cell mixture can be modified with a recombinant nucleic acid vector encoding a CAR for Her 2. The cells can then be formulated into a delivery solution or administered to a subject at different CAR effector cell to target cell ratios. In some embodiments, the ratio of effector to target at the time of formulation or administration is or is about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2; 1. about 1:1, about 1:2, about 1:3, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In illustrative embodiments, the target cells are co-administered subcutaneously or intramuscularly with modified T cells and/or NK cells.

Synthesis of RNA

In another aspect, the delivery solution or cell formulation provided herein includes synthetic RNA. In some embodiments, the synthetic RNA includes an inhibitory RNA, such as siRNA against one or more targets. Targets for these inhibitory RNAs can be any of the siRNA or miRNA targets disclosed elsewhere herein. In some embodiments, the synthetic RNA includes mRNA encoding one or more proteins or peptides. In some embodiments, the mRNA encodes one or more CARs. The CAR may be any CAR composition disclosed herein that includes a bispecific CAR that includes an anti-id ERD as disclosed herein. In some embodiments, the mRNA may encode any of the anti-idiotype polypeptides disclosed herein. In some embodiments, the mRNA encodes a target antibody to an anti-idiotype polypeptide disclosed herein. Such embodiments may have the advantage of providing a target antibody that may be cytotoxic when delivered in soluble form, but is less or non-cytotoxic when taken up by cells after in vivo administration of mRNA encoding the target antibody. Thus, for embodiments in which the anti-idiotype polypeptide has an ICD (ICD that is LE or ICD of a CAR (e.g., a bispecific CAR)), the cells that ingest and express the target antibody can become artificial antigen presenting cells.

In some embodiments, the mRNA encodes one or more cytokines. In some embodiments, the mRNA encodes IL-2 or a functional variant thereof. In some embodiments, the mRNA encodes IL-7 or a functional variant thereof. In some embodiments, the mRNA encodes IL-15 or a functional variant thereof. In some embodiments, the mRNA encodes IL-21 or a functional variant thereof. In some embodiments, the mRNA encodes one or more proteins or polypeptides that bind and activate the CAR. In some embodiments, the mRNA encodes an antigen recognized by the aster of the CAR. In some embodiments, the mRNA encodes HER2 or an extracellular domain of HER 2. In some embodiments, the mRNA encodes EGFR or an extracellular domain of EGFR. In some embodiments, the mRNA encodes Axl or an extracellular domain of Axl. In some embodiments, the mRNA encodes CD19 or an extracellular domain of CD 19. In some embodiments, the mRNA encodes CD22 or an extracellular domain of CD 22. In some embodiments, the mRNA encodes an antibody recognized by the aster of the CAR. In some embodiments, the MRNA encoding an antibody recognized by the astm of the CAR is an antibody to the astm or an anti-idiotype antibody to the scFv. In some embodiments, the mRNA encodes an epitope tag that binds to a CAR and can crosslink both CARs as described elsewhere herein. In some embodiments, the mRNA encodes one or more T and/or NK cell costimulatory proteins. Such co-stimulatory proteins may comprise one or more ligands or antibodies directed against co-stimulatory receptors on T cells and/or NK cells. In some embodiments, the co-stimulatory receptor is CD28. In some embodiments, the co-stimulatory receptor is 4-1BB. In some embodiments, the mRNA encodes a soluble protein or polypeptide. In some embodiments, the mRNA encodes a membrane-bound protein or polypeptide. In some embodiments, the membrane-bound protein or polypeptide is operably linked to a transmembrane domain. In some embodiments, the synthetic RNA includes inhibitory RNA (e.g., siRNA) against one or more targets and mRNA encoding one or more proteins or peptides.

Methods for generating mRNA for delivery of a solution or cell preparation may include in vitro transcription of a template with specifically designed primers followed by the addition of PolyA to generate a construct comprising 3' and 5' untranslated sequences, 5' caps and/or IRES, nucleic acid to be expressed, and PolyA tails (typically 50-200 bases in length). In some embodiments, the synthetic RNA is a naturally occurring endogenous RNA of the nucleic acid of interest. In some embodiments, the RNA is not naturally occurring endogenous RNA of the nucleic acid of interest. In some embodiments, the RNA is modified to alter the stability and/or translation efficiency of the RNA. In some embodiments, the 5'utr, 3' utr, kozak sequence, polyA tail is modified. In some embodiments, the RNA includes a 5' cap. In some embodiments, the RNA is encapsulated in a lipid-based carrier vehicle. A method for assembling lipid nanocarriers includes directly mixing a solution of lipids in ethanol with an aqueous solution of nucleic acid to obtain Lipid Nanoparticles (LNPs). In some embodiments, the LNP comprises a PEG conjugated lipid. PEG conjugated lipids prevent aggregation during particle formation and allow for controlled fabrication of particles having a defined diameter in the range between about 50nm and 150 nm. Pegylation of nanoparticles can have significant drawbacks with respect to safety and activity. Drawbacks associated with the use of pegylated nanoparticles have stimulated the development of PEG substitutes. In some embodiments, the LNP does not comprise PEG. In some embodiments, the LNP comprises poly (glycerol) (PG), poly (oxazoline), a sugar-based system, and a poly (peptide). In some embodiments, the polypeptide comprises poly-sarcosine (pSAR). In some embodiments, the LNP comprises a dendritic cell targeting moiety. In some embodiments, the dendritic cell targeting moiety comprises mannose.

In some embodiments, in the cell preparations and methods provided herein, RNA can be added to or co-administered with a cell preparation comprising modified and/or genetically modified T cells and/or NK cells. In some embodiments, RNA is added to the isolated blood of a subject and processed in parallel with T cells and/or NK cells. In some embodiments, RNA can be formulated separately from modified and/or genetically modified T cells and/or NK cells. The synthetic RNA can be delivered by any means known in the art for therapeutic delivery of RNA. In some embodiments, the RNA is delivered intravenously. In some embodiments, the RNA is delivered intraperitoneally. In some embodiments, the RNA is delivered intramuscularly. In some embodiments, the RNA is delivered intratumorally. In some embodiments, the RNA is delivered intradermally. In an illustrative embodiment, the RNA is delivered subcutaneously. In some embodiments, the RNA is delivered at the same site as the site of administration of the modified and/or genetically modified T cells and/or NK cells. In some embodiments, the RNA is delivered at a site adjacent to the site of administration of the modified and/or genetically modified T cells and/or NK cells. In some embodiments, the RNA is administered once. In some embodiments, the RNA is administered 2, 3, 4, 5, 6 or more times.

Recombinant retroviral particles

Recombinant retroviral particles are disclosed in the methods and compositions provided herein, for example, to modify cells, non-limiting examples being human cells, primary cells, T cells, and/or NK cells to produce genetically modified and/or transduced cells, human cells, primary cells, T cells, and/or NK cells. Recombinant retroviral particles themselves are aspects of the present invention. In general, recombinant retroviral particles included in aspects provided herein are replication defective, meaning that the recombinant retroviral particles cannot replicate as they leave the packaging cell. Indeed, unless otherwise indicated herein, retroviral particles are replication defective, and such retroviral particles are "recombinant retroviral particles" if they include nucleic acids in their genome that are not native to the retrovirus. In an illustrative embodiment, the recombinant retroviral particle is a lentiviral particle.

In some aspects, provided herein are replication defective recombinant retroviral particles for transduction of cells, typically lymphocytes, and in illustrative embodiments T cells and/or NK cells. Replication defective recombinant retroviral particles may comprise envelope proteins. In some embodiments, the envelope protein may be a pseudotyped element. In some embodiments, the envelope protein may be an activating element. In some embodiments, the replication defective recombinant retroviral particle comprises both a pseudotyped element and an activating element. Replication defective recombinant retroviral particles may include any of the pseudotyped elements discussed elsewhere herein. In some embodiments, the replication defective recombinant retroviral particle may include any of the activating elements discussed elsewhere herein. In one aspect, provided herein is a replication defective recombinant retroviral particle (RIP) comprising a polynucleotide having a nucleic acid encoding an anti-idiotype polypeptide provided in any one of the aspects and embodiments herein. Such polypeptides typically include a nucleic acid that further encodes at least one of a CAR, LE, inhibitory RNA, and cytokine. In some embodiments, the RIP comprises a polynucleotide comprising: A. one or more transcriptional units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode one or more of an anti-idiotype polypeptide and an engineered T cell receptor or Chimeric Antigen Receptor (CAR); a pseudotyped element and a T cell activating element on its surface, wherein said T cell activating element is not encoded by a polynucleotide in a replication defective recombinant retroviral particle. In some embodiments, the T cell activating element can be any of the activating elements discussed elsewhere herein. In an illustrative embodiment, the T cell activating element may be an anti-CD 3 scFvFc. In another aspect, provided herein is a RIP comprising a polynucleotide comprising one or more transcriptional units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode an anti-idiotype polypeptide and a first signaling polypeptide comprising an engineered T cell receptor or Chimeric Antigen Receptor (CAR) and a second signaling polypeptide comprising a lymphoproliferative element. In some embodiments, the lymphoproliferative element may be a chimeric lymphoproliferative element. In some embodiments of any of the retroviral particle aspects or embodiments provided herein, or in any other aspect that includes a retroviral particle, the anti-idiotype polypeptide, engineered T cell receptor, CAR, or other transgene is expressed, displayed, and/or otherwise incorporated into the surface of the replication defective retroviral particle at a reduced level of less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the surface expression, as compared to when the transgene is expressed from the EF1-a or PGK promoter and in illustrative embodiments when the transgene is expressed from the EF1-a or PGK promoter in the absence of additional elements that reduce such surface expression (e.g., reducing stator or inhibitory RNA). In illustrative embodiments of any of the polynucleotide vector (e.g., RIP) aspects provided herein or any other aspects including a gene vector, the gene vector is substantially free of protein transcripts encoded by nucleic acids of the gene vector, and/or the RIP does not express or comprise a detectable amount of an engineered T cell receptor or CAR on its surface, or expresses or comprises a reduced amount of an engineered T cell receptor or CAR on its surface.

Various elements and combinations of elements included in the RIP are provided throughout the present disclosure, such as, for example, pseudotyped elements, activating elements, and membrane-bound cytokines, as well as nucleic acid sequences included in the genome of replication-defective recombinant retroviral particles, such as, but not limited to, nucleic acid sequences encoding anti-idiotype polypeptides, nucleic acids encoding CARs; a nucleic acid sequence encoding a lymphoproliferative element; a nucleic acid encoding a cytokine; nucleic acid sequences encoding control elements (e.g., riboswitches); promoters, especially promoters that are constitutively active or inducible in T cells; and nucleic acids encoding inhibitory RNA molecules. In addition, various aspects provided herein (e.g., methods of making recombinant retroviral particles, methods for performing adoptive cell therapy, and methods for transducing T cells) produce and/or include replication defective recombinant retroviral particles. Replication defective recombinant retroviruses produced and/or included in such methods form themselves into replication defective recombinant retroviral particle compositions which are independent aspects of the present invention, which compositions may be in isolated form. Such compositions may be in dry (e.g., lyophilized) form, or may be in the form of a suitable solution or medium as known in the art for storage and use of the retroviral particles.

Recombinant retroviral particle embodiments herein include those wherein the retroviral particle comprises a genome comprising one or more nucleic acids encoding an anti-idiotype polypeptide and one or more inhibitory RNA molecules. Various alternative embodiments of such nucleic acids encoding an inhibitory RNA molecule that may be included in the genome of a retroviral particle (including combinations of such nucleic acids with other nucleic acids encoding CARs or lymphoproliferative elements other than the inhibitory RNA molecule) are included, for example, in the inhibitory RNA portion provided herein and in various other paragraphs that combine these embodiments. In addition, various alternatives to such replication defective recombinant retroviruses can be identified by exemplary nucleic acids disclosed within the packaging cell line aspects disclosed herein. Those of skill in the art will recognize that the disclosure in this section of a recombinant retroviral particle comprising a genome encoding one or more (e.g., two or more) inhibitory RNA molecules can be combined with various alternatives to such nucleic acids encoding the inhibitory RNA molecules provided elsewhere herein. Furthermore, those of skill in the art will recognize that such nucleic acids encoding one or more inhibitory RNA molecules may be combined with various other functional nucleic acid elements provided herein, as disclosed, for example, in the sections herein focusing on inhibitory RNA molecules and nucleic acids encoding such molecules. Furthermore, various embodiments of specific inhibitory RNA molecules provided elsewhere herein can be used in recombinant retroviral particle aspects of the present disclosure.

The necessary elements for the recombination of retroviral vectors, such as lentiviral vectors, are known in the art. These elements are included in the packaging cell line section and in the details provided in the examples section for the preparation of replication defective recombinant retroviral particles and are described in WO 2019/055946. For example, lentiviral particles typically include packaging elements REV, GAG, and POL, which can be delivered to packaging cell lines via one or more packaging plasmids; pseudotyped elements, various embodiments provided herein, that can be delivered to packaging cell lines via pseudotyped plasmids; and a genome produced from a polynucleotide delivered to a host cell via a transfer plasmid. This polynucleotide typically includes the viral LTR and the psi packaging signal. The 5' LTR may be a chimeric 5' LTR fused to a heterologous promoter, including a 5' LTR that is independent of Tat transactivation. The transfer plasmid may be self-inactivating, for example, by removal of the U3 region of the 3' LTR. In some non-limiting embodiments, for any of the composition or method aspects and embodiments provided herein including retroviral particles, vpu (such as a Vpu-containing polypeptide (sometimes referred to herein as a "Vpu polypeptide")) including, but not limited to, src-FLAG-Vpu is packaged within the retroviral particle. In some non-limiting embodiments, vpx (e.g., src-FLAG-Vpx) is packaged within retroviral particles. Without being limited by theory, after transduction of T cells, vpx enters the cytosol of the cells and promotes degradation of SAMHD1, thereby increasing the pool of cytoplasmic dntps available for reverse transcription. In some non-limiting embodiments, vpu and Vpx are packaged within retroviral particles for any composition or method aspects and embodiments comprising retroviral particles provided herein.

Retroviral particles (e.g., lentiviral particles) included in various aspects of the invention are replication defective in the illustrative embodiments, particularly for safety reasons including embodiments in which cells transduced with such retroviral particles are introduced into a subject. When replication-defective retroviral particles are used to transduce cells, the retroviral particles are not produced by the transduced cells. Modifications to the retroviral genome are known in the art to ensure that retroviral particles comprising the genome are replication defective. However, it is to be understood that in some embodiments, replication competent recombinant retroviral particles may be used for any one of the aspects provided herein.

Those of skill in the art will recognize that different types of vectors (e.g., expression vectors) may be used to deliver the functional elements discussed herein to packaging cells and/or T cells. Illustrative aspects of the invention utilize retroviral vectors and in some specific illustrative embodiments lentiviral vectors. Other suitable expression vectors may be used to implement certain embodiments herein. Such expression vectors include, but are not limited to, viral vectors (e.g., poxvirus, poliovirus, adenovirus-based viral vectors (see, e.g., li et al, ophthalmic and visual science research (InvestOpthalmosSci) 35:25432549,1994; borras et al, gene therapy (GeneTher) 6:515 524,1999;Li and Davidson, proc. Natl. Acad. Sci. USA (PNAS) 92:7700 7704,1995;Sakamoto et al, human Gene therapy (H GeneTher) 5:1088 1097,1999;WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., ali et al, 9:8186,1998, flannery et al, journal of national academy of sciences (PNAS) 94:6916 6921,1997;Bennett et al, eye and vision science research (InvestOpthalmosSci) 38:28572863,1997, jomark et al, gene therapy (GeneTher) 4:683 690,1997,Rolling et al, human gene therapy (HumMolTher) 10:641 648,1999;Ali et al, human molecular genetics (HumMolGenet) 5:591 594,1996;Srivastava,WO 93/09239, samulski et al, J.Vir.) (1989) 63:3822-3828, mendelson et al, virology (virol.) 166:154-165, flotte et al, national academy of sciences (Gene Thermol) 4:683 690,1997,Rolling et al, human genome (HumMolGenet al) 10:641 648,1999;Ali, human molecular genet (HumMolGenet) 5:591 594,1996;Srivastava,WO 93/09239, samulski et al, J.V.1068, J.V. J. 10635, J.V.J. J. J.10635, J.V.. J.63:3822-3828, J.V.J.J.J.J.J., A retroviral vector of hawk sarcoma virus (Harvey Sarcoma Virus), avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and breast tumor virus), such as gamma retrovirus; or human immunodeficiency virus (see, e.g., miyoshi et al, proc. Natl. Acad. Sci. USA (PNAS) 94:10319 23,1997;Takahashi et al, J. Virol. JVirol 73:7812 7816,1999), etc.

Replication-defective recombinant retroviral particles are common tools for gene delivery, as disclosed herein (Miller, nature (1992) 357:455-460). The ability of replication defective recombinant retroviral particles to deliver unordered nucleic acid sequences into a wide range of rodent, primate, and human somatic cells makes replication defective recombinant retroviral particles more suitable for gene transfer into cells. In some embodiments, the replication defective recombinant retroviral particle may be derived from alpha retrovirus (Alpharetrovirus genus), beta retrovirus (Betaretrovirus genus), gamma retrovirus (Gammaretrovirus genus), delta retrovirus (Deltaretrovirus genus), epsilon retrovirus (Epsilonretrovirus genus), lentivirus, or foamy virus. There are a number of retroviruses suitable for use in the methods disclosed herein. For example, murine Leukemia Virus (MLV), human Immunodeficiency Virus (HIV), equine Infectious Anemia Virus (EIAV), mouse Mammary Tumor Virus (MMTV), rous Sarcoma Virus (RSV), fuji sarcoma virus (FuSV), moronella leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), moronella sarcoma virus (Mo-MSV), ebensen murine leukemia virus (Abelson murine leukemia virus) (A-MLV), avian myelocytopathic virus-29 (MC 29) and avian erythrocyte proliferation virus (AEV) can be used. A detailed list of Retroviruses can be found in Coffin et al (Retroviruses), 1997, cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press) editions J M Coffin, S M Hughes, H E Varmus, pages 758-763). Details concerning the genomic structure of some retroviruses can be found in the art. For example, details regarding HIV can be found in NCBI Genbank (i.e., genome accession No. AF 033819).

In an illustrative embodiment, the replication defective recombinant retroviral particle may be derived from a lentivirus genus. In some embodiments, the replication defective recombinant retroviral particle may be derived from HIV, SIV or FIV. In further illustrative embodiments, the replication defective recombinant retroviral particle may be derived from Human Immunodeficiency Virus (HIV) in the lentivirus genus. Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. The higher complexity enables lentiviruses to regulate their life cycle, such as during latent infection. Typical lentiviruses are Human Immunodeficiency Virus (HIV), the causative agent of AIDS. In vivo, HIV can infect terminally differentiated cells that divide very rarely, such as lymphocytes and macrophages.

In illustrative embodiments, replication defective recombinant retroviral particles provided herein contain a Vpx polypeptide.

In some embodiments, replication defective recombinant retroviral particles provided herein comprise and/or contain a Vpu polypeptide.

In an illustrative embodiment, the retroviral particle is a lentiviral particle. Such retroviral particles typically comprise a retroviral genome located within a capsid within a viral envelope.

In some embodiments, DNA-containing viral particles are used in place of recombinant retroviral particles. Such viral particles may be adenoviruses, adeno-associated viruses, herpesviruses, cytomegaloviruses, poxviruses, vaccinia viruses, influenza viruses, vesicular Stomatitis Viruses (VSV) or Sindbis viruses (Sindbis viruses). Those skilled in the art will understand how to modify the methods disclosed herein for different viruses and retroviruses or retroviral particles. In the case of using viral particles comprising a DNA genome, one skilled in the art will appreciate that functional units may be included in these genomes to induce integration of all or part of the DNA genome of the viral particle into the genome of T cells transduced with such viruses.

In some embodiments, the HIV RRE and polynucleotide region encoding HIV Rev may be replaced with an N-terminal RGG cassette RNA binding motif and a polynucleotide region encoding ICP 27. In some embodiments, the polynucleotide region encoding HIV Rev may be replaced with one or more polynucleotide regions encoding adenoviruses E1B 55-kDa and E4 Orf 6.

In certain aspects, the replication defective recombinant retroviral particle can include a nucleic acid encoding a self-driven CAR, as disclosed elsewhere herein. As a non-limiting example, such an embodiment is a retroviral particle whose genome comprises one or more first transcription units operably linked to an inducible promoter that is inducible in at least one of T cells or NK cells, and one or more second transcription units operably linked to a constitutive T cell or NK cell promoter, wherein the number of nucleotides between the 5 'end of the one or more first transcription units and the 5' end of the one or more second transcription units is less than the number of nucleotides between the 3 'end of the one or more first transcription units and the 3' end of the one or more second transcription units,

Wherein at least one of the one or more first transcription units encodes a lymphoproliferative element,

and wherein at least one of the one or more second transcriptional units encodes a first Chimeric Antigen Receptor (CAR), wherein the CAR comprises an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain, and wherein a nucleic acid within the first transcriptional unit or the second transcriptional unit encodes an anti-idiotype polypeptide according to one embodiment provided herein.

In some embodiments, the replication defective recombinant retroviral particle may further display T cell activating elements.

Without being limited by theory, T cells contacted with and transduced with these replication-defective recombinant retroviral particles comprising nucleic acid encoding a self-driven CAR can receive an initial enhancement of transcription from the CAR-stimulated inducible promoter, as the T cell activating element can stimulate the induction signal of the CAR-stimulated inducible promoter. Binding of the T cell activating element can induce calcium influx, resulting in dephosphorylation of NFAT and its subsequent nuclear translocation, and binding to the NFAT responsive promoter. The lymphoproliferative elements transcribed and translated by the inducible promoters stimulated by these CARs may initially increase proliferation of these cells. In an illustrative embodiment, the T cell activating element may be a membrane-bound anti-CD 3 antibody and may be GPI linked or otherwise displayed on the virus. In some embodiments, the membrane-bound anti-CD 3 antibody may be fused to a viral envelope protein such as MuLV, VSV-G, henipah virus (Henipah virus) -G such as NiV-G, or variants and fragments thereof.

In some embodiments, the isolated replication-defective retroviral particle is a large-scale batch contained in a large-scale vessel. Such large-scale batches may have, for example, 10 ⁶ -10 ⁸ Titres for TU/ml and 1X 10 ¹⁰ TU to 1X 10 ¹³ TU、1×10 ¹¹ TU to 1X 10 ¹³ TU、1×10 ¹² TU to 1X 10 ¹³ TU、1×10 ¹⁰ TU to 5X 10 ¹² TU, or 1X 10 ¹¹ TU to 5X 10 ¹² Total batch size of TUs. In illustrative embodiments, the retroviral particles of any aspect or embodiment provided herein are substantially pure, as discussed in more detail herein.

Retrovirus genome size

In the methods and compositions provided herein, the recombinant retroviral genome (lentiviral genome in a non-limiting illustrative example) has a limit on the number of polynucleotides that can be packaged into a viral particle. In some embodiments provided herein, the polypeptide encoded by the polynucleotide coding region may be a truncation or other deletion that retains functional activity such that the polynucleotide coding region is encoded by fewer nucleotides than the polynucleotide coding region of the wild-type polypeptide. In some embodiments, the polypeptide encoded by the polynucleotide coding region may be a fusion polypeptide that can be expressed by one promoter. In some embodiments, the fusion polypeptide may have a cleavage signal to produce two or more functional polypeptides from one fusion polypeptide and one promoter. Furthermore, some functions not required after initial ex vivo transduction are not included in the retroviral genome, but are present on the surface of replication defective recombinant retroviral particles via the packaging cell membrane. These different strategies are used herein to maximize the functional elements packaged within replication defective recombinant retroviral particles.

In some embodiments, the recombinant retroviral genome to be packaged may be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides as the low end of the range and 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides as the high end of the range. The retroviral genome to be packaged comprises one or more polynucleotide regions encoding a first engineered signaling polypeptide and a second engineered signaling polypeptide as disclosed in detail herein. In some embodiments, the retroviral genome to be packaged may be less than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides. The functions that may be packaged as discussed elsewhere herein include the retroviral sequences required for retroviral assembly and packaging, such as the retroviral rev, gag and pol coding regions, as well as the 5'LTR and 3' LTR or active truncated fragments thereof, nucleic acid sequences encoding retroviral cis-acting RNA packaging elements and cPPT/CTS elements. Furthermore, in illustrative embodiments, replication defective recombinant retroviral particles herein may comprise any one or more or all of the following, in some embodiments in a reverse orientation relative to the 5 'to 3' orientation established by the retroviral 5 'ltrs and 3' ltrs (as described in WO2019/055946 as a non-limiting example): one or more polynucleotide regions encoding a first engineered signaling polypeptide and a second engineered signaling polypeptide, wherein at least one comprises at least one lymphoproliferative element; a second engineered signaling polypeptide that may include a chimeric antigen receptor; mirnas, control elements, such as riboswitches, that typically regulate expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide; safety switch polypeptides, introns, promoters (which are active in target cells such as T cells), 2A cleavage signals and/or IRES.

Kit and commercial product

In another aspect, provided herein are delivery compositions or suspensions, e.g., for use in the treatment or prevention of a disease, e.g., cancer or tumor growth, comprising a polynucleotide, e.g., a polynucleotide vector (illustrative replication defective recombinant retroviral particle (RIP)), or a modified cell, e.g., a modified lymphocyte, a modified TIL, a modified lymphocyte other than a B cell, or a modified T cell and/or a modified NK cell, as an active ingredient. In another aspect, provided herein are infusion compositions or other cell preparations for treating or preventing cancer or tumor growth comprising a polynucleotide such as a polynucleotide vector (RIP in the illustrative embodiment) or a modified cell such as a modified lymphocyte, modified TIL, a modified lymphocyte other than B-cell or a modified T-cell and/or a modified NK cell. The polynucleotide, such as a polynucleotide vector (RIP in the illustrative embodiment) or modified cell, such as a modified lymphocyte, modified TIL, modified lymphocyte other than B cells or modified T cells and/or modified NK cells, of the delivery or infusion composition may include any of the aspects, embodiments or sub-embodiments discussed above or elsewhere herein, for example, including a nucleic acid encoding an anti-idiotype polypeptide as well as a CAR, LE, cytokine and/or TCR.

Provided herein in one aspect are containers, e.g., commercial containers or packages, or kits comprising the same, comprising polynucleotides, e.g., polynucleotide vectors, e.g., RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells, according to any of the aspects and embodiments provided herein. As non-limiting examples, a polynucleotide such as a polynucleotide vector, e.g., RIP, or a modified cell such as a modified lymphocyte, modified TIL, modified lymphocyte other than B-cell, or modified T-cell and/or modified NK-cell may comprise in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in the T-cell and/or NK-cell. In some embodiments, the nucleic acid sequences of one or more nucleic acid sequences may encode an anti-idiotype polypeptide, an inhibitory RNA, a cytokine, a lymphoproliferative element, and/or a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the nucleic acid sequence of one or more nucleic acid sequences may encode one, two, or more inhibitory RNA molecules directed against one or more RNA targets.

Containers containing polynucleotides such as polynucleotide vectors, e.g., RIP, or modified cells such as modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells in any aspect or embodiment include commercial containers and kits that can be wells or other containers for storing polynucleotides such as polynucleotide vectors, e.g., RIP, or modified cells such as modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells. Indeed, some aspects provided herein comprise a container comprising a polynucleotide, such as a polynucleotide vector, e.g., RIP, or a modified cell, such as a modified lymphocyte, a modified TIL, a modified lymphocyte other than a B cell, or a modified T cell and/or a modified NK cell, wherein such a biologic comprises any of the nucleic acids or other components disclosed herein. Such containers in the illustrative embodiments include substantially pure polynucleotides such as polynucleotide vectors, e.g., RIP, or modified cells such as modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and/or modified NK cells, sometimes referred to herein simply as substantially pure biologies. Typically, the formulation and/or container of the substantially pure biological agent is sterile and negative for mycoplasma, replication competent retroviruses and foreign viruses (adventitious viruses) according to standard protocols (see, e.g., "viral vector characterization: see assay tools (Viral Vector Characterization: A Look at Analytical Tools)"; 10 months at 2018 (available at https:// cellcure dis. Com/viral-vector-replication-analysis-tools /). Exemplary methods for generating substantially pure biological agents and cells for cell therapy are known. For example, for such methods with respect to RIP, the viral supernatant is purified by a combination of depth filtration, TFF, benzonase treatment, diafiltration and formulation. In certain illustrative embodiments, based on the quality control test results, a substantially pure biological agent satisfies all of the following characteristics:

a) Negative for mycoplasma;

b) Endotoxin is less than 25EU/ml, and in certain further illustrative embodiments, less than 10EU/ml;

c) The absence of replication competent retroviruses (e.g., lentiviruses) of the same type as purposefully detected in the detected container; and

d) No foreign virus was detected;

furthermore, in the exemplary embodiment, if the biological agent is a viral particle, such as a retroviral particle, it meets the following quality control test results:

a) Less than 1pg host cell DNA/viral TU, and in certain further illustrative embodiments, less than 0.3pg/TU;

b) Less than 100 residual plasmid copies per viral TU, and in certain further illustrative embodiments, less than 10 copies per viral TU of any plasmid used to make recombinant retroviral particles;

c) Less than 1ng HEK protein/TU, and in certain further illustrative embodiments, less than 50pg HEK protein/TU; and

d) Greater than 100TU/ng P24 protein, and in certain further illustrative embodiments, greater than 10,000TU/ng P24 protein.

Before retroviral particles are delivered to a customer, they are typically tested against delivery specifications, including some or all of the above. The titer of each particle can be determined by ELISA from p24 viral capsid protein, by q-RT PCR from viral RNA genome copy number, by qPCR based Product Enhanced RT (PERT) assay from measurement of reverse transcriptase activity, but can all be converted to infectious titer by measuring functional gene Transfer Units (TUs) in bioassays.

Determination of infection titres of purified bulk retroviral material and end products by bioassay and qPCR is an exemplary analytical test method for determining infection titres of retroviruses. The indicator cell bank (e.g., F1 XT) can be grown in, for example, serum-free medium, seeded at 150,000 cells per well, and then exposed to serial dilutions of the retroviral product. Purified retroviral particles are diluted on indicator cells, for example from 1:200 to 1:1,600. Reference standard viruses may be added for system applicability. After 4 days of incubation with retrovirus, the cells were harvested, and the DNA was extracted and purified. The standard curve of the human genome (e.g., 100-10,000,000 copies/well) and the unique retroviral genome sequence plasmid pDNA amplicon are used, followed by the addition of genomic DNA from a cell sample exposed to the retroviral particles. For each PCR reaction, cq values for both the retroviral amplicon and the endogenous control (e.g., human RNAseP) are extrapolated back to the copy number of each reaction. From these values, the integrated genome copy number was calculated. In some cases, an indicator cell such as 293T has been characterized as triploid, so 3 copies of a single copy gene per cell should be used in the calculation. Using the initial viable cell count per well, the volume and genomic copy number ratio of retrovirus added to the cells, i.e., the Transduction Units (TUs) per ml of retrovirus particles, can be determined.

Efficacy testing may include efficacy testing for release profiles with purity and specific activity. For example, potency release testing of the end product may include measuring the number of Transduction Units (TUs) that can be compared to the amount of viral particles, e.g., by performing ELISA for viral proteins, e.g., for lentiviruses, by performing p24 capsid protein ELISA with a cutoff of at least 100, 1,000, 2,000, or 2,500TU/ng p24, and CAR functionality, e.g., by measuring interferon gamma release from a reporter cell line exposed to genetically modified cells.

In any of the kits or isolated replication defective recombinant retroviral particles hereinIt includes a container of such retroviral particles in which there is sufficient recombinant retroviral particles present to obtain an MOI (number of transducing units, or TU for each cell) of 0.1 to 50, 0.5 to 20, 0.5 to 10, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 2 to 15, 2 to 10, 2 to 7, 2 to 3, 3 to 10, 3 to 15 or 5 to 15 or at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15, or an MOI of at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15 in a reaction mixture prepared using the retroviral particles. The transduction units of the viral particles provided in the kit should be able to use a MOI that prevents the production of too many integrants in individual cells, with an average of less than 3 slow genome copies per cell genome, and more preferably 1 copy per cell. For the kit and isolated retroviral particle embodiments, such MOI can be based on 1, 2.5, 5, 10, 20, 25, 50, 100, 250, 500 or 1,000ml of the reaction mixture, assuming 1X 10 ⁶ Target cells/ml, for example in the case of whole blood, 1X 10 is assumed ⁶ PBMC/ml blood. Thus, the container for retroviral particles may comprise 1X 10 ⁵ Up to 1X 10 ⁹ 、1×10 ⁵ Up to 1X 10 ^8、 1×10 ⁵ Up to 5X 10 ⁷ 、1×10 ⁵ Up to 1X 10 ⁷ 、1×10 ⁵ Up to 1X 10 ⁶ ；5×10 ⁵ Up to 1X 10 ⁹ ；5×10 ⁵ Up to 1X 10 ⁸ 、5×10 ⁵ Up to 5X 10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁷ 、5×10 ⁵ Up to 1X 10 ⁶ Or 1X 10 ⁷ Up to 1X 10 ⁹ 、1×10 ⁷ Up to 5X 10 ⁷ 、1×10 ⁶ Up to 1X 10 ⁷ To 1X 10 ⁶ Up to 5X 10 ⁶ TU (TU). In certain illustrative embodiments, the container may comprise 1X 10 ⁷ Up to 1X 10 ⁹ 、5×10 ⁶ Up to 1X 10 ⁸ 、1×10 ⁶ Up to 5X 10 ⁷ 、1×10 ⁶ Up to 5X 10 ⁶ Or 5X 10 ⁷ Up to 1X 10 ⁸ Is a retroviral transduction unit of (a). Without being limited by theory, such amounts of particles will support 1 to 100ml of blood at an MOI of 1 to 10. In some illustrative embodiments, as described herein, one mayTo treat as little as 10ml, 5ml, 3ml or even 2.5ml of blood for T cell and/or NK cell modification and optionally the subcutaneous and/or intramuscular administration methods provided herein. Thus, the present methods have the advantage that in some illustrative embodiments they require significantly fewer retroviral particle transduction units than existing methods involving nucleic acids encoding a CAR (e.g., CAR-T methods).

Each container containing retroviral particles may, for example, have a volume of 0.05ml to 5ml, 0.05ml to 1ml, 0.05ml to 0.5ml, 0.1ml to 5ml, 0.1ml to 1ml, 0.1ml to 0.5ml, 0.1ml to 10ml, 0.5ml to 5ml, 0.5ml to 1ml, 1.0ml to 10.0ml, 1.0ml to 5.0ml, 10ml to 100ml, 1ml to 20ml, 1ml to 10ml, 1ml to 5ml, 1ml to 2ml, 2ml to 20ml, 2ml to 10ml, 2ml to 5ml, 0.25ml to 10ml, 0.25 to 5ml, or 0.25 to 2 ml.

In some embodiments in which the kit comprises modified cells such as modified lymphocytes, modified TILs, modified non-B-cell lymphocytes such as modified T cells and/or modified NK cells in a cell suspension in a commercial container such as a cryopreservation infusion bag, the container such as a cryopreservation infusion bag can hold 5ml, 10ml, 15ml, 20ml, 25ml, 50ml, 75ml, 100ml, 150ml, 200ml, 250ml, 300ml, 400ml or 500ml or less blood. In some embodiments, a container, such as a cryopreservation infusion bag, can hold at least 5ml, 10ml, 15ml, 20ml, 25ml, 50ml, 75ml, 100ml, 150ml, 200ml, 250ml, 300ml, 400ml, or 500ml of blood. In some embodiments, a container such as a cryopreservation infusion bag may hold 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml, 25ml, and 50ml of blood as the low end of the range to 10ml, 15ml, 20ml, 25ml, 50ml, 75ml, 100ml, 150ml, 200ml, 250ml, 300ml, 400ml, and 500ml of blood as the high end of the range. In some embodiments, a container such as a cryopreservation infusion bag may hold 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml, 25ml, and 50ml of blood as the low end of the range to 10ml, 15ml, 20ml, 25ml, 50ml, 75ml, 100ml, 150ml, 200ml, 250ml, 300ml, 400ml, and 500ml of blood as the high end of the range. For example, a container such as a cryopreservation infusion bag may hold 1ml to 10ml, 5ml to 25ml, 10ml to 50ml, 25ml to 100ml, 50ml to 200ml, or 100ml to 500ml of blood. In some embodiments, the container, such as a cryopreservation infusion bag, may comprise heparin. In other embodiments, the container, such as a cryopreservation infusion bag, does not include heparin.

In some embodiments in which the kit includes modified cells such as modified lymphocytes, modified TILs, modified non-B cell lymphocytes such as modified T cells and/or modified NK cells in cell suspensions in a commercial container such as a cryopreservation infusion bag, the number of cells delivered may be sufficient to provide 1x10 ⁵ Individual cells to 1x10 ⁹ Individual cells, 1x10 ⁶ Individual cells to 1x10 ⁹ Individual cells, or 1x10 ⁶ Individual cells to 5x10 ⁸ Individual cells, e.g., CAR positive living T cells and/or NK cells/kg body weight of the subject of the cell to be delivered. Thus, in some embodiments, the commercial container may comprise the above-described range x50-150kg or 50-100kg. In some embodiments, the commercial container comprises 1x10 ⁷ Individual cells to 1x10 ¹¹ Individual cells, 1x10 ⁸ Individual cells to 1x10 ¹¹ Individual cells or 1x10 ⁸ Individual cells to 5x10 ¹⁰ Individual cells, such as CAR positive living T cells and/or NK cells, or cells positive for an anti-idiotype extracellular recognition domain in the illustrative embodiment.

In an illustrative embodiment, a polynucleotide encoding an anti-idiotype polypeptide and, in an illustrative embodiment, a CAR, is located in the genome of a retroviral particle (typically a substantially pure retroviral particle) according to any replication defective recombinant retroviral particle aspects and embodiments provided herein. In an illustrative embodiment, according to any of the embodiments provided herein, the replication defective recombinant retroviral particle in the kit comprises a polynucleotide comprising one or more transcriptional units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more first transcriptional units encode a first polypeptide comprising an anti-idiotype polynucleotide and CAR or LE, and optionally a second polypeptide comprising the other of CAR or LE.

Kits provided herein can include containers containing polynucleotides, such as polynucleotide vectors, e.g., RIP, or modified cells, such as modified lymphocytes, modified TILs, modified lymphocytes other than B cells, such as modified T cells and/or NK cells, and accessory kits. The accessory kit component may include one or more of the following:

a) One or more containers comprising a delivery solution that is compatible with, in an illustrative embodiment is effective, and in a further illustrative embodiment is suitable for, subcutaneous and/or intramuscular administration as provided herein;

b) One or more containers of hyaluronidase as provided herein;

c) One or more blood bags, such as a blood collection bag, in the illustrative embodiment, containing anticoagulant, a blood processing buffer bag, a blood processing waste collection bag, and a blood processing cell sample collection bag in the bag or in a separate container;

d) One or more sterile syringes compatible with, in an illustrative embodiment effective for, and in a further illustrative embodiment suitable for, subcutaneous or intramuscular delivery of T cells and/or NK cells;

e) T cell activating elements as disclosed in detail herein, such as anti-CD 3 provided in a solution in a container containing the retroviral particles, or provided in a separate container, or in an illustrative embodiment associated with the surface of the replication defective retroviral particles;

f) One or more leukoreduction filter assemblies;

g) One or more receptacles containing a solution or medium that is compatible with, in an illustrative embodiment is effective, and in a further illustrative embodiment is suitable for, transduction of T cells and/or NK cells;

h) One or more receptacles containing a solution or medium compatible with, in an illustrative embodiment effective to flush T cells and/or NK cells, and/or in a further illustrative embodiment suitable for flushing T cells and/or NK cells;

i) One or more containers containing a pH adjusting pharmaceutical agent;

j) One or more containers containing polynucleotides, typically substantially pure polynucleotides (e.g., found in recombinant retroviral particles according to any embodiment herein), comprising one or more second transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more second transcription units encode a polypeptide comprising a second CAR directed against a different target epitope, and in certain embodiments, a different antigen, in illustrative embodiments an antigen found on the same target cancer cell (e.g., B cell);

k) One or more containers comprising homologous antigens of the first CAR and/or the second CAR encoded by a nucleic acid (e.g., a retroviral particle); and

l) instructions physically or digitally associated with other kit parts for use thereof, e.g. for modifying T cells and/or NK cells, for delivering the modified T cells and/or NK cells subcutaneously or intramuscularly to a subject, and/or for treating tumor growth or cancer in a subject.

In any of the kit aspects and embodiments herein including an antigen or cognate antigen, less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the polypeptides in the kit are non-human, i.e., are produced by a non-human source.

In some embodiments, the kit may be a single package/single use kit, but in other embodiments the kit is a multi-package or multi-use kit for treating more than one blood sample from contact with a nucleic acid encoding a CAR, optionally by subcutaneous administration. Typically, the containers of nucleic acids encoding the CAR (and optionally the pair of containers of nucleic acids encoding the second CAR in certain embodiments) in the kit are used for one implementation of the method of modifying T cells and/or NK cells and optionally subcutaneous administration. Containers containing nucleic acids encoding the CAR and optionally the second CAR are typically stored frozen and transported. Thus, the kit may comprise sufficient containers (e.g., vials) of nucleic acids encoding the CAR (and optionally pairs of containers encoding the second CAR in certain embodiments) for 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 implementations of the methods of modifying T cells and/or NK cells provided herein, and thus may comprise containers (e.g., vials) of 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 nucleic acids encoding the CAR (e.g., retroviral particles), and are similarly considered as 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 packages, implementations, administrations, or X kits, respectively. Similarly, accessory components in the kit will be provided for use in similar number of implementations of methods of modifying T cells and/or NK cells and optionally subcutaneous administration using the kit.

If present in such kits, the one or more leukoreduction filter assemblies typically include one or more leukoreduction filters or sets of leukoreduction filters, each of which is typically located within a filter housing, and a plurality of connected sterile tubes connected or adapted to be connected thereto, and a plurality of valves connected or adapted to be connected thereto, which are adapted for use in a single use, closed blood processing system. Typically, there is one leukoreduction filter assembly for each container of nucleic acid encoding the CAR in the kit. Thus, in an illustrative embodiment, a 20-pack kit comprises 20 vials of CAR-encoding nucleic acid and 20 leukoreduction filter assemblies. In some embodiments, the kits herein comprise one or more containers containing nucleic acids and one or more leukoreduction filter assemblies. Such kits may optionally be intended for administration to a subject by any route, including, for example, infusion or intramuscular in the illustrative embodiments and/or subcutaneous delivery in the further illustrative embodiments. Thus, such kits optionally include other accessory components intended for use with such routes of administration. One or more containers for subcutaneous or intramuscular delivery of solutions are discussed in more detail herein, are generally sterile, and may include 100ml to 5L, 1ml to 1L, 1ml to 500ml, 1ml to 250ml, 1ml to 200ml, 1ml to 100ml, 1ml to 10ml, or 1ml to 5ml; a total combined volume of 5ml to 1L, 5ml to 500ml, 5ml to 250ml, 5ml to 100ml, 5ml to 10ml, or about 5ml, or the volume is individual for each container. In some illustrative embodiments, the kit comprises a plurality of containers of subcutaneous delivery solution, wherein each container has a volume of 10ml to 200ml, 10ml to 100ml, 1ml to 20ml, 1ml to 10ml, 1ml to 5ml, 1ml to 2ml, 2ml to 20ml, 2ml to 10ml, 2ml to 5ml, 0.25ml to 10ml, 0.25 to 5ml, or 0.25 to 2 ml. In an illustrative embodiment, there is one container of delivery solution for each container of nucleic acid encoding a CAR in the kit. Thus, in an illustrative embodiment, a 20-pack kit comprises 20 vials of CAR-encoding nucleic acid and 20 containers of sterile delivery solution.

In certain kit aspects, provided herein are embodiments in which one or both of the containers containing a nucleic acid encoding a first CAR and optionally a nucleic acid encoding a second CAR are nucleic acids according to any of the self-driven CAR embodiments provided herein. In such embodiments, the accessory component of the kit may further comprise one or more of the following:

one or more containers containing a delivery solution suitable for, compatible with, and/or effective for intravenous or intraperitoneal administration as provided herein; and

instructions physically or digitally associated with the other kit parts for use thereof, e.g., intravenous or intraperitoneal delivery of modified T cells and/or NK cells to a subject.

In certain aspects, provided herein is a use of RIP in the manufacture of a kit for modifying T cells or NK cells, wherein the use of the kit comprises: contacting T cells or NK cells ex vivo with a replication defective recombinant retroviral particle, wherein the replication defective recombinant retroviral particle comprises a pseudotyped element on the surface and a T cell activating element on the surface, wherein the contacting facilitates transduction of the T cells or NK cells by the replication defective recombinant retroviral particle, thereby producing modified and in the illustrative embodiment genetically modified T cells or NK cells.

In some aspects, provided herein are aspects that include the use of RIP in the preparation of a kit for modifying T cells or NK cells. Details about polynucleotides and replication defective recombinant retroviral particles containing such polynucleotides are disclosed in more detail herein and in the illustrative examples section. In some embodiments, the T cell or NK cell may be from a subject. In some embodiments, the T cell activating element may be membrane-bound. In some embodiments, the contacting may be performed for 1, 2, 3, 4, 5, 6, 7, or 8 hours as the low end of the range to 4, 5, 6, 7, 8, 10, 12, 15, 18, 21, and 24 hours, e.g., 1 to 12 hours, as the high end of the range. Replication-defective recombinant retroviral particles for use in making the kit may comprise any of the aspects, embodiments or sub-embodiments discussed elsewhere herein.

Further, in another aspect, provided herein is a container (e.g., a commercial container or package) or a kit comprising the container, comprising an isolated packaging cell according to any of the packaging cell and/or packaging cell line aspects provided herein, in an illustrative embodiment, an isolated packaging cell from a packaging cell line. In some embodiments, the kit comprises additional containers comprising additional reagents, such as buffers or reagents for use in the methods provided herein. Furthermore, in certain aspects, provided herein is the use of any of the replication-defective recombinant retroviral particles provided herein in any aspect for the preparation of a kit for modifying a T cell or NK cell according to any aspect provided herein and in an illustrative embodiment genetically modifying a T cell or NK cell according to any aspect provided herein. Furthermore, in certain aspects, provided herein is the use of any packaging cell or packaging cell line provided herein in any aspect, in the preparation of a kit for producing replication defective recombinant retroviral particles according to any aspect provided herein.

Self-driven CAR methods and compositions

Provided herein in certain aspects are polynucleotides, referred to herein as "self-driven CARs," that encode a membrane-bound lymphoproliferative element whose expression in T cells or NK cells is under the control of an inducible promoter that is induced by binding of an antigen to an extracellular binding pair member polypeptide expressed by the T cells or NK cells and functionally linked to an intracellular activation domain, such as the cd3ζ intracellular activation domain or any of the intracellular activation domains disclosed elsewhere herein. In the illustrative embodiments herein, the anti-idiotype polypeptide is co-expressed by a T cell or NK cell to provide additional functional options for the self-driven CAR. In an illustrative embodiment, the co-expressed anti-idiotype polypeptide is a safety switch. In an illustrative embodiment, such binding pair member polypeptide is a CAR. In other embodiments, such binding pair member polypeptides are TCRs. Thus, in certain embodiments, provided herein are polynucleotides comprising an inducible promoter operably linked to a nucleic acid encoding a membrane-bound lymphoproliferative element that is induced by binding of a CAR to its target. Expression of lymphoproliferative elements can induce proliferation of T cells or NK cells. Provided herein in certain aspects are genetically modified or transduced T cells, referred to herein as "self-driven CAR-T cells," which include self-driven CARs. Any embodiment comprising a self-driven CAR-T cell may comprise a "self-driven CAR NK cell," which is a genetically modified or transduced NK cell comprising a self-driven CAR. In some embodiments, there are self-driven CAR NK cells in addition to the self-driven CAR-T cells. In other embodiments, there are self-driven CAR NK cells instead of self-driven CAR-T cells. Various embodiments including self-driven CARs are disclosed in the illustrative embodiments section herein and may be combined with any of the embodiments or details of that section.

Illustrative cell handling methods for genetically modified T cells and/or NK cells

Methods provided herein in illustrative aspects include methods for modifying T cells and/or NK cells, or related methods of making cell preparations, comprising contacting blood cells comprising lymphocytes (e.g., NK cells and/or T cells) in a reaction mixture ex vivo with a recombinant vector, such as or comprising a replication defective recombinant retroviral particle of a polynucleotide encoding a CAR. In illustrative embodiments, the reaction mixture includes T cell activating elements in solution or on the surface of the recombinant retroviral particle to facilitate genetic modification of T cells in the reaction mixture. Some methods provided herein include the optional step of collecting (110) blood from a subject. Blood may be collected or obtained from a subject by any suitable method known in the art, as discussed in more detail herein. For example, blood may be collected by venipuncture, apheresis, or any other blood collection method that collects a sample of blood. In some embodiments, the volume of blood collected is 1ml to 120ml. In the illustrative embodiments, particularly those in which the subject from which blood is obtained has normal levels of NK cells and in the illustrative embodiments normal levels of T cells, the volume of blood collected is 1ml to 25ml.

Notably, the methods provided herein for modification, and in illustrative embodiments for genetic modification, do not include the step of collecting blood from a subject in some embodiments. Regardless of whether blood is collected from a subject, in illustrative method aspects provided herein for modifying lymphocytes (e.g., T cells and/or NK cells), lymphocytes are contacted with an encapsulated nucleic acid vector (e.g., replication defective retroviral particles) in a reaction mixture. In illustrative embodiments, such contacting and the reaction mixture in which the contacting occurs are performed in a closed cell processing system, as discussed in more detail herein. Such closed processing systems and methods used in some aspects and embodiments of the systems and methods provided herein may be any systems and methods known in the art. As non-limiting examples, the system or method may be a conventional closed cell processing system and method, or a system or method referred to herein as a "newer" method or system (see, e.g., WO2018/136566 and WO2019/055946, each of which is incorporated herein by reference in its entirety). In traditional closed cell processing methods involving ex vivo lymphocyte gene modification and/or transduction, especially in methods for autologous cell therapy, many steps are performed within days, such as PBMC enrichment, washing, cell activation, transduction, expansion, collection and optionally reintroduction. In the latest methods, some of the steps and time involved in such ex vivo cell treatments have been reduced (see e.g. WO 2018/136566). In other more recent methods (see fig. 1A), some of the steps and time involved in the ex vivo cell treatment have been further reduced or eliminated as has, for example, been the ex vivo expansion step (see, for example, WO 2019/055946). These recent methods (and other improved cell handling methods provided herein) also use rapid ex vivo transduction processes, such as processes that do not include or include minimal pre-activation (e.g., contacting lymphocytes (e.g., T cells and/or NK cells) with an activator for less than 30, 15, 10, or 5 minutes before contacting the lymphocytes with a retroviral particle). In certain embodiments of such methods, T cells and/or NK cell activating elements are present in the reaction mixture in which the contacting step is performed. In an illustrative embodiment, the T cell and/or NK cell activating element is associated with the surface of a retroviral particle present in the reaction mixture. In illustrative embodiments, such methods using rapid ex vivo genetic modification without the need for an ex vivo expansion step are used in rapid point of care (rPOC) autologous cell therapy methods. However, such latest methods still involve PBMC enrichment steps/procedures (120A), which typically take at least about 1 hour in a closed system, followed by cell counting, transfer and medium addition, which takes at least about 45 minutes again, and then contacting lymphocytes with retroviral particles to form a transduction reaction mixture (130A). Following the "viral transduction" step (which is typically a contact step and incubation as discussed in detail herein), lymphocytes are typically washed out of retroviral particles retained in suspension (140A), e.g., using Sepax, and collected by re-suspending PBMCs in a delivery solution to form a cell preparation (150A), typically in an infusion bag for re-infusion, a syringe for injection, or a cryopreservation vial for storage (160A). As discussed in further detail herein, conventional PBMC enrichment procedures typically involve a ficoll density gradient and centrifugation (e.g., centrifugation) or centripetal (e.g., sepax) force or enrichment of PBMCs using leukocyte infiltration (leucophoresis).

The inventors have observed that subcutaneous administration has shown surprising results in which the implantation of modified and/or genetically modified lymphocytes is increased relative to modified and/or genetically modified lymphocytes introduced by intravenous infusion. This results in more effective CAR-dependent tumor reduction and elimination in animals. In an illustrative embodiment, the modified lymphocytes (e.g., T cells and/or NK cells) in solution are introduced and in an illustrative embodiment reintroduced into the subject by subcutaneous administration, delivery or injection. In some examples of these embodiments involving contacting lymphocytes in a reaction mixture with retroviral particles, such as those illustrated in fig. 1, including those illustrative embodiments that include at least some other blood components that are not normally present after separation of lymphocytes in a PBMC enrichment procedure, the resulting cell preparation as a separate aspect provided herein is optionally administered (e.g., re-administered) into a subject. In the illustrative embodiment (fig. 1D) in which the PBMC enrichment procedure is not used after lymphocytes are contacted with retroviral particles, the cell preparation produced there can be reintroduced back into the subject using subcutaneous or intramuscular administration. Thus, as discussed in more detail herein, some aspects provided herein are cell preparations, and delivery solutions (i.e., excipients) for preparing such cell preparations, which in illustrative embodiments are compatible with and in further illustrative embodiments are effective against cell preparations suitable for subcutaneous delivery. Without being limited by theory, it is believed that the presence of additional blood cells (particularly neutrophils) during concentration and/or washing of lymphocytes using only a cell handling filter (e.g., a hemaltate filter) makes the cell preparation easier to deliver subcutaneously to avoid some of the additional risks that would exist if these other blood cell types, particularly neutrophils or aggregated T cells, were directly infused back into the patient's blood. For example, a subcutaneous formulation of retrovirus reconstituted with total nucleated cells on a lymphopenia filter may contain neutrophils (or more generally granulocytes) in addition to lymphocytes. In an illustrative embodiment, the cell preparation comprises neutrophils, B cells, monocytes, erythrocytes, basophils, eosinophils and/or macrophages, and modified T cells (CAR-T cells) and/or NK cells (CAR-NK cells). Subcutaneous or intramuscular formulations and administration are preferred over intravenous formulations and administration because formulations (suspensions) of retroviruses reconstituted with lymphocytes may further contain cell aggregates and express adhesion receptors that can be introduced into pulmonary congestion by intravenous delivery.

Methods for subcutaneous administration are well known in the art and generally involve administration into the fat layer beneath the skin. It should be noted that it is contemplated that any embodiment herein involving subcutaneous delivery may alternatively be intramuscular delivery (which is delivery into a muscle), intradermal delivery, or intratumoral delivery. In some embodiments, subcutaneous administration may be performed on the upper thigh, upper arm, abdomen, or upper hip of the subject. Subcutaneous administration is distinguished from intraperitoneal administration, which penetrates the fat layer used in subcutaneous administration and delivers a formulation or drug into the peritoneum of a subject.

Steps and reaction mixtures for use in the methods herein

In certain aspects, provided herein is a method of administering a modified cell to a subject, which method may include the step of transducing, transfecting, genetically modifying, and/or modifying the cell prior to delivering the modified cell to the subject. The cells may be lymphocytes, such as Peripheral Blood Mononuclear Cells (PBMCs) (typically a population of PBMCs), typically T cells and/or NK cells, and in certain illustrative embodiments resting T cells and/or resting NK cells. The transduction, transfection, modification, and/or genetic modification steps may include contacting lymphocytes with a population of recombinant nucleic acid/polynucleotide vectors, which in certain illustrative embodiments includes a nucleic acid encoding an anti-idiotype polypeptide, and in illustrative embodiments is a replication defective recombinant retroviral particle (RIP), wherein the contacting (and incubating under contacting conditions) promotes membrane association, membrane fusion, or endocytosis by the recombinant nucleic acid vector, and optionally promotes transduction or transfection of cells, such as resting T cells and/or resting NK cells, to produce modified and in illustrative embodiments genetically modified T cells and/or NK cells. It is noted that while many aspects and embodiments provided herein are discussed in terms of RIP, it is contemplated that one of skill in the art will recognize that many different recombinant nucleic acid vectors (including but not limited to those provided herein) may be used and/or included in such methods and compositions. In the illustrative embodiment in which the recombinant nucleic acid vector is a RIP, the RIP typically comprises on its surface a fusogenic element and a binding element, which may be part of a pseudotyped element. In some embodiments, T cells and/or NK cells are activated prior to contact with RIP or other polynucleotide vectors. In illustrative embodiments, preactivation of T cells and/or NK cells is not required, and an activating element, which may be any of the activating elements provided herein, is present in the reaction mixture that is contacted. In further illustrative embodiments, the activating element is present on the surface of a replication defective recombinant retroviral particle. In illustrative embodiments, the activating element is anti-CD 3, e.g., anti-CD 3 scFv, or anti-CD 3 scFvFc.

Many of the method aspects provided herein include one or more of the following steps: 1) A step of collecting blood from a subject; 2) A step of contacting cells (e.g., NK cells and/or in an illustrative embodiment T cells, which may be from collected blood) with a recombinant vector (typically multiple copies thereof) encoding a CAR and/or a lymphoproliferative element in a reaction mixture, in an illustrative embodiment RIP, wherein the contacting may comprise an optional incubation; 3) Typically, the step of washing unbound recombinant vector from the cells in the reaction mixture; 4) A step of collecting modified cells, e.g., modified NK cells and/or modified T cells in an illustrative embodiment, in a solution, which in an illustrative embodiment may be a delivery solution, to form a cell suspension, which in an illustrative embodiment is a cell preparation; and 5) a step of delivering the cell preparation to a subject, in an illustrative embodiment, the subject is a subject from whom blood is collected, such as by infusion, or in certain illustrative embodiments, intradermally, intramuscularly or intratumorally, or in further illustrative embodiments, subcutaneously. Notably, in certain illustrative embodiments, the reaction mixture includes unfractionated whole blood or includes one or more cell types that are not PBMCs, and may include all or many of the cell types found in whole blood, including Total Nucleated Cells (TNCs). Notably, in certain embodiments, the recombinant vector comprises a self-driven CAR that encodes the CAR and the lymphoproliferative element.

As a non-limiting example, in some embodiments, 10 to 120ml of blood is collected (or white blood cells are isolated in 10 to 120ml by performing a leukopenia on a total blood volume of 0.5 to 2.0); passing the collected, unfractionated blood/isolated cells through a leukoreduction filter to separate TNC at the top of the filter; adding replication-defective recombinant retroviral particles to the TNC on top of the leukoreduction filter to a total reaction mixture volume of 500 μl to 10ml to form a reaction mixture and initiating contact; optionally incubating the reaction mixture for any of the contact times provided herein, as non-limiting examples, for example, 1-4 hours; washing the unassociated replication-defective recombinant retroviral particles from the cells in the reaction mixture by filtering the reaction mixture with 10 to 120ml of washing solution; and cells (including modified T cells and NK cells) retained on the TNC filter are eluted from the filter with 2ml to 10ml of the delivery solution, thereby forming a cell preparation suitable for introduction or reintroduction into the subject.

Some embodiments of any method used in any aspect provided herein (which may include a method for modifying and in an illustrative embodiment genetically modifying lymphocytes, PBMCs, and in an illustrative embodiment NK cells, and/or in a further illustrative embodiment T cells) may include the step of collecting blood from a subject. Blood includes blood components that can be used in the methods and compositions provided herein, including blood cells, such as lymphocytes (e.g., T cells and NK cells). In certain illustrative embodiments, the subject is a human subject suffering from cancer (i.e., a human cancer subject). Notably, certain embodiments do not include such steps. However, in embodiments that include collecting blood from a subject, blood may be collected or obtained from the subject by any suitable method known in the art as discussed in more detail herein, and thus the collected blood or blood-derived component may be referred to as a "blood-derived product" and is typically a "peripheral blood-derived product" because it is typically separated from peripheral blood. For example, blood-derived products may be collected by venipuncture or any other blood collection method known in the art by which a sample of unfractionated whole blood is collected in a vessel (e.g., a blood bag), or by which white blood cells and lymphocytes are separated from the blood, such as by apheresis (e.g., white blood apheresis or lymphoplasmacytoid apheresis). In some embodiments, the volume of blood collected (e.g., unfractionated whole blood) is 1 to 5ml, 5 to 10ml, 10 to 15ml, 15 to 20ml, 20 to 25ml, 5 to 25ml, 25 to 250ml, 25 to 125ml, 50 to 100ml, or 50 to 250ml, 75 to 125ml, 90 to 120ml, or 95 to 110ml. In some embodiments, the volume of blood collected may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900ml as the low end of the range to 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900ml or 1L as the high end of the range. In some embodiments, the volume of blood collected is less than 250ml, 100ml, 75ml, 20ml, 15ml, 10ml, or 5ml. In some embodiments, lymphocytes (e.g., T cells and/or NK cells) can be obtained by apheresis. In some embodiments, the volume of blood obtained and processed during an apheresis procedure (e.g., a white blood cell apheresis procedure or a lymphoplasmacysis procedure) can be 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 times the total blood volume of the subject as the low end of the range to 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, or 2.5 times the total blood volume of the subject as the high end of the range, e.g., 0.5 to 2.5, 0.5 to 2, 0.5 to 1.5, or 1 to 2 times the total blood volume of the subject. The total blood volume of humans is typically in the range of 4.5 to 6L, so that more blood is typically obtained and processed during apheresis than when unfractionated whole blood is collected. Whether the target blood cells (e.g., T cells) are obtained by apheresis or the unfractionated whole blood is collected, e.g., in a blood bag, it is contemplated that the target blood cells (e.g., T cells) therein will be treated according to the methods provided herein, which in certain illustrative embodiments results in the target blood cells becoming modified, genetically modified, and/or transduced. When apheresis (e.g., white blood cell apheresis or lymphoplasmacytoid apheresis) is used to collect a cell fraction comprising T cells and/or NK cells (e.g., to provide white blood cells or lymphoplasmacytes), such cells are resuspended in solution, either directly or after one or more washes, to which a recombinant vector encoding a CAR is added to form a reaction mixture provided herein. Such reaction mixtures may be used in any of the methods herein. In some illustrative methods in which a subject or blood sample from a subject has a low cd3+ blood count, blood cells (e.g., white blood cells or lymphocytes) are collected using apheresis (e.g., white blood cell apheresis or lymphoplasmacytoid apheresis) for inclusion in the methods provided herein.

Regardless of whether blood is collected from a subject or obtained by apheresis, in any method aspect provided herein that includes a step of modifying cells such as lymphocytes (e.g., T cells and/or NK cells), a population of cells such as lymphocytes (e.g., T cells and/or NK cells) is typically contacted in a reaction mixture with multiple copies of a recombinant vector, which in some embodiments is a copy of a non-viral vector, and in illustrative embodiments is the same RIP. The contacting in any of the embodiments provided herein can be performed in a chamber, e.g., within a blood bag, of a closed system suitable for processing blood cells, e.g., as discussed in more detail herein. In some embodiments, the blood bag may have 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500ml or less of blood during contact. In some embodiments, the blood bag may have at least 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500ml of blood during the contacting. In some embodiments, the blood bag may have 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50ml of blood as the low end of the range to 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500ml of blood as the high end of the range during contact. For example, the blood bag may have 1 to 10ml, 5 to 25ml, 10 to 50ml, 25 to 100ml, 50 to 200ml, or 100 to 500ml of blood during the contacting. In some embodiments, the mixture inside the blood bag may include an anticoagulant, such as heparin. In other embodiments, the mixture inside the blood bag does not include an anticoagulant, or does not include heparin. The transduction reaction mixture may include one or more buffers, ions, and a culture medium.

Regarding the retroviral particles in certain exemplary reaction mixtures provided herein and in illustrative embodiments, the lentiviral particles, there is 0.1 to 50, 0.5 to 20, 0.5 to 10, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 2 to 15, 2 to 10, 2 to 7, 2 to 3, 3 to 10, 3 to 15, or 5 to 15 infection rates (MOI); or at least 1 and less than 6, 11 or 51MOI; or in some embodiments, 5 to 10MOI units of replication defective recombinant retroviral particles. In some embodiments, the MOI may be at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15. With respect to compositions and methods for transducing lymphocytes in blood, in certain embodiments, higher MOI may be used than in methods in which PBMC are isolated and used in reaction mixtures. For example, illustrative embodiments of compositions and methods for transducing lymphocytes in whole blood, assume 1 x 10 ⁶ Individual PBMC/ml bloodRetroviral particles may be used such that the MOI is 1 to 50, 2 to 25, 2.5 to 20, 2.5 to 10, 4 to 6 or about 5 and in some embodiments, 5 to 20, 5 to 15, 10 to 20 or 10 to 15.

The contacting steps included in some methods provided herein or the reaction mixture aspects of the reaction mixture in some embodiments comprise at least 10% unfractionated whole blood (e.g., at least 10%, 20%, 25%, 50%, 60%, 70%, 80%, 90%, 95% or 99% whole blood) and optionally an effective amount of an anticoagulant; or the reaction mixture further comprises at least one other blood or blood preparation component that is not a PBMC, e.g. the reaction mixture comprises an effective amount of an anticoagulant and one or more non-PBMC type blood preparation components. The percentage of whole blood is the volume percentage of the reaction mixture prepared using unfractionated whole blood. For example, where the reaction mixture is formed by adding replication defective recombinant retroviral particles to whole blood, and in the illustrative embodiment unfractionated whole blood, the percentage of whole blood in the reaction mixture is the volume of whole blood divided by the total volume of the reaction mixture multiplied by 100. In illustrative embodiments, such non-PBMC type blood or blood preparation components are one or more (e.g., at least one, two, three, four, or five) or all of the following other components:

a) Erythrocytes, wherein erythrocytes comprise 1% to 60% of the volume of the reaction mixture;

b) Neutrophils, wherein the neutrophils comprise at least 10% of the leukocytes in the reaction mixture, or wherein the reaction mixture comprises at least 10% neutrophils and the same amount of T cells;

c) Basophils, wherein basophils comprise at least 0.05% of the leukocytes in the reaction mixture;

d) Eosinophils, wherein the reaction mixture comprises at least 0.1% of the leukocytes in the reaction mixture;

e) Plasma, wherein the plasma comprises at least 1% by volume of the reaction mixture; and

f) Anticoagulant

(such blood or blood formulation components a-f above are referred to herein as ("notable non-PBMC blood or blood formulation components")).

In any aspect disclosed herein that includes a percentage of whole blood, the percentage is based on volume. For example, in certain embodiments, at least 25% of the volume of the reaction mixture may be whole blood. Thus, in such embodiments, at least 25ml of 100ml of such reaction mixture will be whole blood.

The inventors have observed that when such cells are contacted with a polynucleotide vector (e.g., RIP), the surface expression of TCR complexes (including tcra, tcrp, and CD 3) on CD4 positive (cd4+) cells and CD8 positive (cd8+) cells is reduced or "darkened," thereby showing binding polypeptides, e.g., T cell activating elements, that bind to the TCR complexes, as in certain illustrative embodiments herein. This darkening is primarily a result of internalization of the TCR complex upon activation. Furthermore, the extent of such darkening increases with increasing concentration of a given gene vector in the reaction mixture and is related to the ability of the gene vector to activate and enter cells. Similarly, internalization of other surface polypeptides upon binding to polypeptides on the surface of a gene vector results in darkening of the surface polypeptides on cells in contact with the gene vector, and may be common during transduction using other binding polypeptides. Thus, in some embodiments, the percent reduction in surface polypeptide expression on cells contacted with a gene vector comprising a binding polypeptide as compared to surface polypeptide expression on cells not contacted with a gene vector comprising a binding polypeptide is used to quantify the efficacy of the gene vector and determine the appropriate dose of gene vector for modifying the population of cells. In an illustrative embodiment, the percent reduction in expression of the surface TCR complex on cells contacted with the gene vector as compared to expression of the surface TCR complex on cells not contacted with the gene vector is used to quantify the efficacy of the gene vector and determine an appropriate dose of the gene vector for modifying the population of cells. As used herein, a "darkening unit" (DU) is a unit that is at 37℃and 5% CO ₂ After the following 4 hours of contact with the gene vector, compared to the surface expression of the surface polypeptide in a mixture of cells under similar conditions but not in contact with the gene vector,the surface expression of the surface polypeptide is reduced by 50% by the amount of the gene vector (e.g., RIR retroviral particles) in 1ml of the cell mixture. The surface polypeptide is typically a binding partner of a binding polypeptide present on the surface of the gene vector. In some embodiments, the surface polypeptide is a TCR complex polypeptide. In some embodiments, the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCR a or tcrp. In an illustrative embodiment, the binding partner is CD3 and the binding polypeptide is anti-CD 3.

Because the level of expression of a binding polypeptide on the surface of a polynucleotide vector (sometimes referred to herein as a "gene vector") will vary between different binding polypeptides and polynucleotide vector formulations, the ability of a polynucleotide vector to reduce the surface expression of a surface polypeptide should be determined for each formulation of the polynucleotide vector. In some embodiments, the ability of the polynucleotide vector to reduce surface expression of the surface polypeptide is determined based on the number of target cells. In some embodiments, the ability of the polynucleotide vector to reduce surface expression of a surface polypeptide is based on the volume of the cell. In any of the aspects and embodiments herein, the reduction in surface expression of the surface polypeptide may be referred to as darkening the surface polypeptide. For example, if the surface expression of CD3 on a cell is reduced, CD3 darkens on the cell, and the cell may be referred to as CD3-, even though the cell may still contain CD3 that is not expressed on its surface. Without being limited by theory, T cells that temporarily internalize CD3 and darken CD3 are T cells, and eventually will re-express CD3 on their cell surfaces, making them cd3+ again.

Thus, in one aspect, provided herein is a method of determining the amount of a polynucleotide vector formulation that darkens surface expression of a surface polypeptide by darkening percentage on cells in a darkened volume, comprising:

a) Forming a plurality of reaction mixtures comprising a plurality of volumes of a polynucleotide vector formulation and a plurality of volumes of a cell mixture, wherein at least two of the plurality of reaction mixtures comprise different volumes of a polynucleotide vector formulation and/or a cell mixture, wherein the cell mixture comprises a plurality of cells comprising the surface polypeptide on their surfaces, and wherein the polynucleotide vector formulation comprises a plurality of polynucleotide vectors comprising binding polypeptides capable of binding the surface polypeptide on their surfaces;

b) Incubating the reaction mixture;

c) Measuring surface expression of the surface polypeptide in the reaction mixture and in an untouched volume of the cell mixture, wherein the untouched volume of the cell mixture is not contacted with the polynucleotide vector formulation; and

d) Using the measured surface expression of the surface polypeptide in the reaction mixture, the measured surface expression of the surface polypeptide in the non-contact volume of the cell mixture, and the amounts of the polynucleotide vector formulation and the cell mixture in the reaction mixture, the amount of the polynucleotide vector formulation is determined to darken the darkening percentage of cells in the darkening volume.

In some embodiments, the amount of the cell mixture in the reaction mixture is based on volume. In some embodiments, the amount of the cell mixture in the reaction mixture is based on the number of target cells. In some embodiments, the polynucleotide vector formulation is a viral formulation. In an illustrative embodiment, the viral formulation is a replication defective recombinant retroviral particle formulation. In some embodiments, the percentage darkening (percentage of cells darkened) is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%. In illustrative embodiments, the darkening percentage is at least or about 80%, 85%, 90%, or 95%. In some embodiments, the darkening volume is 0.25ml, 0.5ml, 0.75ml, 1ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml, 20ml, or 25ml. In some embodiments, the surface polypeptide may be CD3D, CD3E, CD3G, CD3Z, TCR a, TCR β, CD16A, NKp46, 2B4, CD2, DNAM, or NKG2C, NKG2D, NKG2E, NKG2F and/or NKG2H. In some embodiments, the surface polypeptide is a TCR complex polypeptide. In some embodiments, the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCR a or tcrp. In an illustrative embodiment, the surface polypeptide is CD3E. In some embodiments, the binding polypeptide can be any of the activating elements disclosed in the activating element section herein. In such embodiments, the surface polypeptide may be a binding partner of the activating element.

In an illustrative embodiment, the cell mixture is whole blood. In further illustrative embodiments, the cell mixture has undergone a red blood cell depletion procedure. In some embodiments, whole blood is collected from a healthy subject, e.g., a subject that does not have or is unaware of or suspected of having a disease, disorder, or condition associated with elevated expression of an antigen. In some embodiments, whole blood is collected from a subject having a disease, disorder, or condition associated with elevated expression of an antigen, wherein the polynucleotide vector is to be administered to one subject or other subject having the disease, disorder, or condition. In some embodiments, whole blood is collected from each subject, and a darkening unit is calculated for each subject separately.

In some embodiments, the reaction mixture may be incubated for less than or about 24, 12, 10, 8, 6, 4, or 2 hours or 60, 45, 30, 15, 10, or 5 minutes, or only for initial contact. In some embodiments, the reaction mixture may be incubated for 10 minutes to 24 hours, or 10 minutes to 8 hours, or 1 hour to 6 hours, or in illustrative embodiments, 3.5 to 4.5 hours, or 4 hours. In some embodiments, the reaction mixture may be incubated at about 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃, or 42 ℃. In some embodiments, in the absence of CO ₂ The reaction mixture was incubated under conditions. In an illustrative embodiment, the reaction mixture is reacted with 5% CO ₂ Incubation was performed.

In some embodiments, surface expression of the surface polypeptide is measured by Fluorescence Activated Cell Sorting (FACS) methods. In some embodiments, the antibody used in the FACS method is GMP. In some embodiments, CD3 antibodies are used to determine surface expression of the surface polypeptides. In some embodiments, the CD3 antibody is UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030 (BW 264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, TR66.Opt, huM291, WT31, WT32, SPv-T3B, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2, F101.01, and SK7. In an illustrative embodiment, the CD3 antibody is PerCP mouse anti-human CD 3-clone SK7 (BD, 347344). In some embodiments, cells present in the cell mixture are separated from unbound polynucleotide vector in the incubated reaction mixture prior to measuring surface expression of the surface polypeptide.

In the illustrative embodiment of the above method, the polynucleotide vector preparation is a replication defective recombinant retroviral particle preparation, the percentage darkening is 50%, the darkening volume is 1ml, the surface polypeptide is CD3, the cell mixture is whole blood collected from healthy subjects, and the reaction mixture is at 37℃and 5% CO ₂ Incubate for 4 hours and the method was used to calculate darkening units.

Such methods can be used to determine the amount of retroviral particles in a polynucleotide vector formulation that reduces surface polypeptide expression on a cell by a specific percentage. This amount can then be used to determine the amount of preparation of retroviral particles for subsequent transduction of whole blood, isolated PBMCs or isolated TNC. In any of the aspects and embodiments provided herein that include genetically modified and/or transduced lymphocytes, the amount of a preparation of a polynucleotide vector (e.g., replication defective recombinant retroviral particle) added to the lymphocyte can be determined using the methods described above.

A Darkening Unit (DU) may be used in any aspect or embodiment herein that includes a contacting step to determine the amount of polynucleotide vector to be added. Since the 1DU polynucleotide vector reduced the surface expression of the surface polypeptide by 50% in a 1ml volume of cells, the 10DU polynucleotide vector reduced the surface expression of the surface polypeptide by 50% in 10ml of cell mixture. In some embodiments, sufficient DU is added to a volume of cells to reduce surface expression of a surface polypeptide, such as CD3, by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96% or 97% after contact with a polynucleotide vector as compared to surface expression of the surface polypeptide in a mixture of cells under similar conditions but not contacted with the polynucleotide vector. In an illustrative embodiment, after contact with the polynucleotide vector, sufficient DU is added to a volume of cells to reduce surface expression of the surface polypeptide by greater than 80%, 85%, 90% or 95% as compared to surface expression of the surface polypeptide in a mixture of cells under similar conditions but not contacted with the polynucleotide vector. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20DU is added per ml of cell mixture. In illustrative embodiments, 5 to 20DU, 5 to 15DU, 10 to 20DU, or 13 to 18DU is added per ml of cell mixture. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20DU is added per 1,000,000 target cells. In some embodiments, the target cell is a lymphocyte, such as a T cell or NK cell. In illustrative embodiments, the cells are in whole blood, isolated PBMCs, or isolated TNCs. In further illustrative embodiments, the cells are the remaining fraction of whole blood after the red blood cells are cut. In some embodiments, sufficient DU is added to darken the population of cells by a specific percentage, e.g., darken CD3 on the population of T cells by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96% or 97%. In some embodiments, there is a sufficient darkening unit of the polynucleotide vector (RIP in the illustrative embodiment) to increase the percentage of surface darkened surface polypeptide of the cells, and in the illustrative embodiment the population of T cells, and in the illustrative embodiment the percentage of darkened surface CD3 "to at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96% or 97%. In any aspect and embodiment herein that includes cells contacted with a polynucleotide vector, a composition comprising cells can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20DU per ml of cells, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20DU per ml of blood, cell preparation, population of cells.

In illustrative embodiments, such contacting and the reaction mixture in which the contacting occurs are performed in a closed cell processing system, as discussed in more detail herein. Packaging cells and in illustrative embodiments, packaging cell lines, and in particular illustrative embodiments, packaging cells provided in certain aspects herein, can be used to produce replication defective recombinant retroviral particles. The cells in the reaction mixture may be PBMCs or TNCs, and/or in providing the reaction mixture aspects herein of compositions and methods for transducing lymphocytes in whole blood, anticoagulants and/or other blood components may be present, including other types of non-PBMC type blood cells, as discussed herein. Indeed, in illustrative embodiments of these compositions and method aspects for transducing lymphocytes in whole blood, the reaction mixture may be substantially whole blood and is typically an anticoagulant, retroviral particles, and a relatively small amount of a solution in which the retroviral particles are delivered into the whole blood.

In the reaction mixtures regarding the compositions and methods provided herein for modifying lymphocytes in whole blood, lymphocytes (including NK cells and T cells) can be present in the reaction mixture at a lower percentage of blood cells and a lower percentage of white blood cells than methods involving PBMC enrichment procedures prior to forming the reaction mixture. For example, in some embodiments of these aspects, more granulocytes or neutrophils are present in the reaction mixture than NK cells or even T cells. Details regarding the composition of the anticoagulant and one or more other blood components present in the reaction mixture for modifying aspects of lymphocytes in whole blood are provided in detail in other sections herein. In some of the reaction mixtures provided herein, T cells may comprise, for example, 10, 20, 30, or 40% of lymphocytes in the reaction mixture as the low end of the range to 40, 50, 60, 70, 80, or 90% of lymphocytes in the reaction mixture as the high end of the range. In illustrative embodiments, T cells comprise 10 to 90%, 20 to 90%, 30 to 90%, 40 to 80%, or 45 to 75% of lymphocytes. In such embodiments, for example, NK cells may comprise 1, 2, 3, 4, or 5% of lymphocytes in the reaction mixture as the low end of the range to 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% of lymphocytes in the reaction mixture as the high end of the range. In illustrative embodiments, the T cells comprise 1 to 14%, 2 to 14%, 3 to 14%, 4 to 14%, 5 to 13%, 5 to 12%, 5 to 11%, or 5 to 10% of the lymphocytes in the reaction mixture. In some embodiments, T cells can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the reaction mixture. As disclosed herein, the compositions and method aspects for transducing lymphocytes in whole blood generally do not involve any blood fractionation, such as a PBMC enrichment step of a blood sample, prior to contacting lymphocytes in the blood sample with recombinant nucleic acid vectors (e.g., retroviral particles) in the reaction mixtures disclosed herein for these aspects. Thus, in some embodiments, lymphocytes from unfractionated whole blood are contacted with recombinant retroviral particles. However, in some embodiments, particularly for certain aspects of the self-driven CAR methods and compositions section herein, the neutrophils/granulocytes are isolated from other blood cells prior to contacting the cells with the replication-defective recombinant retroviral particles. In some embodiments, peripheral Blood Mononuclear Cells (PBMCs), including Peripheral Blood Lymphocytes (PBLs) such as T cells and/or NK cells, are separated from other components in the blood sample using, for example, a PBMC enrichment procedure, prior to combining the peripheral blood mononuclear cells with the retroviral particles into a reaction mixture. Those skilled in the art will appreciate that various methods known in the art may be used to enrich different blood fractions containing T cells and/or NK cells.

The PBMC enrichment procedure is one in which PBMCs are enriched at least 25-fold, and typically at least 50-fold, from other blood cell types. For example, PBMCs are believed to account for less than 1% of the blood cells in whole blood. At least 30% and in some examples, up to 70% of the cells isolated in the PBMC eluate are PBMCs following the PBMC enrichment procedure. It is even possible to achieve higher PBMC enrichment using some PBMC enrichment procedures. Known in the artVarious PBMC enrichment procedures. For example, the PBMC enrichment procedure is a fei-kol density gradient centrifugation process that separates a major cell population, such as lymphocytes, monocytes, granulocytes and erythrocytes, throughout the density gradient medium. In such methods, the aqueous medium comprises fei kol, a hydrophilic polysaccharide that forms a high density solution. Whole blood stratification above or below the density medium (without mixing the two layers), followed by centrifugation will disperse cells according to their density and PBMC fractions form a thin white layer at the interface between plasma and density gradient medium (see e.g. Panda and ravindoran (2013), isolation of human PBMC (Isolation ofHuman PBMCs), biological protocol (BioProtoc.), volume 3 (3)). Furthermore, using the rotational force of the Sepax cell processing system, centripetal force can be used in fecol to separate PBMCs from other blood components.

In some embodiments, apheresis, e.g., white blood apheresis, can be used to isolate cells, such as PBMCs. For example, AMICUS RBCX (Fresenius-Kabi) and Trima Accel (Terumo BCT) apheresis devices and kits can be used. Cells isolated by apheresis typically contain T cells, B cells, NK cells, monocytes, granulocytes, other nucleated leukocytes, erythrocytes and/or platelets. Cells collected by apheresis can be washed to remove the plasma fraction and the cells placed in an appropriate buffer or medium, such as Phosphate Buffered Saline (PBS) or a washing solution that is devoid of calcium and possibly magnesium, or possibly devoid of multiple (if not all) divalent cations, for use in subsequent processing steps. In some embodiments, cells collected by apheresis can be genetically modified by any of the methods provided herein. In some embodiments, cells collected by apheresis can be used to prepare any of the cell preparations provided herein. In some embodiments, cells collected by apheresis can be resuspended in a variety of biocompatible buffers (e.g., such as Ca-free, mg-free PBS). Alternatively, undesirable groups in the sample containing cells collected by apheresis may be removed The cells were separated and resuspended in medium. In some embodiments, leukopenia may be used to isolate cells, such as lymphocytes. In any of the embodiments provided herein that include PBMCs, leukocyte apheresis (leukopak) can be used. In any embodiment including TNC, a buffy coat may be used. In another PBMC enrichment method, an automatic leukocyte removal collection system (e.g., SPECTRA

APHERESIS SYSTEM from Terumo BCT, inc.Lakewood, CO 80215, usa), the influent whole blood is separated from the target PBMC fraction using high speed centrifugation while effluent substances (such as plasma, red blood cells and granulocytes) are typically returned to the donor, although such return would be optional in the methods provided herein. Additional processing may be required to remove residual red blood cells and granulocytes. Both methods involve time-intensive purification of PBMCs, whereas the leukopenia method requires that the patient be present and involved during the PBMC enrichment step.

As other non-limiting examples of PBMC enrichment procedures, in some embodiments of the transduction, genetic modification, and/or modification methods herein, PBMCs are isolated using Sepax or Sepax 2 cell processing system (BioSafe). In some embodiments, PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec). In some embodiments, an automated apheresis separator is used that collects blood from a subject, passes the blood through a device that picks out a particular cell type (e.g., PBMCs), and returns the remainder to the subject. Density gradient centrifugation may be performed after apheresis. In some embodiments, PBMCs are isolated using a leukoreduction filter assembly. In some embodiments, magnetic bead activated cell sorting is then used to purify specific cell populations, such as PBLs or subsets thereof, from PBMCs according to cell phenotype (i.e., positive selection), which are then used in the reaction mixtures herein.

Other purification methods, such as substrate adhesion, which utilize substrates that mimic the environment encountered by T cells during recruitment to purify T cells prior to their addition to the reaction mixture, or negative selection may be used, wherein undesired cells are targeted for removal with an antibody complex that targets the undesired cells to be removed prior to formation of the reaction mixture for the contacting step. In some embodiments, red blood cells may be removed using a red blood cell rosette process prior to forming the reaction mixture. In other embodiments, the hematopoietic stem cells may be removed prior to the contacting step and thus in these embodiments, the hematopoietic stem cells are absent during the contacting step. In some embodiments herein, particularly for compositions and methods for transducing lymphocytes in whole blood, there is no ABC transporter inhibitor and/or substrate (i.e., is not present in the reaction mixture used to make the contact) prior to, during, or prior to and during the contact, with or without any step of optional incubation or method.

In certain embodiments of any aspect provided herein, the lymphocytes are modified with prior activation and/or stimulation and in illustrative embodiments genetically modified and/or transduced, and cultured ex vivo until the desired number of cells to be delivered is reached. In certain illustrative embodiments of any aspect provided herein, the modification and in illustrative embodiments the genetically modifying and/or transducing of lymphocytes is performed without pre-activation or stimulation and/or without pre-activation or stimulation, whether in vivo, in vitro, or ex vivo; and/or further, in some embodiments, after initial contact (with or without optional incubation), without ex vivo or in vitro activation or stimulation, or after initial contact (with or without optional incubation), without ex vivo or in vitro activation or stimulation. In certain illustrative embodiments, the cells are activated during the contacting, and are not activated at all or are not activated more than 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours prior to the contacting. In certain illustrative embodiments, for modifying, genetically modifying, and/or transducing cells, activation by elements not present on the surface of the retroviral particle is not required. Thus, before, during or after contact, except in the reverse direction Such activating or stimulating elements are not required beyond the transcriptome particle. Thus, as discussed in more detail herein, these illustrative embodiments, which do not require pre-activation or stimulation, provide the ability to rapidly conduct in vitro experiments that are intended to better understand T cells and the biological agent mechanisms therein. In addition, such methods provide for more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells or NK cells, as well as the development of such commercial production methods. Finally, such methods provide for more rapid ex vivo treatment of lymphocytes (e.g., NK cells, particularly T cells) for adoptive cell therapy, such as by providing a point of care (rPOC) method, which fundamentally simplifies delivery of such therapies. In illustrative embodiments, some, most, at least 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% or all of the lymphocytes are quiescent when combined with the retroviral particles to form a reaction mixture, and are typically quiescent when contacted with the retroviral particles in the reaction mixture. In a method for modifying lymphocytes (e.g., T cells and/or NK cells) in blood or components thereof, the lymphocytes may be contacted in their normally resting state when present in the collected blood immediately prior to collection. In some embodiments, T cells and/or NK cells are composed of 95% to 100% resting cells (Ki-67 ^- ) Composition is prepared. In some embodiments, the T cells and/or NK cells contacted with the replication defective recombinant retroviral particle comprise 90, 91, 92, 93, 94, and 95% resting cells as the low end of the range to 96, 97, 98, 99, or 100% resting cells as the high end of the range. In some embodiments, the T cells and/or NK cells comprise primordial cells. In some illustrative embodiments, the composition and method aspects for transducing lymphocytes in whole blood include the sub-embodiments of this paragraph.

In illustrative embodiments of aspects herein that include replication defective recombinant retroviral particles, contact between T cells and/or NK cells and the replication defective recombinant retroviral particles can facilitate transduction of T cells and/or NK cells by the replication defective recombinant retroviral particles. Without being bound by theory, during contact, replication defective recombinant retroviral particles recognize and bind to T cells and/or NK cells, and the T cells and NK cells are "modified" as that term is used herein. At this point the retroviral and host cell membranes begin to fuse and any retroviral pseudotyped elements and/or T cell activating elements, including anti-CD 3 antibodies, become integrated into the surface of the modified T cells and/or NK cells. Next, as a next step in the transduction process, genetic material from the replication defective recombinant retroviral particle enters the T cell and/or NK cell, where the T cell and/or NK cell is "genetically modified" as that term is used herein. Notably, such a process can be performed hours or even days after the start of the contact, and even after washing away unassociated retroviral particles. The genetic material is then typically integrated into the genomic DNA of the T cells and/or NK cells, at which point the T cells and/or NK cells are now "transduced," as that term is used herein. Similarly, the cells may be modified, genetically modified, and/or transduced with recombinant vectors other than replication defective recombinant retroviral particles. Cells may also internalize and integrate genetic material into the genomic DNA of T cells and/or NK cells after transfection, at which point T cells and/or NK cells are now "stably transfected" as that term is used herein. Thus, in an illustrative embodiment, any of the methods herein for modifying and/or genetically modifying lymphocytes (e.g., T cells and/or NK cells) are methods for transducing lymphocytes (e.g., T cells and/or NK cells). It is believed that the vast majority of modified cells and genetically modified cells have been transduced at day 6 (in vivo or ex vivo) after the initial contact. Lentiviral transduction methods are known. Exemplary methods are described, for example, in Wang et al (2012) [ J.Immunetither.) ] 35 (9): 689-701; cooper et al (2003) Blood 101:1637-1644; verhoeyen et al (2009) [ Methods of molecular biology ] 506:97-114; and Cavalieri et al (2003) blood 102 (2): 497-505 throughout the present disclosure, transduced or, in some embodiments, stably transfected T cells and/or NK cells include progeny of an ex vivo transduced cell that retains at least some nucleic acid or polynucleotide incorporated into the cell genome during ex vivo transduction. In the methods herein that reference is made to "reintroducing" transduced cells, it is to be understood that such cells are not typically in a transduced state at the time of their collection from the subject's blood.

Although in the illustrative embodiments, in the methods herein, T cells and/or NK cells are not activated prior to contact with the recombinant retrovirus, in the illustrative embodiments, T cell activating elements are present in the reaction mixture in which the initial contact of the recombinant retrovirus and lymphocytes is made. For example, such T cell activating elements may be in solution in the reaction mixture. For example, during contact and subsequent optional incubation, soluble anti-CD 3 antibodies may be present in the reaction mixture at 25-200, 50-150, 75-125, or 100 ng/ml. In illustrative embodiments, the soluble anti-CD 3 antibody is multivalent, such as bivalent, tetravalent, or higher order valency. In an illustrative embodiment, the T cell activating element is associated with a retroviral surface. The T cell activating element may be any T cell activating element provided herein. In illustrative embodiments, the T cell activating element can be an anti-CD 3, such as an anti-CD 3 scFv or an anti-CD 3 scFvFc. Thus, in some embodiments, the replication defective recombinant retroviral particle may further comprise a T cell activating element that associates with the outside of the surface of the retrovirus in other illustrative examples.

The contacting step of the transduction methods and/or methods for modifying or genetically modifying lymphocytes in whole blood provided herein generally comprises an initial step in which retroviral particles (typically a population of retroviral particles) are contacted with blood cells (typically a population of blood cells comprising an anticoagulant and/or other blood components other than PBMCs that are not present after a PBMC enrichment procedure) in a suspension in a liquid buffer and/or culture medium to form a transduction reaction mixture. As in other aspects provided herein, an optional incubation period may be performed in this reaction mixture after this contacting, the reaction mixture comprising retroviral particles and blood cells comprising lymphocytes (e.g., T cells and/or NK cells) in suspension. In a method for modifying T cells and/or NK cells in blood or components thereof, the reaction mixture may include at least one, two, three, four, five, or all other blood components as disclosed herein and in illustrative embodiments, one or more anticoagulants.

Following initial contact of the retroviral particles and lymphocytes, the transduction reaction mixture in any aspect provided herein can be incubated at 23 to 39 ℃ and in some illustrative embodiments, at 37 ℃. In certain embodiments, the transduction reaction may be performed at 37-39 ℃ to achieve faster fusion/transduction. In some embodiments, the contacting step is a cold contacting step as discussed elsewhere herein, with an optional incubation step. In some embodiments, the cold contacting step is performed at a temperature below 37 ℃, for example at 1 ℃ to 25 ℃ or 2 ℃ to 6 ℃. The optional incubation associated with the contacting step at these temperatures may be performed for any of the lengths of time discussed herein (e.g., in the illustrative examples section). In illustrative embodiments, the optional incubation associated with these temperatures is performed for 8 hours, 6 hours, 4 hours, 2 hours, and in illustrative embodiments 1 hour or less.

In some embodiments (including illustrative embodiments in which the contacting is performed on a filter), the contacting is performed at a lower temperature, e.g., at 2 ℃ to 25 ℃, referred to herein as cold contacting, and then the retroviral particles that remain unassociated in suspension are removed from the reaction mixture, e.g., by washing the reaction mixture on a filter (e.g., a leukopenia filter) that retains leukocytes including T cells and NK cells, but does not retain free unassociated viral particles. When contacted in the transduction reaction mixture, the cells and retroviral particles can be immediately treated to remove from the cells the retroviral particles which remain free in suspension and unassociated with the cells. Optionally, the cells and retroviral particles in suspension, whether free in suspension or associated with the cells in suspension, are incubated for different lengths of time, as provided herein for use in the contacting step in the methods provided herein. Washing may be performed prior to other steps, whether such cells are to be studied in vitro, ex vivo or introduced into a subject. Such suspensions may include allowing the cells and retroviral particles to settle, or causing such settlement by applying a force, such as centrifugal force, to the bottom of a container or chamber, as discussed in further detail herein. In the illustrative embodiment, such g-forces are lower than those successfully used in the centrifugal inoculation procedure (spinoculation procedure). Further contact times and discussions regarding contact and optional incubation will be discussed further herein (e.g., in the illustrative embodiments section).

Current methods requiring ex vivo expansion of genetically modified lymphocytes for extended periods of time prior to formulation and reintroduction into a subject may be employed in some embodiments of the methods herein, including modification of such cells with a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide. There has long been a need for effective point-of-care adoptive cell therapies that allow subjects to achieve blood drawing (collection), modification of lymphocytes, and reintroduction in a single visit. In some embodiments, the methods provided herein allow for rapid ex vivo treatment of lymphocytes and in some illustrative embodiments PBMCs and in other illustrative embodiments Total Nucleated Cells (TNCs) without the need for ex vivo expansion steps, such as by providing such point-of-care methods, and in some illustrative embodiments, in a shorter period of time (rapid point-of-care (rPOC)), radically simplifying the delivery of adoptive cell therapies. Disclosed herein are illustrative methods for modifying lymphocytes, particularly NK cells, and in illustrative embodiments, T cells, which are significantly faster and simpler than previous methods. Thus, in some embodiments, any of the methods provided herein for transducing, genetically modifying and/or modifying PBMCs or lymphocytes, typically T cells and/or NK cells, can be performed (or can occur) for any of the time periods provided in the present description, including (but not limited to) the time periods provided in the illustrative embodiments section. For example, the contacting can be performed for less than 24 hours, such as less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, but in each case there is at least an initial contacting step in which the retroviral particles and cells are contacted in suspension in the transduction reaction mixture, followed by separation and typically discarding of the retroviral particles that remain in suspension that are not associated with the cells, as discussed in further detail herein. It should be noted, but not wishing to be bound by theory, that contact is initiated when the retroviral particles are combined with lymphocytes, typically by adding a solution containing the retroviral particles to a solution containing lymphocytes (e.g., T cells and/or NK cells).

After initial contact (including initial cold contact), in some embodiments, the reaction mixture containing the cells and recombinant nucleic acid vector (which in the illustrative embodiments includes nucleic acid encoding an anti-idiotype polypeptide, and in further illustrative embodiments, a retroviral particle) is incubated in suspension for a specified period of time without removing the recombinant nucleic acid vector (e.g., retroviral particle) that remains free in solution and unassociated with the cells. This incubation is sometimes referred to herein as optional incubation. Thus, in illustrative embodiments, contacting (including initial contacting and optional incubation) can occur (or can occur) for 15 minutes to 12 hours, 15 minutes to 10 hours, or 15 minutes to 8 hours, or any time included in the illustrative embodiments section. In certain embodiments including a cold contact step, the recombinant nucleic acid vector and in the illustrative embodiments the retroviral particles not associated with the cells are washed away by suspending the cells for a second incubation after the optional washing step. In an illustrative embodiment, the secondary incubation is performed at a temperature between 32 ℃ and 42 ℃ (e.g., at 37 ℃). The optional secondary incubation may be performed for any length of time described herein. In illustrative embodiments, the optional secondary incubation is performed for 6 hours or less. Thus, in illustrative embodiments, the contacting (including initial contacting and optional incubation) may be performed (or may occur) (where as indicated generally herein, the low end of the selected range is less than the high end of the selected range) for 30 seconds or 1, 2, 5, 10, 15, 30, or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours to 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours as the low end of the range. Thus, in some embodiments, after formation of the reaction mixture by addition of retroviral particles to lymphocytes, the reaction mixture may be incubated for 5 minutes as the low end of the range to 10, 15 or 30 minutes as the high end of the range, or 1, 2, 3, 4, 5, 6, 8, 10 or 12 hours. In other embodiments, the reaction mixture may be incubated for 15 minutes to 12 hours, 15 minutes to 10 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1 hour, 15 minutes to 45 minutes, or 15 minutes to 30 minutes. In other embodiments, the reaction mixture may be incubated for 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, or 30 minutes to 45 minutes. In other embodiments, the reaction mixture may be incubated for 1 hour to 12 hours, 1 hour to 8 hours, 1 hour to 4 hours, or 1 hour to 2 hours. In another illustrative embodiment, the contacting is performed only between an initial contacting step (in the reaction mixture, including free retroviral particles in suspension and cells in suspension without any further incubation) and without any further incubation in the reaction mixture, or an incubation in the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour.

After the indicated period of time for initial contact and optional incubation which may be part of the contacting step, the blood cells or fractions thereof containing T cells and/or NK cells in the reaction mixture are separated from the retroviral particles not associated with such cells. For example, this may be done using a PBMC enrichment procedure (e.g., ficoll gradient in a Sepax unit), or in certain illustrative embodiments provided herein, by filtering the reaction mixture with a leukocyte depletion filter assembly and then collecting leukocytes (which include T cells and NK cells). In another embodiment, this can be done by centrifuging the reaction mixture at a relative centrifugal force of less than 500g, such as 400g, or 300 to 490g, or 350 to 450 g. Such centrifugation for separating the retroviral particles from the cells may be carried out for example for 5 minutes to 15 minutes, or for 5 minutes to 10 minutes. In the illustrative embodiment where centrifugal force is used to separate cells from retroviral particles not associated with the cells, such g-forces are typically lower than those successfully used in centrifugal seeding procedures.

In some illustrative embodiments, the methods provided herein do not involve, in any way, performing centrifugal inoculation. In such embodiments, one or more cells are not subjected to centrifugal seeding of at least 400g, 500g, 600g, 700g, or 800g for at least 15 minutes. In some embodiments, one or more cells are not subjected to centrifugal seeding for at least 10, 15, 20, 25, 30, 35, 40, or 45 minutes. In some embodiments, centrifugal inoculation is included as part of the contacting step. In an illustrative embodiment, when centrifugal inoculation is performed, there is no other incubation as part of the contact, as the time of centrifugal inoculation provides the incubation time of the optional incubation discussed above. In other embodiments, additional incubations for 15 minutes to 4 hours, 15 minutes to 2 hours, or 15 minutes to 1 hour follow centrifugal inoculation. Centrifugal inoculation can be performed for example for 30 minutes to 120 minutes, typically at least 60 minutes, for example 60 minutes to 180 minutes, or 60 minutes to 90 minutes. Centrifugal inoculation is typically performed in a centrifuge having a relative centrifugal force of at least 800g, more typically at least 1200g, for example 800g to 2400g, 800g to 1800g, 1200g to 2400g, or 1200g to 1800 g. After centrifugal seeding, such methods typically involve an additional step of re-suspending the pelleted cells and retroviral particles and then removing the retroviral particles that are not associated with the cells according to the steps discussed above when centrifugal seeding is not performed.

In embodiments including centrifugal inoculation, the contacting step including optional incubation and centrifugal inoculation may be performed at 4 ℃ to 42 ℃, or 20 ℃ to 37 ℃. In certain illustrative embodiments, no centrifugal inoculation is performed and the contacting and associated optional incubation is performed at 20-25 ℃ for 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or 15 minutes to 2 hours, 15 minutes to 1 hour, or 15 minutes to 30 minutes.

Methods of genetically modifying lymphocytes provided according to any of the methods herein generally comprise inserting into the cell a polynucleotide comprising one or more transcriptional units encoding any transgene, e.g., one, two, three, or all of an anti-idiotype polypeptide, cytokine, CAR, and lymphoproliferative element, and in illustrative embodiments include a nucleic acid encoding an anti-idiotype polypeptide according to any of the embodiments provided herein. Such anti-idiotype polypeptides, CARs and lymphoproliferative elements may be provided to support the shorter and simpler methods provided herein, which may support expansion of modified, genetically modified and/or transduced T cells and/or NK cells after contact and optional incubation. Thus, in exemplary embodiments of any of the methods provided herein, lymphoproliferative elements can be delivered from the genome of the internal retroviral particles of genetically modified and/or transduced T cells and/or NK cells such that these cells are characterized by increased proliferation and/or viability as disclosed in the lymphoproliferative element section herein. In exemplary embodiments of any of the methods provided herein, the genetically modified T cells or NK cells are capable of transplantation in vivo in mice and/or enrichment in vivo in mice for at least 7, 14, or 28 days. Those skilled in the art will recognize that such mice can be treated or otherwise genetically modified such that any immunological differences between genetically modified T cells and/or NK cells do not elicit an immune response in the mice against any component of lymphocytes transduced by replication defective recombinant retroviral particles.

The medium that may be included in the contacting step (e.g., when the cells and retroviral particles are initially contacted) or in any aspect provided herein (during optional subsequent incubation with a reaction mixture comprising retroviral particles and cells in suspension in a medium) may include or may be cultured in the cellsThe medium used during and/or during the various washing steps in any of the aspects provided herein may include a basic medium, such as a commercially available medium for ex vivo T cell and/or NK cell culture. Non-limiting examples of such media include X-VIVO ^TM 15 chemically defined serum-free hematopoietic cell Medium (Lonza) (2018 catalog No. BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q or 04-418Q), immunoCurt ^TM XF T cell expansion Medium (STEMCELL Technologies) (2018 catalog 10981),

T-cell expansion XSFM (Irvine Scientific) (2018 catalog No. 91141), AIM +.>

Medium CTS ^TM (therapeutic grade) (Thermo Fisher Scientific (referred to herein as "Thermo Fisher"), or CTS ^TM Optimizer ^TM Culture media (Thermo Fisher) (2018 catalog number A10221-01 (basal medium (bottle)) and A10484-02 (supplementation), A10221-03 (basal medium (bag)), A1048501 (basal medium and supplementation kit (bottle)) and A1048503 (basal medium and supplementation kit (bag)). Such culture media may be chemically defined serum-free formulations manufactured in accordance with cGMP, as described herein for kit components. Culture media may be exogenously and completely. Basal medium has been approved by regulatory authorities for use in ex vivo cell processing, such as FDA510 (k) -approved devices in some embodiments, culture media is with or without 2018 catalog number A1048501 (CTS) ^TM OpTmizer ^TM T cell expanded SFM, bottle format) or A1048503 (CTS) ^TM OpTmizer ^TM T cell expansion SFM, bag format) (both available from Thermo Fisher (Waltham, MA)) as a basal medium for supplied T cell expansion supplements. Additives such as human serum albumin, human ab+ serum, and/or serum derived from the subject may be added to the transduction reaction mixture. The supportive cytokine (e.g. IL2, IL7 or IL15, or fines found in human serum)Cytokine) is added to the transduction reaction mixture. In certain embodiments, dGTP may be added to the transduction reactant.

In some embodiments of any of the methods herein that include a step of modifying lymphocytes (e.g., T cells and/or NK cells), the cells can be contacted with the retroviral particles without prior activation. As a non-limiting example, a retroviral particle may include a nucleic acid encoding an anti-idiotype polypeptide. In some embodiments of any of the methods herein comprising the step of genetically modifying T cells and/or NK cells, in one embodiment, the T cells and/or NK cells are not incubated on a substrate that adheres to monocytes for more than 4 hours, or in another embodiment, more than 6 hours, or in another embodiment, more than 8 hours, prior to transduction. In one illustrative embodiment, T cells and/or NK cells are incubated overnight on an adherent substrate to remove monocytes prior to transduction. In another embodiment, the method may comprise incubating the T cells and/or NK cells on an adherent substrate that binds monocytes for no more than 30 minutes, 1 hour, or 2 hours prior to transduction. In another embodiment, prior to the transduction step, the T cells and/or NK cells are not exposed to a step of removing monocytes by incubation on an adherent substrate. In another embodiment, the T cells and/or NK cells are not incubated with or exposed to bovine serum (e.g., cell culture bovine serum, such as fetal bovine serum) prior to or during the contacting step and/or the modifying and/or genetic modification and/or transduction step.

In illustrative embodiments, some or all of the steps in the methods provided herein for modification or the use of such methods are performed in a closed system. Thus, the reaction mixtures formed in such methods, as well as modified, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) prepared by such methods, can be included within such closed systems. A closed system is a cell handling system that is typically closed or completely closed to the environment outside the system's tubes and chambers (e.g., the environment in a room or even the environment in a protective enclosure) for handling and/or transporting cells. One of the greatest risks for safety and regulation in cell handling procedures is the risk of contamination due to frequent exposure to the environment, as found in conventional open cell culture systems. To mitigate this risk, some commercial approaches have been developed that address the use of disposable (single use) devices, particularly in the absence of antibiotics. However, even when used under sterile conditions, there is always a risk of contamination by opening the flask to sample or add other growth medium. To address this problem, the methods provided herein are typically performed within a closed system, which is typically an ex vivo method. Such methods are designed and can be operated such that the product is not exposed to the external environment. The transfer of material is performed through sterile connectors, such as sterile tubing and sterile welded connectors. Air for gas exchange may be present through the gas permeable membrane, through the 0.2 μm filter to prevent environmental exposure. In some illustrative embodiments, the methods are performed on T cells, for example, to provide T cells that are modified and in illustrative embodiments genetically modified.

Such closed system methods can be performed using commercially available devices. Different closed system devices may be used at different steps in the method and tubes and connectors (such as welds, luer, spike or clavulanic) ports may be used to transfer cells between these devices to prevent exposure of cells or culture medium to the environment. For example, blood may be collected into an IV bag or syringe, optionally including an anticoagulant, and in some aspects transferred into a Sepax2 device (Biosafe) for PBMC enrichment and isolation. In other embodiments, the whole blood may be filtered using a leukoreduction filter assembly to collect leukocytes. Isolated PBMCs or isolated leukocytes may be transferred into a chamber of a G-Rex device for optional activation, transduction, and optional expansion. Alternatively, the collected blood may be transduced within a blood bag, such as a bag for collecting blood. Finally, the cells can be collected and harvested into another bag using the Sepax2 device. The method may be performed in any device or combination of devices suitable for closed system T cell and/or NK cell production. Non-limiting examples of such devices include G-Rex devices (Wilson Wolf), gatheRex (Wilson Wolf), sepax2 (Biosafe), WAVE bioreactor (General Electric), cultiLife cell culture bag (Takara), permaLife bag (OriGen), cliniMACS Prodigy (Miltenyi Biotec), and VueLife bag (Saint-Gobain). In illustrative embodiments, the optional activation, transduction, and optional amplification may be performed in the same chamber or vessel in a closed system. For example, in an illustrative embodiment, the chamber may be a chamber of a G-Rex device and PBMCs or leukocytes may be transferred into the chamber of the G-Rex device after enrichment and isolation, and may remain in the same chamber of the G-Rex device until collection.

The methods provided herein may include transferring blood and cells therein and/or fractions thereof and lymphocytes between containers within a closed system, either before or after the blood and cells and/or fractions thereof and lymphocytes are contacted with the retroviral particles, so that no environmental exposure occurs. The container used in the closure system may be, for example, a tube, bag, syringe, or other container. In some embodiments, the container is a container for a research facility. In some embodiments, the container is a container for commercial production. In other embodiments, the container may be a collection container for a blood collection procedure. The methods used for modification herein generally involve a contacting step in which lymphocytes are contacted with replication defective recombinant retroviral particles. In some embodiments, the contacting may be performed in a container (e.g., within a blood bag). Blood and its various lymphocyte-containing fractions may be transferred from one container to another (e.g., from a first container to a second container) for contact within a closed system. The second container may be a cell processing chamber of a closed device (e.g., a G-Rex device). In some embodiments, after contacting, the modified and in the illustrative embodiments genetically modified (e.g., transduced) cells can be transferred to a different container within a closed system (i.e., not exposed to the environment). Prior to or after this transfer, the cells are typically washed within a closed system to remove substantially all or all of the retroviral particles. In some embodiments, the methods disclosed herein (from collecting blood to contacting (e.g., transduction), optional incubation and post-incubation separation, and optional washing) are performed for 15 minutes, 30 minutes, or 1, 2, 3, or 4 hours as the low end of the range to 4, 8, 10, or 12 hours as the high end of the range.

Various embodiments of the method, as well as other aspects, such as the use of NK cells and T cells prepared by such methods, are disclosed in detail herein. Furthermore, various elements or steps for transduction, genetic modification, and/or modification of such method aspects of PBMCs, lymphocytes, T cells, and/or NK cells are provided herein, e.g., in the present section and the illustrative examples section, and such methods include examples provided throughout the present specification, as further discussed herein. For example, embodiments of any aspect for transduction, genetic modification, and/or modification of PBMCs or lymphocytes (e.g., NK cells or T cells in the illustrative embodiments), which are provided, for example, in this section and the illustrative embodiments section, can include any of the embodiments of the TIPs provided herein, including those that include one or more of the anti-idiotype polypeptides, lymphoproliferative elements, CARs, pseudotyped elements, control elements, activating elements, membrane-bound cytokines, mirnas, kozak-type sequences, WPRE elements, triple stop codons, and/or other elements disclosed herein, and can be combined with methods of producing retroviral particles using packaging cells. In certain illustrative embodiments, the retroviral particle is a lentiviral particle. Such methods for modifying, genetically modifying and/or transducing PBMCs or lymphocytes such as T cells and/or NK cells may be performed in vitro or ex vivo. Those of skill in the art will recognize that the details provided herein for transduction, genetic modification, and/or modification of PBMCs or lymphocytes (e.g., T cells and/or NK cells) may be applicable in any aspect including such steps.

In the methods provided herein, introducing and reintroducing (also referred to herein as administering and re-administering) modified cells, such as modified lymphocytes and in the illustrative embodiments genetically modified lymphocytes or in some embodiments RIP, into a subject can be by any means known in the art. Such introduction or reintroduction of genetically modified lymphocytes typically involves suspending i) modified and/or ii) genetically modified and/or iia) transduced or iiib) transfected cells in a delivery solution to form a cell preparation that can be introduced or reintroduced into a subject, as discussed in further detail herein. For example, such introduction of RIP may involve suspending the RIP in a delivery solution to form a transduction formulation that may be introduced into a subject. For example, the introduced or RIPS, lymphocyte or modified lymphocyte, or reintroduction of the lymphocyte or modified lymphocyte, can be delivered into the blood vessel of the subject by infusion. In some embodiments, for lymphocytes or modified lymphocytes, RIPS or modified lymphocytes (e.g., T cells and/or NK cells) are administered by intraperitoneal administration, intratumoral administration, intramuscular administration, or in illustrative embodiments by subcutaneous administration or otherwise introduced or reintroduced back into the subject.

Some of the administered cells are modified with nucleic acid encoding a lymphoproliferative element. Without being bound by theory, in a non-limiting illustrative method, ex vivo delivery of a polynucleotide encoding a lymphoproliferative element (which may be integrated into the genome of a T cell and/or NK cell) to a resting T cell and/or NK cell provides a cell with a driver for in vivo expansion without subjecting the host to lymphocyte depletion. Thus, in illustrative embodiments, the subject is not exposed to the lymphocyte depleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21 or 28 days or 1 month, 2 months, 3 months or 6 months of exposure, during exposure, and/or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21 or 28 days or 1 month, 2 months, 3 months or 6 months after reintroducing the modified T cells and/or NK cells back into the subject. Furthermore, in non-limiting illustrative embodiments, the methods provided herein can be performed without exposing the subject to lymphocyte depleting agents during the step in which the replication defective recombinant retroviral particle is contacted with resting T cells and/or resting NK cells of the subject and/or during the entire ex vivo method. Thus, methods of expanding T cells and/or NK cells in vivo in a subject that are modified, and in illustrative embodiments genetically modified, are features of some embodiments of the present disclosure. In certain embodiments, such methods involve ex vivo expansion of the modified cells, and in illustrative embodiments, such methods are ex vivo expansion-free or substantially expansion-free.

This entire method/process in non-limiting illustrative embodiments of any aspect provided herein (from the time the subject draws blood to reintroduce the modified and in illustrative embodiments genetically modified lymphocytes back into the subject after ex vivo transduction of T cells and/or NK cells) can be performed for a period of time of less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours. In any of the embodiments disclosed herein, the introduction or reintroduction of the modified lymphocytes may be by intravenous injection, intraperitoneal administration, subcutaneous administration, intratumoral administration, or intramuscular administration. In other embodiments, the entire method/process of the non-limiting illustrative embodiments herein (a period of time from the drawing/collection of blood from the subject to reintroducing the modified lymphocytes back into the subject after ex vivo transduction of T cells and/or NK cells) is performed for 1 hour to 12 hours, 2 hours to 8 hours, 1 hour to 3 hours, 2 hours to 4 hours, 2 hours to 6 hours, 4 hours to 12 hours, 4 hours to 24 hours, 8 hours to 36 hours, 8 hours to 48 hours, 12 hours to 24 hours, 12 hours to 36 hours, or 12 hours to 48 hours, or a period of time from 15, 30, 60, 90, 120, 180 and 240 minutes as the low end of the range to 120, 180 and 240, 300, 360, 420 and 480 minutes as the high end of the range. In other embodiments, the entire method/process (from the subject to draw/collect blood to reintroduce modified and in the illustrative embodiment genetically modified lymphocytes back into the subject after ex vivo transduction of T cells and/or NK cells) is performed for a period of 1, 2, 3, 4, 6, 8, 10 and 12 hours as the low end of the range to 8, 9, 10, 11, 12, 14, 18, 24, 36 or 48 hours as the high end of the range. In some embodiments, the modified and genetically modified T cells and/or NK cells are isolated from unassociated replication-defective recombinant retroviral particles after a period of time in which contact is made.

Because the illustrative embodiments of the methods for modifying lymphocytes and related methods for performing adoptive cell therapies provided herein can be performed in significantly shorter times as compared to previous methods, it is possible to radically improve patient care and safety as well as product manufacturability. Thus, such methods are expected to be advantageous in view of the regulatory authorities responsible for approving such methods for therapeutic purposes in vivo. For example, a subject in any non-limiting example provided herein including aspects of the subject may remain in the same building (e.g., infusion clinic) or room as the instrument that processes its blood or sample throughout sample processing, prior to reintroducing the modified T cells and/or NK cells into the patient. In a non-limiting illustrative embodiment, the subject remains within the location line and/or within a distance of 100, 50, 25 or 12 feet or arms from the blood or cells it is treating throughout the method/process of reintroducing blood into the subject after ex vivo transduction of T cells and/or NK cells from the subject. In other non-limiting illustrative embodiments, the subject remains awake and/or at least one person can continuously monitor the subject's blood or cells being treated during and/or continuously during the entire method/process of reintroducing blood into the subject after blood withdrawal/collection from the subject to transduce T cells and/or NK cells ex vivo. Because of the improvements provided herein, the entire method/process of blood withdrawal/collection from a subject for adoptive cell therapy and/or transduction of resting T cells and/or NK cells to reintroduce blood into the subject after ex vivo transduction of T cells and/or NK cells can be performed with continuous monitoring of humans. In other non-limiting illustrative embodiments, blood cells are not incubated in the room where no human exists at any point in the overall method/process of reintroducing blood into the subject after blood withdrawal/collection from the subject to transduce T cells and/or NK cells ex vivo. In other non-limiting illustrative embodiments, the entire method/process of blood withdrawal/collection from a subject to reintroduce blood to the subject after ex vivo transduction of T cells and/or NK cells is performed next to the subject and/or in the same room as the subject and/or next to the bed or chair of the subject. Thus, confusion of sample consistency can be avoided, as well as long and expensive incubations exceeding days or weeks. This advantage is further demonstrated by the fact that the methods provided herein are readily adaptable to closed and automated blood processing systems, wherein the blood sample and components thereof to be reintroduced into the subject are contacted with only disposable, single-use components.

Methods for modifying, genetically modifying, and/or transducing cells provided herein, such as lymphocytes, and in illustrative embodiments T cells and/or NK cells, can be part of a method for delivering a polynucleotide, such as a polynucleotide vector, to a subject, for delivering a modified cell to a subject, or for performing adoptive cell therapy. Typically, these methods include the steps of collecting blood from a subject and returning modified, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) to the subject. The present disclosure provides various methods of treatment using a CAR and, in an illustrative embodiment, an anti-idiotype polypeptide, which may be a CAR having an anti-idiotype extracellular recognition domain. When present in T lymphocytes or NK cells, the CARs of the present disclosure can mediate cytotoxicity against target cells. The CARs of the present disclosure bind to an antigen present on a target cell, thereby mediating killing of the target cell by T lymphocytes or NK cells that are genetically modified to produce the CAR. The ASTR of the CAR binds to an antigen present on the surface of the target cell. The present disclosure provides methods for killing or inhibiting the growth of target cells involving contacting cytotoxic immune effector cells (e.g., cytotoxic T cells or NK cells) genetically modified to produce a subject CAR such that the T lymphocytes or NK cells recognize antigens present on the surface of the target cells and mediate killing of the target cells. For example, the target cell may be a cancer cell and in some illustrative embodiments, the autologous cell therapy methods herein may be methods for treating cancer. In these embodiments, the subject may be an animal or human suspected of having cancer, or more typically, a subject known to have cancer. In some embodiments for treating PDL-1 positive cancer, and in illustrative embodiments of PDL-1 positive Diffuse Large B Cell Lymphoma (DLBCL), genetically modified cells may be administered in combination with an anti-PDL-1 antibody or antibody mimetic.

In some illustrative embodiments, cells are introduced or reintroduced into a subject by infusion into a vein or artery, particularly when neutrophils are not present in a preparation of lymphocytes that have been contacted with a retroviral particle and are ready for reintroduction, or by subcutaneous, intratumoral, or intramuscular administration, for embodiments in which at least 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, or 25% of the cells in the cell preparation to be administered are neutrophils, or 1% to 90%, 1% to 75%, 1% to 50%, 1% to 25%, 1% to 20%, 1% to 10%, 5% to 90%, 5% to 75%, 5% to 50%, 5% to 25%, 5% to 20%, 5% to 10%, 10% to 90%, 10% to 75%, 10% to 50%, 10% to 25%, or 10% to 20% of the cells in the cell preparation to be administered are neutrophils. Such embodiments may include co-administration or sequential administration with a hyaluronidase, as discussed in further detail herein. In any of the embodiments disclosed herein, the number of lymphocytes present in the cell preparations provided herein and optionally reinfused or in the illustrative embodiments delivered subcutaneously into the subject, and in the illustrative embodiments the number of modified T cells and/or NK cells can be at 1 x 10 as the lower end of the range ³ 、2.5×10 ³ 、5×10 ³ 、1×10 ⁴ 、2.5×10 ⁴ 、5×10 ⁴ 、1×10 ⁵ 、2.5×10 ⁵ 、5×10 ⁵ 、1×10 ⁶ 、2.5×10 ⁶ 、5×10 ⁶ And 1X 10 ⁷ Individual cells/kg to 5X 10 as the upper end of the range ⁴ 、1×10 ⁵ 、2.5×10 ⁵ 、5×10 ⁵ 、1×10 ⁶ 、2.5×10 ⁶ 、5×10 ⁶ 、1×10 ⁷ 、2.5×10 ⁷ 、5×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ And 1X 10 ¹⁰ Between individual cells/kg. In certain embodiments, the number of lymphocytes present in the cell preparations herein and optionally reinfused or otherwise delivered into a subject, and in illustrative embodiments the number of modified T cells and/or NK cells can be at 1 x 10 as the low end of the range ⁴ 、2.5×10 ⁴ 、5×10 ⁴ And 1X 10 ⁵ Individual cells/kg to 2.5X10 as the upper end of the range ⁴ 、5×10 ⁴ 、1×10 ⁵ 、2.5×10 ⁵ 、5×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、2.5×10 ⁷ 、5×10 ⁷ And 1X 10 ⁸ Between individual cells/kg, or at 1X 10 as the lower end of the range ⁴ Individual cells/kg to 2.5X10 as the upper end of the range ⁴ 、5×10 ⁴ 、1×10 ⁵ 、2.5×10 ⁵ 、5×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、2.5×10 ⁷ 、5×10 ⁷ And 1X 10 ⁸ Between individual cells/kg. In some embodiments, the number of lymphocytes present in the cell preparations herein and optionally reinfused or intratumorally, intramuscularly, subcutaneously or otherwise delivered into a subject, and in illustrative embodiments the number of T cells and/or NK cells can be 5 x 10 as the low end of the range ⁵ 、1×10 ⁶ 、2.5×10 ⁶ 、5×10 ⁶ 、1×10 ⁷ 、2.5×10 ⁷ 、5×10 ⁷ And 1X 10 ⁸ To 2.5X10 as the upper end of the range ⁶ 、5×10 ⁶ 、1×10 ⁷ 、2.5×10 ⁷ 、5×10 ⁷ 、1×10 ⁸ 、2.5×10 ⁸ 、5×10 ⁸ And 1X 10 ⁹ Between individual cells. In some embodiments, the number of lymphocytes present in the cell preparations herein and useful for infusion, reinfusion, or other delivery means (e.g., subcutaneous delivery) into a 70kg subject or patient, and in illustrative embodiments the number of T cells and/or NK cells is 7 x 10 ⁵ Up to 2.5X10 ⁸ Individual cells. In other embodiments, the number of lymphocytes present in the cell preparations herein and available for transduction, and in illustrative embodimentsThe number of T cells and/or NK cells in a medium is about 7X 10 ⁶ Plus or minus 10%.

In any of the embodiments and aspects provided herein that include T cells, NK cells, B cells, or stem cells, the cells may be autologous cells or allogeneic cells. In some embodiments, the allogeneic cells may be genetically engineered allogeneic cells. Allogeneic cells, such as allogeneic T cells, and methods for genetically engineering allogeneic cells are known in the art. In some embodiments, wherein the allogeneic cells are T cells, the T cells have been genetically engineered such that at least one component of the TCR complex is functionally impaired and/or at least partially deleted. In some embodiments, the T cells have been genetically engineered such that expression of at least one component of the TCR complex has been reduced or eliminated. In some embodiments, the allogeneic cells may be modified such that they lack all or part of the B2 microglobulin gene. In some embodiments, the allogeneic cells may include any lymphoproliferative element and/or CLE disclosed herein. The use of lymphoproliferative elements and CLE can reduce the number of cells desired and can facilitate cell manufacturing of T cells, NK cells, B cells or stem cells. In some embodiments, the allogeneic cells may be immortalized cells. In any aspect or embodiment herein that includes allogeneic cells, steps that include collecting blood or contacting the cells with replication defective recombinant retroviral particles may be eliminated. For example, to treat a subject with allogeneic CAR-T cells, the T cells may have been previously genetically modified, and the genetically modified allogeneic CAR-T cells are administered to the subject without blood collection from the subject. In some embodiments, the allogeneic cells are administered subcutaneously. In some embodiments, the allogeneic cells are administered intravenously. In some embodiments, the allogeneic cells are administered intraperitoneally.

In some embodiments of any of the methods for modifying lymphocytes (e.g., T cells and/or NK cells) provided herein and aspects related to the use of replication-defective recombinant retroviral particles (RIP) to make a kit for modifying T cells and/or NK cells in a subject, RIP in the modified, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or a population thereof or a cell-free composition provided herein (e.g., GMP RIP composition) is introduced or reintroduced into the subject. The modified and in the illustrative embodiments genetically modified lymphocytes may be introduced or reintroduced into the subject by any means known in the art. For example, the introducing or reintroducing may be by infusion into a blood vessel of the subject. Intratumoral, intraperitoneal, intramuscular, and in certain illustrative embodiments, subcutaneous. In some embodiments, the modified, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or a population thereof undergo 4 or less cell divisions ex vivo prior to being introduced or reintroduced into a subject. In some embodiments, the lymphocytes used in such methods are resting T cells and/or resting NK cells, which are contacted with the replication defective recombinant retroviral particle for 1 hour to 12 hours. In some embodiments, the time at which blood is collected from the subject is no more than 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour from the time at which the modified and/or genetically modified T cells and/or NK cells are formulated for delivery and/or reintroduction into the subject. In some embodiments, all steps after collection and before reintroduction of the blood are performed in a closed system, which is monitored manually throughout the treatment.

In some embodiments of the methods and compositions disclosed herein, T cells and/or NK cells modified, and in illustrative embodiments genetically modified, are introduced back, reintroduced or reinfused or otherwise delivered into a subject without further ex vivo manipulation, such as stimulation and/or activation of T cells and/or NK cells. In prior art methods, ex vivo manipulation is used to stimulate/activate T cells and/or NK cells and to expand the genetically modified T cells and/or NK cells prior to introducing the genetically modified T cells and/or NK cells into a subject. In prior art methods, this typically takes days or weeks, and requires the subject to return to the clinic for days or weeks of blood infusion after initial blood draw. In some embodiments of the methods and compositions disclosed herein, T cells and/or NK cells are not stimulated ex vivo by exposure to anti-CD 3 alone or in combination with co-stimulation by, for example, anti-CD 28, in solution or attached to a solid support (e.g., anti-CD 3/anti-CD 28 coated beads) prior to contacting the T cells and/or NK cells with the replication-defective recombinant retroviral particle. Thus, an ex vivo propagation-free method is provided herein. In other embodiments, T cells and/or NK cells modified and in the illustrative embodiments genetically modified are not expanded ex vivo, or only a small number of cell divisions (e.g., 1, 2, 3, 4, or 5 rounds of cell division), but instead are expanded in vivo (i.e., within a subject) or expanded primarily in vivo. In some embodiments, no additional medium is added to allow further expansion of the cells. In some embodiments, cell production of Primary Blood Lymphocytes (PBLs) does not occur when PBLs are contacted with replication defective recombinant retroviral particles. In illustrative embodiments, cell production of PBLs does not occur when PBLs are ex vivo. In traditional methods of adoptive cell therapy, a subject experiences lymphocyte depletion prior to reinfusion of genetically modified T cells and/or NK cells. In some embodiments, the patient or subject does not experience lymphocyte depletion prior to infusion or reinfusion of the modified and or genetically modified T cells and/or NK cells. However, embodiments of the methods and compositions disclosed herein can also be used with T cells and/or NK cells that are pre-activated or pre-stimulated. In some embodiments, T cells and/or NK cells can be stimulated ex vivo by exposure to anti-CD 3 with or without an anti-CD 28 solid support prior to contacting the T cells and/or NK cells with the replication-defective recombinant retroviral particle. In some embodiments, T cells and/or NK cells may be exposed to the anti-CD 3/anti-CD 28 solid support for less than 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, or 24 hours, including no exposure, prior to contacting the T cells and/or NK cells with the replication-defective recombinant retroviral particle. In illustrative embodiments, T cells and/or NK cells may be exposed to the anti-CD 3/anti-CD 28 solid support for less than 1, 2, 3, 4, 6, or 8 hours prior to contacting the T cells and/or NK cells with the replication-defective recombinant retroviral particle.

Enrichment of T cells and/or NK cells by positive selection

In some embodiments, any cell in the cell mixture, cell preparation, or reaction mixture that is useful for adoptive cell therapy, referred to herein as a desired cell, such as one or more cell populations of T cells and/or NK cells, may be enriched prior to formulation for delivery. In some embodiments, the desired cells may be enriched by positive selection prior to contact with a recombinant nucleic acid vector, such as a replication-defective retroviral particle. In other embodiments, the desired cells may be enriched by positive selection after the cell mixture, cell preparation, or reaction mixture is contacted with a recombinant nucleic acid vector, such as a replication-defective retroviral particle. In some embodiments, enriching one or more cell populations may be performed concurrently with any of the methods of genetic modification disclosed herein, and in illustrative embodiments, genetic modification with replication defective retroviral particles.

Monocytes (e.g., PBMCs) or TNCs may be separated from more complex cell mixtures, such as whole blood, by density gradient centrifugation or reverse perfusion of a leukoreduction filter assembly, respectively, as described in more detail herein. In some embodiments, the desired cells may have a particular cell lineage, e.g., NK cells, T cells, and/or T cell subsets, including naive

Effector, memory, suppressor T cells, and/or regulatory T cells, and may be enriched by selecting cells expressing one or more surface molecules. In illustrative embodiments, the one or more surface molecules may include CD4, CD8, CD16, CD25, CD27, CD28, CD44, CD45RA, CD45RO, CD56, CD62L, CCR7, KIR, foxP3, and/or TCR components such as CD3. Methods using beads conjugated with antibodies to one or more surface molecules can be used to enrich for desired fines using magnetic, density, and size-based separationsAnd (5) cells.

Engineered signaling polypeptides

In some embodiments, replication-defective recombinant retroviral particles for contacting T cells and/or NK cells have a polynucleotide or nucleic acid with one or more transcriptional units encoding one or more engineered signaling polypeptides. In some embodiments, the engineered signaling polypeptide includes any combination of an extracellular domain (e.g., an antigen specific targeting region or astm), a stem, and a transmembrane domain, in combination with one or more intracellular activation domains, optionally one or more regulatory domains (e.g., co-stimulatory domains), and optionally one or more T cell survival motifs. In an illustrative embodiment, at least one, two, or all of the engineered signaling polypeptides are Chimeric Antigen Receptors (CARs) or Lymphoproliferative Elements (LEs), such as Chimeric Lymphoproliferative Elements (CLE). In some embodiments, at least one, two, or all of the engineered signaling polypeptides are engineered T Cell Receptors (TCRs). In some embodiments, when two signaling polypeptides are utilized, one encodes a lymphoproliferative element and the other encodes a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. With respect to any domain of the engineered signaling polypeptides disclosed herein, exemplary sequences can be found in WO 2019/055946, which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that such engineered polypeptides may also be referred to as recombinant polypeptides. The engineered signaling polypeptides provided herein (e.g., CARs, engineered TCRs, LEs, and CLE) are generally transgenic with respect to lymphocytes, particularly T cells and NK cells, and most particularly T cells and/or NK cells, engineered with the methods and compositions provided herein to express such signaling polypeptides.

Extracellular domain

In some embodiments, the engineered signaling polypeptide includes an extracellular domain that is a member of a specific binding pair. For example, in some embodiments, the extracellular domain may be an extracellular domain of a cytokine receptor or a mutant thereof or a hormone receptor or a mutant thereof. Such mutant extracellular domains are reported in some embodiments to be constitutively active when expressed in at least some cell types. In illustrative embodiments, such extracellular domains and transmembrane domains do not include a ligand binding region. It is believed that such domains do not bind to ligands when present in the engineered signaling polypeptide and expressed in B cells, T cells, and/or NK cells. Mutations in such receptor mutants may occur in the extracellular juxtamembrane region. Without being bound by theory, mutations in at least some of the extracellular domains (and some of the extracellular-transmembrane domains) of the engineered signaling polypeptides provided herein are responsible for signaling of the engineered signaling polypeptides in the absence of the ligand by bringing together the activation chains that are not normally together. Additional examples of extracellular domains comprising mutations in the extracellular domain can be found, for example, in the lymphoproliferative element section herein.

In certain illustrative embodiments, the extracellular domain comprises a dimerization motif. In an illustrative embodiment, the dimerization motif comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. Other examples of extracellular domains comprising dimerization motifs can be found, for example, in the lymphoproliferative element section herein.

In certain embodiments, the extracellular domain is an Antigen Specific Targeting Region (ASTR), sometimes referred to herein as an antigen binding domain. Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; ligand-receptor binding pairs; etc. Thus, members of specific binding pairs suitable for use in the engineered signaling polypeptides of the present disclosure include astm, which is an antibody, antigen, ligand, receptor binding domain of ligand, receptor, ligand binding domain of receptor, and alternative non-antibody scaffolds, also referred to herein as antibody mimics. In any aspect or embodiment provided herein that includes an ASTR, the ASTR may be a suitable antibody mimetic. In some embodiments, the antibody mimetic may beIs an affibody, affimer, affibody, alpha body, alphamab, anti-carrier protein, peptide aptamer, armadillo repeat protein, trimer, affimer (also known as avid multimer), C-lectin domain, cysteine knot microgrotein, cyclic peptide, cytotoxic T lymphocyte-associated protein-4, DARPin (designed ankyrin repeat protein), fibrinogen domain, fibronectin binding domain (FN 3 domain) (e.g., an attachment protein or monoclonal antibody), fynomer, kink bacteria, kunitz domain peptide, nanofitin, leucine-rich repeat domain, lipocalin domain, mAb 2 or Fcab ^TM Nanobodies, nanomembers, OBody, pronectin, single chain TCRs, triangular tetrapeptide repeat domains, VHH or V-like domains. In any aspect or embodiment provided herein that includes an astm (e.g., scFv) as an antibody, a suitable antibody mimetic can be used in place of the antibody.

An ASTR suitable for use in the engineered signaling polypeptides of the present disclosure may be any antigen binding polypeptide. In certain embodiments, the astm is an antibody, such as a full length antibody, a single chain antibody, a Fab fragment, a Fab 'fragment, (Fab') 2 fragment, an Fv fragment, and a bivalent single chain antibody or a bifunctional antibody.

In some embodiments, the ASTR is a single chain Fv (scFv). In some embodiments, the heavy chain is located N-terminal to the light chain in the engineered signaling polypeptide. In other embodiments, the light chain is located N-terminal to the heavy chain in the engineered signaling polypeptide. In any of the disclosed embodiments, the heavy and light chains can be separated by a linker, as discussed in more detail herein. In any of the disclosed embodiments, the heavy or light chain can be N-terminal to the engineered signaling polypeptide and typically is C-terminal to another domain (e.g., a signal sequence or signal peptide).

Other antibody-based recognition domains (cAb VHH (camelid antibody variable domain) and humanized versions, igNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domain) are suitable for use with engineered signaling polypeptides and in methods of using the engineered signaling polypeptides of the present disclosure. In some cases, the T Cell Receptor (TCR) recognition domain is based.

Naturally occurring T cell receptors include the alpha and beta subunits, which are produced by unique recombination events in the genome of T cells, respectively. Libraries of TCRs can be screened for selectivity for a target antigen (e.g., any of the antigens disclosed herein). Screening for native and/or engineered TCRs may identify TCRs that have high affinity and/or reactivity for the target antigen. Such TCRs may be selected, cloned, and polynucleotides encoding such TCRs may be included in replication defective recombinant retroviral particles to genetically modify lymphocytes, or in an illustrative embodiment, T cells or NK cells, such that the lymphocytes express the engineered TCRs. In some embodiments, the TCR may be a single chain TCR (scTv, single chain double domain TCR comprising vαvβ).

Certain embodiments of any aspect or embodiment herein that includes a CAR include a CAR having an extracellular domain engineered to co-select for an endogenous TCR signaling complex and a CD3Z signaling pathway. In one embodiment, the chimeric antigen receptor astm is fused to an endogenous TCR complex chain (e.g., TCR α, CD3E, etc.) to facilitate incorporation into the TCR complex and signaling through the endogenous CD3Z chain. In other embodiments, the CAR contains a first scFv or protein that binds to a TCR complex and a second scFv or protein that binds to an antigen of interest (e.g., a tumor antigen). In another embodiment, the TCR may be a single chain TCR (scTv, single chain double domain TCR containing vαvβ). Finally, scFv can also be generated to recognize specific MHC/peptide complexes, thereby acting as an alternative TCR. Such peptide/MHC scFv conjugates can be used in a number of configurations similar to CARs.

In some embodiments, the ASTR may be multispecific, e.g., bispecific antibodies. Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one antigen of interest and the other is for another antigen of interest. In certain embodiments, the bispecific antibody can bind to two different epitopes of the antigen of interest. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing an antigen of interest. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. In some embodiments, any of the CARs of the disclosure can be multispecific. In some embodiments, one of the ASTRs on a multispecific, e.g., bispecific, may be an anti-idiotype extracellular recognition domain as disclosed elsewhere herein.

ASTRs suitable for use in the engineered signaling polypeptides or engineered TCRs of the present disclosure may have a variety of antigen binding specificities. In some cases, the antigen binding domain is specific for an epitope present in an antigen expressed by (synthesized by) a target cell. In one example, the target cell is a cancer cell-associated antigen. The antigen associated with the cancer cell may be an antigen associated with: for example, breast cancer cells, B-cell lymphoma cells, such as diffuse large B-cell lymphoma (DLBCL) cells, hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), lymphoma cells, non-hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, leukemia cells, chronic Myelogenous Leukemia (CML) cells, chronic Lymphocytic Leukemia (CLL) cells, acute Myelogenous Leukemia (AML) cells, acute Lymphoblastic Leukemia (ALL) cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colorectal cancer cells, and the like. Cancer cell-associated antigens may also be expressed by non-cancer cells. In some embodiments, the cancer cell is a PDL-1 positive cancer cell. In an illustrative embodiment, the cancer cell is a PDL-1 positive DLBCL cell. In some embodiments, the cancer cell is a PDL-1 negative cell. In an illustrative embodiment, the cancer cell is a PDL-1 negative DLBCL cell.

In any aspect or embodiment herein including an astm or recombinant TCR, the antigen can be a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the tumor-associated antigen or tumor-specific antigen is Axl, ROR1, ROR2, her2 (ERBB 2), prostate Stem Cell Antigen (PSCA), PSMA (prostate specific membrane antigen), B Cell Maturation Antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calomel, chromogranin, protein melanin-a (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, myo-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), MUC-1, epithelial membrane protein (EMA), epithelial Tumor Antigen (ETA), tyrosinase, melanomA-Associated antigen (MAGE), MAGE-Al, high molecular weight-melanomA-Associated antigen (HMW-MAA), placental alkaline phosphatase, synaptotagmin, thyroglobulin, thyroid transcription factor-1, dimeric form M2 of the pyruvate kinase isozymes (tumor M2-PK), CD19, CD20, CD22, CD23, CD24, CD27, CD30, CD33, CD34, CD37, CD38, CD40, CD44v6, CD44v7/8, CD45, CD70, CD99, CD117, CD123, CD138, CD171, GD2 (ganglioside G2), ephA2, CSPG4, FAP (fibroblast activation protein), kappa, lambda, 5T4, αvβ6 integrin, integrin αvβ3 (CD 61), galactose lectin, alpha vβ3 (CD 61), K-Ras (V-Ki-Ras 2 Kirsten rat sarcoma viral oncogene), ral-B, B7-H3, B7-H6, CAIX, EGFR, EGP2, EGP40, epCAM, fetal AchR, FR alpha, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, HLA-DR, IL-11R alpha, IL-13R alpha 2, lewis-Y, muc16, NCAM, NKG2D ligand, PRAME, survivin, TAG72, TEMs, FR2, EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp 17), mesothelin, PAP (prostaacid phosphatase), prostaglandin, TARP (T cell receptor gamma alternate reading frame protein), trp-p8, STEP 1 (six transmembrane epithelial antigen of prostate 1), abnormal Ras protein, abnormal p53 protein, squamous cell carcinoma antigen of New Youzhou esophagus (NYESO 1), PDL-1, etc.

In any aspect or embodiment herein including an astm or recombinant TCR, as disclosed elsewhere herein, the astm or recombinant TCR can recognize, bind to, or otherwise interact with the idiotype of the target antibody or antibody mimetic. In such embodiments, the anti-idiotype polypeptide is a CAR or TCR, wherein astm is the anti-idiotype external recognition domain of the anti-idiotype polypeptide. In some embodiments, an ASTR can be any extracellular recognition domain of an anti-idiotype polypeptide disclosed herein. For example, the number of the cells to be processed, ASTR can recognize idiotcetuximab, molozumab-CD 3, efalizumab, tositumomab-i 131, nebulomab, ibritumomab, cetuximab, daclizumab, olaparimab, aciumab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, timobenzab, omalizumab, bevacizumab natalizumab, panitumumab, ranibizumab, eculizumab, cetuximab, you-tertraumab, kanamab, golimumab, ofatuzumab, tobulumab, denomumab, belimumab, ipilimumab, bentuximab, pertuzumab, enmettrastuzumab, risperiduzumab, oxuzumab, cetuximab, ramucirumab, vedolizumab Nafiizumab, pembrolizumab, bonauzumab, alemtuzumab, avokumumab, idazomib, valitumumab, dituzumab, secuzumab, mevallizumab, alikumumab, ai Luozhu, exenatide, rayleimumab, bei Luotuo Shu Shan, atrazumab, ottomab, brodamuzumab, dulciton Li Youshan, oxtuzumab, coumarone, sha Lilu, abamzumab, eimerisemab, orebanzumab, benralizumab, de watt Lu Shankang, getuzumab, eremizumab-aoue, galbanzumab-lm, broso You Shan (burosum-tbza), lamab (fluambizumab), gammagumab-35-67, gammagumab-35 kumamab, the pharmaceutical composition comprises (a) rebamipramizumab (freemanizumab-vfrm), eculizumab (ravulizumab-cvvz), cimetidine Li Shan antibody (cemiplimab-rwlc), ibamzumab (ibalizumab-uiyk), epratuzumab (emapalumab-lzsg), panitumumab (moxetumomab pasudotox-tdfk), karaxizumab (capplacizumab-yhdp), risatuzumab (risenkezumab-rzaa), vitamin-poisuotuzumab (polatuzumab vedotin-piiq), luo Moshan antibody (romisozumab-aqqg), blosaizumab (brolizumab-dbilb), fuuzumab (crizalizumab-tmca), enrouzumab (enfortumab vedotin-ejfv), dezamizumab (38-49-nxsimuzumab) tetuzumab (tetuzumab-trbw), ai Punai bead mab (eptimezumab-jjmr), ai Satuo bead mab (isatuximab-irfc), facituzumab govitecan (sacituzumab govitecan-hziy), infliximab (inebilizumab-cdon), tavalvulizumab (tafasitamab-cxix), bei Lan Tatuzumab Mo Futing (belantamab mafodotin-blmf), saririzumab (satralizumab-mwge), atemiweimab, aleweimab-ebgn, naxitamab-gqgk, margetuximab-cmkb, asuwimb-zykl, ivermectin, multi-tarolimab (dostarlimab-gxχ), telbizumab (loncastuximab tesirine-lpyl), eltuximab-vjw), adantiumab (amammaab-avuju), atavisuab-av-ju), alemtuzumab-gqgk, margetuximab-umbum Qu Luolu mab, anistuzumab-fnia, moxidomab, statin-tetrituximab, bimetalizumab, naloxone Li Shan, terstuzumab, melezit Li Shan, enomomab, butirimumab, rituximab, tereprunomumab Li Shan, obutyzumab, pi An Puli mab, tanitumumab, farnesimab, su Timo mab, terstuzumab, and refafar Li Shan.

In some embodiments, an astm suitable for use in an engineered signaling polypeptide in which the member of the specific binding pair is a ligand for the receptor. Ligands include (but are not limited to): hormones (e.g., erythropoietin, growth hormone, leptin, etc.); cytokines (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., regulatory proteins; vascular Endothelial Growth Factor (VEGF), etc.); integrin binding peptides (e.g., peptides comprising the sequence Arg-Gly-Asp (SEQ ID NO: 1)), and the like.

When a member of a specific binding pair in the engineered signaling polypeptide is a ligand, the engineered signaling polypeptide can be activated in the presence of a second member of the specific binding pair, wherein the second member of the specific binding pair is a receptor for the ligand. For example, when the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor including a soluble VEGF receptor.

As described above, in some cases, the member of the specific binding pair included in the engineered signaling polypeptide is an ASTR, which is a receptor, e.g., a receptor for a ligand, a co-receptor, etc. The receptor may be a ligand binding fragment of the receptor. Suitable receptors include (but are not limited to): growth factor receptors (e.g., VEGF receptor); killer lectin-like receptor subfamily K; member 1 (NKG 2D) polypeptide (receptor for MICA, MICB, ULB 6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor, etc.); CD27; natural Cytotoxic Receptors (NCR) (e.g., NKP30 (NCR 3/CD 337) polypeptides (HLA-B associated transcript 3 (BAT 3) and B7-H6) receptors, etc.), and the like.

In certain embodiments including any aspect provided herein of astm, the astm can be located at an intermediate protein that links the astm with a target molecule expressed on the target cell. The intermediate protein may be expressed endogenously or introduced exogenously, and may be naturally, engineered or chemically modified. In certain embodiments, the ASTR may be an anti-tagged ASTR such that at least one tagged intermediate (typically an anti-tag conjugate) is included between the tag recognized by the ASTR and the target molecule (typically a protein target expressed on the target cell). Thus, in such embodiments, the ASTR binds to the label and the label binds to an antibody directed against an antigen on a target cell (e.g., a cancer cell). Non-limiting examples of labels include Fluorescein Isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, polymethylchlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein. Thus, astm comprises a molecule that binds to the label.

Handle

In some embodiments, the engineered signaling polypeptide comprises a handle in a portion of the engineered signaling polypeptide that is extracellular and interposed between the ASTR and the transmembrane domain. In some embodiments, the handle has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type CD8 handle region (TTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 2)), at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type CD28 handle region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3)), or at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type immunoglobulin heavy chain handle region. In the engineered signaling polypeptide, the handle used allows the antigen-specific targeting region, and typically the entire engineered signaling polypeptide, to remain bound to the antigen of interest in an increased amount.

The handle region may be 4 to 250 amino acids, 10 to 250 amino acids, 4 to 200 amino acids, 4 to 100 amino acids, or 4 to 75 amino acids in length. In some embodiments, the stem region length may be, for example, from 4aa to about 10aa, from about 10aa to about 15aa, from about 15aa to about 20aa, from about 20aa to about 25aa, from about 25aa to about 30aa, from about 30aa to about 40aa, or from about 40aa to about 50aa. In some embodiments, the handle separates the anti-id ERD from the cell membrane to which it is attached, far enough to allow the anti-id ERD to bind to its target antibody upon contact. In certain embodiments, the handle is a means to effectively isolate the anti-id ERD from the cell membrane to which it is attached, allowing the anti-id ERD and its target antibody to bind when in contact with each other.

In some embodiments, the handle of the engineered signaling polypeptide comprises at least one cysteine. For example, in some embodiments, the handle may include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 4). If present, a cysteine in the stem of the first engineered signaling polypeptide may be capable of forming a disulfide bond with the stem in the second engineered signaling polypeptide.

The handle may comprise an immunoglobulin hinge region amino acid sequence known in the art; see, e.g., tan et al (1990) Proc. Natl. Acad. Sci. USA, 87:162; and Huck et al (1986) nucleic acid research (nucleic acids Res.) 14:1779. As non-limiting examples, an immunoglobulin hinge region may comprise a domain having at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO: 5); CPPC (SEQ ID NO: 4); CPEPKSCDTPPPCPR (SEQ ID NO: 6) (see, e.g., glaser et al (2005), "J.Biol.chem.)," 280:41494); ELKTPLGDTTHT (SEQ ID NO: 7); KSCDKTHTCP (SEQ ID NO: 8); KCCVDCP (SEQ ID NO: 9); KYGGPPCP (SEQ ID NO: 10); EPKSCDKTHTCPPCP (SEQ ID NO: 11) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 12) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 13) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 14) (human IgG4 hinge), etc. The handle may include a hinge region having the amino acid sequence of a human IgG1, igG2, igG3 or IgG4 hinge region. The handle may comprise one or more amino acid substitutions and/or insertions and/or deletions compared to the wild-type (naturally occurring) hinge region. For example, his229 of a human IgG1 hinge may be substituted with Tyr such that the handle includes the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 15) (see, e.g., yan et al (2012) J.Biol.chem.) (287:5891). The handle may comprise an amino acid sequence derived from human CD 8; for example, the handle may comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16), or a variant thereof.

Transmembrane domain

The engineered signaling polypeptides of the present disclosure may include a transmembrane domain for insertion into a eukaryotic cell membrane. The transmembrane domain may be interposed between the ASTR and the costimulatory domain. The transmembrane domain may be interposed between the handle and the costimulatory domain such that the chimeric antigen receptor comprises, in order from the amino terminus (N-terminus) to the carboxy terminus (C-terminus: ASTR, handle, transmembrane domain and activation domain.

Any Transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use in the aspects and embodiments disclosed herein. In some embodiments, a TM domain provided herein that includes any aspect of the CAR can include a transmembrane domain from: IL3RA, IL 4A, CD 5RA, IL 6A, CD 6ST, IL7RA, IL7RAins PPCL, IL 9A, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD RA, IL31RA, ITGA1, ITGA4, IL20RA, IL20RB IL3RA, IL 4A, CD 5RA, IL 6A, CD 6ST, IL7RA, IL7RAIns PPCL, IL 9A, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD27 RA, IL31RA, ITGA1, ITGA4, IL13RA1, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD RA ITGA6, A, CD1, ITGB2, ITGB7, kirs 2, LEPR, LFA-1 (CD 11a, CD 18), A, CD (KLRF 1), OSMR, PAG/A, CD1, SLAM (SLAMF 1, CD150, IPO-3), SLAMF4 (CD 244, 2B 4), SLAMF6 (NTB-A, CD 108), SLAMF7, SLAMF8 (BLAME), TNFR2, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, TNFRSF18, VLA1 or VLA-6, or a functional mutant and/or fragment thereof.

Non-limiting examples of TM domains suitable for use in any aspect or embodiment provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a segment of at least 10, 15, 20, or all amino acids of the following TM domains or any of the combined handle and TM domains: a) CD 8. Alpha. TM (SEQ ID NO: 17); b) CD 8. Beta. TM (SEQ ID NO: 18); c) CD4 handle (SEQ ID NO: 19); d) CD3Z TM (SEQ ID NO: 20); e) CD28 TM (SEQ ID NO: 21); f) CD134 (OX 40) TM (SEQ ID NO: 22); g) CD7 TM (SEQ ID NO: 23); h) CD8 handle and TM (SEQ ID NO: 24); and i) the CD28 handle and TM (SEQ ID NO: 25).

As non-limiting examples, the transmembrane domain of aspects of the invention may have at least 80%, 90% or 95% sequence identity to the transmembrane domain of SEQ ID NO:17 or may have 100% sequence identity to any one of the transmembrane domains from the following genes: a CD8 beta transmembrane domain, a CD4 transmembrane domain, a cd3ζ transmembrane domain, a CD28 transmembrane domain, a CD134 transmembrane domain, or a CD7 transmembrane domain.

Intracellular activation domains

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present disclosure generally induce the production of one or more cytokines upon activation; increase cell death; and/or increase CD8 ⁺ T cells, CD4 ⁺ Proliferation of T cells, NKT cells, γδ T cells and/or neutrophils. The activation domain may also be referred to herein as an activation domain. The activation domain can be used in a CAR or in a lymphoproliferative element provided herein.

In some embodiments, the intracellular activation domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular activation domain of one aspect of the invention may have at least 80%, 90% or 95% or 100% sequence identity to the CD3Z, CD D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, ZAP70, NKp30 (B7-H6), NKG2D, NKp44, NKp46, fcry (FCER 1G), fcrβ (FCER 1B), fcγri, fcγriia, fcγriic, fcγriiia, and FcRL5 domains as described below.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present disclosure include intracellular signaling polypeptides comprising an immunoreceptor tyrosine-based activation motif (ITAM). ITAM motif is YX ₁ X ₂ L/I, wherein X ₁ X is X ₂ Independently any amino acid. In some embodiments, the intracellular activation domain of the engineered signaling polypeptide comprises 1, 2, 3, 4, or 5 ITAM motifs. In some embodiments, the ITAM motif is repeated twice in the intracellular activation domain, wherein the first and second instances of the ITAM motif are repeated from 6 to 8 amino acids from each other (e.g., (YX) ₁ X ₂ L/I)(X ₃ ) _n (YX ₁ X ₂ L/I), wherein n is an integer of 6 to 8, and 6 to 8X ₃ Each of which may be any amino groupAcid) is separated. In some embodiments, the intracellular activation domain of the engineered signaling polypeptide comprises 3 ITAM motifs.

Suitable intracellular activation domains may be those portions containing an ITAM motif derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular activation domain may be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular activation domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include (but are not limited to): CD3Z (CD 3 ζ); CD3D (CD 3 delta); CD3E (CD 3 epsilon); CD3G (CD 3 γ); CD79A (antigen receptor complex associated protein alpha chain); CD79B (antigen receptor complex-associated protein beta chain) DAP12; FCERlG (fcepsilon receptor iγ chain).

In some embodiments, the intracellular activation domain may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in the following ITAM motif-containing polypeptides, or to an contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 160aa of any of the following ITAM motif-containing polypeptides: CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD 3-zeta, CD3H, CD3Q, T3Z, TCRZ, etc.) having the exemplary sequence MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL [ YNELNLGRREEYDVL ] DKRRGRDPEMGGKPRRKNPQEGL [ YNELQKDKMAEAYSEI ] GMKGERRRGKGHDGL [ YQGLSTATKDTYDAL ] HMQALPPR (SEQ ID NO: 26), MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL [ YNELNLGRREEYDVL ] DKRRGRDPEMGGKPQRRKNPQEGL [ YNELQKDKMAEAYSEI ] GMKGERRRGKGHDGL [ YQGLSTATKDTYDAL ] HMQALPPR (SEQ ID NO: 27); RVKFSRSADAPAYQQGQNQL [ YNELNLGRREEYDVL ] DKRRGRDPEMGGKPRRKNPQEGL [ YNELQKDKMAEAYSEI ] GMKGERRRGKGHDGL [ YQGLSTATKDTYDAL ] HMQALPPR (SEQ ID NO: 28), RVKFSRSADAPAYQQGQNQL [ YNELNLGRREEYDVL ] DKRRGRDPEMGGKPQRRKNPQEGL [ YNELQKDKMAEAYSEI ] GMKGERRRGKGHDGL [ YQGLSTATKDTYDAL ] HMQALPPR (SEQ ID NO: 29), NQL [ YNELNLGRREEYDVL ] DKR (SEQ ID NO: 30); EGL [ YNELQKDKMAEAYSEI ] GMK (SEQ ID NO: 31) and DGL [ YQGLSTATKDTYDAL ] HMQ (SEQ ID NO: 32); t cell surface glycoprotein CD3 DELTA chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, DELTA subunit; CD3 DELTA; CD3D antigen, DELTA polypeptide (TiT 3 complex); OKT3, DELTA chain; T cell receptor T3 DELTA chain; T cell surface glycoprotein CD3 DELTA chain; etc.), having exemplary sequences: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV [ YQPLRDRDDAQYSHL ] GGNWARNK (SEQ ID NO: 33), MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV [ YQPLRDRDDAQYSHL ] GGNWARNK (SEQ ID NO: 34) and DQV [ YQPLRDRDDAQYSHL ] GGN (SEQ ID NO: 35); the CD3 epsilon chain of a T cell surface glycoprotein (also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3 epsilon, T3e, etc.), which has an exemplary sequence: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD [ YEPIRKGQRDLYSGL ] NQRRI (SEQ ID NO: 36) and NPD [ YEPIRKGQRDLYSGL ] NQR (SEQ ID NO: 37); the T cell surface glycoprotein cd3γ chain (also known as the CD3G, T cell receptor T3 γ chain, CD3- γ, T3G, γ polypeptide (TiT complex), etc.), which has an exemplary sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL [ YQPLKDREDDQYSHL ] QGNQLRRN (SEQ ID NO: 38) and DQL [ YQPLKDREDDQYSHL ] QGN (SEQ ID NO: 39); CD79A (also known as B cell antigen receptor complex associated protein alpha chain; CD79A antigen (immunoglobulin associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane bound immunoglobulin associated protein; surface IgM associated protein; etc.), which has exemplary sequences: MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL [ YEGLNLDDCSMYEDI ] SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 40), MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL [ YEGLNLDDCSMYEDI ] SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 41) and ENL [ YEGLNLDDCSMYEDI ] SRG (SEQ ID NO: 42); CD79B, having the exemplary sequence: LDKDDSKAGMEEDHT [ YEGLDIDQTATYEDI ] VTLRTGEVKWSVGEHPGQE (SEQ ID NO: 211); DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-related protein; TYRO protein tyrosine kinase binding protein; killer activating receptor-related protein; etc.), having exemplary sequences: MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP [ YQELQGQRSDVYSDL ] NTQRPYYK (SEQ ID NO: 43), MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP [ YQELQGQRSDVYSDL ] NTQ (SEQ ID NO: 44), MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP [ YQELQGQRSDVYSDL ] NTQRPYK (SEQ ID NO: 45), MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP [ YQELQGQRSDVYSDL ] NTQRPYK (SEQ ID NO: 46) and ESP [ YQELQGQRSDVYSDL ] NTQ (SEQ ID NO: 47); and FCERlG (also known as FCRG; fcepsilon receptor iγ chain; fcreceptor γ chain; fc-epsilon RI- γ; fcR γ; fceRI γ; high affinity immunoglobulin epsilon receptor subunit γ; immunoglobulin E receptor, high affinity, γ chain; etc.), having exemplary sequences: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV [ YTGLSTRNQETYETL ] KHEKPPQ (SEQ ID NO: 48) and DGV [ YTGLSTRNQETYETL ] KHE (SEQ ID NO: 49), wherein the ITAM motif is set forth in brackets.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present disclosure include the DAP10/CD28 type signaling chain. An example of a DAP10 signal conducting chain is the amino acid SEQ ID NO:50. In some embodiments, suitable intracellular activating domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO 50.

An example of a CD28 signal transduction chain is the amino acid sequence SEQ ID NO. 51. In some embodiments, suitable intracellular activating domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO. 51.

Intracellular activating domains suitable for use in the engineered signaling polypeptides of the present disclosure include ZAP70 polypeptides, e.g., suitable intracellular activating domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all amino acids in SEQ ID No. 52, or to a stretch of about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, or about 500 amino acids to about 619 amino acids of the following amino acid sequence.

Regulatory domain

The regulatory domain may alter the effect of an intracellular activation domain in the engineered signaling polypeptide, including enhancing or inhibiting downstream effects of the activation domain or altering the nature of the reaction. Regulatory domains suitable for use in the engineered signaling polypeptides of the present disclosure include co-stimulatory domains. The length of a regulatory domain suitable for inclusion in an engineered signaling polypeptide may be from about 30 amino acids to about 70 amino acids (aa), for example, the length of the regulatory domain may be from about 30aa to about 35aa, from about 35aa to about 40aa, from about 40aa to about 45aa, from about 45aa to about 50aa, from about 50aa to about 55aa, from about 55aa to about 60aa, from about 60aa to about 65aa, or from about 65aa to about 70aa. In other cases, the length of the regulatory domain may be about 70aa to about 100aa, about 100aa to about 200aa, or greater than 200aa.

The costimulatory domain generally enhances and/or alters the nature of the response of the activation domain. The costimulatory domains useful in the engineered signaling polypeptides of the present disclosure are typically receptor-derived polypeptides. In some embodiments, the costimulatory domain homodimerizes. The subject co-stimulatory domain may be intracellular of the transmembrane protein (i.e., the co-stimulatory domain may be derived from the transmembrane protein). In some embodiments, any of the CARs provided herein can include a co-stimulatory domain. In some embodiments, the co-stimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an intracellular domain of a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids or less: IL7RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1 IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL RA, IL31RA, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, LAT, LEPR, LFA-1 (CD 11a/CD 18), LIGHT, LIFR, LMP1, LTBR, MPL, MYD88 IL7RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23R, IL RA, IL31RA, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB, ITGB2, ITGB7, LAT, LEPR, LFA-1 (CD 11a/CD 18), LIGHT, LIFR, LMP1, LTBR, MPL, MYD88, IL18R1 NKG2C, NKP80 (KLRF 1), OSMR, OX40, PD-1, PRLR, PSGL1, PAG/Cbp, SLAM (SLAMF 1, CD150, IPO-3), SLAMF4 (C244, 2B 4), SLAMF6 (NTB-A, ly 108), SLAMF7, SLAMF8 (BLAME), SLP-76, TILR2, TILR4, TILR7, TILR9, TNFR2, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, TNFRSF18, TRANCE/RANKL, VLA1, or VLA-6, or a functional mutant and/or fragment thereof.

The length of a costimulatory domain suitable for inclusion in an engineered signaling polypeptide can be about 30 amino acids to about 70 amino acids (aa), for example, the costimulatory domain can be about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length. In other cases, the co-stimulatory domain may be about 70aa to about 100aa, about 100aa to about 200aa, or greater than 200aa in length.

In some embodiments of the present invention, in some embodiments, the co-stimulatory domain may include a domain having at least 50%, 60%, 70%, 80%, 95% identity to at least 10, 15, 20 or all amino acids of the intracellular portion or about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, or about 160aa to about 185aa depending on how long the intracellular portion of the protein) of at least 50%, 60%, 70%, 80%, 95%, 98%, 95%, or 100% identity to the domain. CD137 (also known as TNFRSF9; CD137, 4-1BB, CDwl37, ILA, etc.), such as SEQ ID NO:53, CD28 (also called Tp 44) such as SEQ ID NO:54, CD28 (IC. DELTA.) deleted for Lck binding such as SEQ ID NO:55, ICOS (also called AILIM, CD278 and CVIDl) such as SEQ ID NO:56, OX40 (also called TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGPlL) such as SEQ ID NO:57, CD27 (also called S152, T14, TNFRSF7 and Tp 55) such as SEQ ID NO:58, BTLA (also called BTLAl and CD 272), such as SEQ ID NO:59, CD30 (also called TNFRSF8, dlS E and Ki-1), such as SEQ ID NO:60, GIFRSF 18, tgL (also called TNFRSF18 RP5-902P8.2, AITR, CD357 and GITR-D), for example SEQ ID NO 61 or HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR and TR 2), for example SEQ ID NO 62.OX40 contains a p85 PI3K binding motif at residues 34-57 of each of SEQ ID NOS 296 (of Table 1) and a TRAF binding motif at residues 76-102. In some embodiments, the costimulatory domain may comprise the p85 PI3K binding motif of OX 40. In some embodiments, the costimulatory domain may comprise the TRAF binding motif of OX 40. Lysine corresponding to amino acids 17 and 41 of SEQ ID NO. 296 is a potential negative regulatory site that serves as part of the ubiquitin targeting motif. In some embodiments, one or both of these lysines in the co-stimulatory domain of OX40 is a mutant arginine or another amino acid.

Connector

In some embodiments, the engineered signaling polypeptide includes a linker between any two adjacent domains. For example, the linker can be between the transmembrane domain and the first stimulatory domain. As another example, an ASTR may be an antibody, and a linker may be between the heavy and light chains. As another example, the linker may be between the ASTR and the transmembrane and costimulatory domains. As another example, the linker may be between the costimulatory domain and the intracellular activation domain of the second polypeptide. As another example, the linker may be between the astm and the intracellular signaling domain.

The linker peptide may have any of a variety of amino acid sequences. Proteins may be linked by spacer peptides which are generally flexible, but other chemical bonds are not excluded. The linker may be a peptide between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be generated by coupling the proteins using synthetic oligonucleotides encoding the linkers. Peptide linkers with a degree of flexibility may be used. The linker peptide may have virtually any amino acid sequence, provided that a suitable linker will have a sequence that results in a generally flexible peptide. The use of small amino acids such as glycine and alanine is useful in the production of flexible peptides. The creation of such sequences is conventional to those skilled in the art.

Suitable linkers may be readily selected and may be any of a variety of suitable lengths, such as 1 amino acid (e.g., gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G) _n Glycine-serine polymers (including, for example (GS) _n 、(GSGGS) _n 、(GGS) _n 、(GGGS) _n And (GGGGS) _n Where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore can act as neutral chains between components. Glycine polymers are of particular interest because glycine has significantly more phi-psi space than even alanine and is less restricted than residues with longer side chains (see Scheraga, review of computational chemistry (rev. Computational chem.)) 11173-142 (1992). Exemplary flexible linkers include, but are not limited to, GGGGSGGGGS (SEQ ID NO: 674), GGGGSGGGGSGGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 372), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 675), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), GGSSRSS (SEQ ID NO: 673), GGGGSGGGSGGGGS (SEQ ID NO: 65), GGSG (SEQ ID NO: 66), GGSGG (SEQ ID NO: 67), GSGSGSG (SEQ ID NO: 68), GSGGG (SEQ ID NO: 69), GGGSG (SEQ ID NO: 70), GSSSG (SEQ ID NO: 71), and the like. Those skilled in the art will recognize that the design of a peptide that binds to any of the elements described above may include a linker that is wholly or partially flexible, such that the linker may include a flexible linker and one or more portions that impart less flexibility to the structure.

Combination of two or more kinds of materials

In some embodiments, the polynucleotide provided by the replication defective recombinant retroviral particle has one or more transcriptional units encoding some combination of one or more engineered signaling polypeptides. In some of the methods and compositions provided herein, after transcription of the T cells by replication defective recombinant retroviral particles, the T cells that are modified, and in the illustrative embodiments genetically modified, include a combination of one or more engineered signaling polypeptides. It will be understood that references to a first polypeptide, a second polypeptide, a third polypeptide, etc. are for convenience, and that elements on "first polypeptide" and those on "second polypeptide" means that the elements are on different polypeptides, referred to as first or second, for ease of reference and convenience only in the other elements or steps of a specific polypeptide in general.

In some embodiments, the first engineered signaling polypeptide comprises an extracellular antigen binding domain capable of binding an antigen, and an intracellular signaling domain. In other embodiments, the first engineered signaling polypeptide further comprises a T cell survival motif and/or a transmembrane domain. In some embodiments, the first engineered signaling polypeptide does not include a co-stimulatory domain, while in other embodiments, the first engineered signaling polypeptide does include a co-stimulatory domain.

In some embodiments, the second engineered signaling polypeptide comprises a lymphoproliferative gene product and optionally an extracellular antigen binding domain. In some embodiments, the second engineered signaling polypeptide further comprises one or more of the following: a T cell survival motif, an intracellular signaling domain, and one or more co-stimulatory domains. In other embodiments, when two engineered signaling polypeptides are used, at least one is a CAR.

In one embodiment, one or more engineered signaling polypeptides are expressed under the same transcript under a T cell specific promoter or a general promoter, wherein in the transcript the nucleic acids encoding the engineered signaling polypeptides are separated by nucleic acids encoding one or more Internal Ribosome Entry Sites (IREs) or one or more protease cleaving peptides.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein a first engineered signaling polypeptide comprises a first extracellular antigen binding domain capable of binding to a first antigen and a first intracellular signaling domain other than a co-stimulatory domain, and a second engineered signaling polypeptide comprises a second extracellular antigen binding domain capable of binding to VEGF and a second intracellular signaling domain, such as a signaling domain of a co-stimulatory molecule. In a certain embodiment, the first antigen is PSCA, PSMA or BCMA. In a certain embodiment, the first extracellular antigen-binding domain comprises an antibody or fragment thereof (e.g., scFv), such as an antibody or fragment thereof specific for PSCA, PSMA, or BCMA. In a certain embodiment, the second extracellular antigen-binding domain that binds VEGF is a receptor for VEGF, i.e., VEGFR. In certain embodiments, the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In one embodiment, the VEGFR is VEGFR2.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, and the second engineered signaling polypeptide comprises an antigen binding domain (wherein the antigen is an angiogenic or angiogenic factor), and one or more costimulatory molecule signaling domains. The angiogenic factor may be, for example, VEGF. The one or more costimulatory molecule signaling motifs can comprise, for example, costimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1 BB.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, the second polypeptide comprises an antigen binding domain capable of binding to an antigen binding domain of VEGF, and a costimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1 BB. In another embodiment, the first signaling polypeptide or the second signaling polypeptide further has a T cell survival motif. In some embodiments, the T cell survival motif is or is derived from an intracellular signaling domain of the IL-7 receptor (IL-7R), an intracellular signaling domain of the IL-12 receptor, an intracellular signaling domain of the IL-15 receptor, an intracellular signaling domain of the IL-21 receptor, or an intracellular signaling domain of a transforming growth factor beta (TGF-beta) receptor or a TGF-beta decoy receptor (TGF-beta-dominant-negative receptor II (DNRII)).

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, and the second engineered signaling polypeptide comprises an antigen binding domain capable of binding VEGF, an IL-7 receptor intracellular T cell survival motif, and a costimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1 BB.

In some embodiments, more than two signaling polypeptides are encoded by the polynucleotide. In certain embodiments, only one of the engineered signaling polypeptides comprises an antigen binding domain that binds to a tumor-associated antigen or a tumor-specific antigen; each of the remaining ones of the engineered signaling polypeptides comprises an antigen binding domain that binds to a non-tumor associated antigen or a non-tumor specific antigen. In other embodiments, two or more of the engineered signaling polypeptides comprise an antigen binding domain that binds to one or more tumor-associated antigens or tumor-specific antigens, wherein at least one of the engineered signaling polypeptides comprises an antigen binding domain that does not bind to a tumor-associated antigen or tumor-specific antigen.

In any aspect or embodiment herein including ASTR, the antigen may be a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the tumor-associated antigen or tumor-specific antigen is Axl, ROR1, ROR2, her2 (ERBB 2), prostate Stem Cell Antigen (PSCA), PSMA (prostate specific membrane antigen), B Cell Maturation Antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calomel, chromogranin, protein melanin-a (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, myo-specific actin (MSA), neurofilament, neuronal Specific Enolase (NSE), MUC-1, epithelial membrane protein (EMA), epithelial Tumor Antigen (ETA), tyrosinase, melanomA-Associated antigen (MAGE), MAGE-Al, high molecular weight-melanomA-Associated antigen (HMW-MAA), placental alkaline phosphatase, synaptotagmin, thyroglobulin, thyroid transcription factor-1, dimeric form M2 of pyruvate kinase isozymes (tumor M2-PK), CD19, CD20, CD22, CD23, CD24, CD27, CD30, CD33, CD34, CD37, CD38, CD40, CD44v6, CD44v7/8, CD45, CD70, CD99, CD117, CD123, CD138, CD171, GD2 (ganglioside G2), ephA2, CSPG4, FAP (fibroblast activation protein), kappa, lambda, 5T4, αvβ6 integrin, integrin αvβ3 (CD 61), galectin, K-Ras (V-Ki-Ras 2 Kirsten rat sarcoma viral oncogene), ral-B, B7-H3, B7-H6, CAIX, EGFR, EGP2, EGP40, epCAM, fetal AchR, FR alpha, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, HLA-DR, IL-11R alpha, IL-13R alpha 2, lewis-Y, muc16, NCAM, NKG2D ligand, PRAME, survivin, TAG72, TEMs, FR2, EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp 17), mesothelin, PAP (prostaacid phosphatase), prostaglandin, TARP (T cell receptor gamma alternate reading frame protein), trp-p8, STEP 1 (six transmembrane epithelial antigen of prostate 1), abnormal Ras protein, abnormal p53 protein, NYESO1 or PDL-1.

In some embodiments, the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain that binds the first antigen, and a first intracellular signaling domain; and the second engineered signaling polypeptide comprises a second extracellular antigen-binding domain that binds to a second antigen or a receptor that binds to a second antigen, and a second intracellular signaling domain, wherein the second engineered signaling polypeptide does not comprise a co-stimulatory domain. In a certain embodiment, the first antigen binding domain and the second antigen binding domain are independently an antigen binding portion of a receptor or an antigen binding portion of an antibody. In a certain embodiment, one or both of the first antigen binding domain or the second antigen binding domain is an scFv antibody fragment. In certain embodiments, the first engineered signaling polypeptide and/or the second engineered signaling polypeptide additionally comprises a transmembrane domain. In a certain embodiment, the first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, e.g., any of the T cell survival motifs described herein.

In another embodiment, the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain that binds HER2 and the second engineered signaling polypeptide comprises a second extracellular antigen-binding domain that binds MUC-1.

In another embodiment, the second extracellular antigen-binding domain of the second engineered signaling polypeptide binds interleukin.

In another embodiment, the second extracellular antigen-binding domain of the second engineered signaling polypeptide binds to a damage-associated molecular pattern molecule (DAMP; also known as alertin). In other embodiments, the DAMP is a heat shock protein, a chromatin-associated protein high mobility kit 1 (HMGB 1), S100A8 (also known as MRP8 or calgranulin a), S100A9 (also known as MRP14 or calgranulin B), serum Amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

In certain embodiments, the second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.

In some embodiments, the activation of signal transduction via the second engineered signaling polypeptide is non-antigenic, but is related to hypoxia. In certain embodiments, hypoxia is induced by activation of hypoxia inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α or HIF-3β.

In some embodiments, for example, for modifying, genetically modifying, and/or transducing lymphocytes to be introduced or reintroduced by subcutaneous injection, expression of one or more engineered signaling polypeptides is regulated by a control module disclosed in more detail herein.

Other sequences

An engineered signaling polypeptide (e.g., CAR) may further comprise one or more additional polypeptide domains, wherein such domains include, but are not limited to, signal sequences, epitope tags, affinity domains, and polypeptides whose presence or activity can be detected (detectable tags), e.g., by antibody analysis or as a result thereof, for the production of a detectable signal. Non-limiting examples of additional domains for any aspect or embodiment provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following sequences as described below: a signal sequence, an epitope tag, an affinity domain, or a polypeptide that produces a detectable signal.

Signal sequences suitable for use in the subject CAR, e.g., the first polypeptide of the subject CAR, include any eukaryotic signal sequence, including naturally occurring signal sequences, synthetic (e.g., artificial) signal sequences, and the like. In some embodiments, the signal sequence may be, for example, CD8 signal sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 72).

Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 73), FLAG (e.g., DYKDDDK; SEQ ID NO: 74), c-myc (e.g., EQKLISEEDL; SEQ ID NO: 75), and the like.

Affinity domains include peptide sequences suitable for recognition or purification that can interact with a binding partner (e.g., a binding partner immobilized on a solid support). DNA sequences encoding multiple consecutive single amino acids (e.g., histidine) when fused to the expressed protein can be used for one-step purification of recombinant proteins bound to a resin column (e.g., agarose gel) by high affinity. Exemplary affinity domains include His5 (hhhhhhh; SEQ ID NO: 76), hisX6 (HHHHH; SEQ ID NO: 77), C-myc (EQKLISEEDL; SEQ ID NO: 75), flag (DYKDDDDK; SEQ ID NO: 74), streptococcal tag (WSHPQFEK; SEQ ID NO: 78), hemagglutinin, e.g., HA tag (YPYDVPDYA; SEQ ID NO: 73), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 79), phe-His-His-Thr (SEQ ID NO: 80), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 81), metal binding domains, e.g., zinc binding domain or calcium binding domain (e.g., from calmodulin (e.g., calmodulin, troponin C, calcineurin B, myosin light chain, restorer protein, S-regulatory protein, opsin, VILIP, troponin, penicillin, calpain large subunit, S100 protein, small albumin, calbindin D9K, calprotectin D28 and calprotectin D, and the amino acid sequence of Myo-linked chains, the biological binding domain, the amino acid sequence of Myo-gamma binding domain, the amino acid sequence of the amino acid sequence, and the amino acid sequence of Myo-linked to the amino acid sequence.

Suitable detectable signal producing proteins include, for example, fluorescent proteins, enzymes that catalyze reactions that produce a detectable signal as a product, and the like.

Suitable fluorescent proteins include, but are not limited to, green Fluorescent Protein (GFP) or variants thereof, blue fluorescent variants (BFP) of GFP, cyan fluorescent variants (CFP) of GFP, yellow fluorescent variants (YFP) of GFP, enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, emerald, yellow precious stone (TYFP), golden star, yellow crystal, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, sky blue, T-sapphire, cyPet, YPet, mKO, hcRed, T-HcRed, dsRed, dsRed2, dsRed-monomer, J-Red, dimer2, T-dimer2 (12), mPL, cupped coral colors, renilla GFP, monter GFP, paGFP, kaede proteins and proteins, phycoerythrin and phycoerythrin conjugates, including B-phycoerythrin, R-phycoerythrin and other phycoerythrin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape, mPlum (Shaner et al (2005) Nature methods (Nat. Methods) 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from coral species are suitable for use, as described, for example, in Matz et al (1999) Nature Biotechnology (Nature Biotechnol.) 17:969-973.

Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline Phosphatase (AP), beta-Galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetaminophenosidase, beta-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose Oxidase (GO), and the like.

Chimeric antigen receptor

In some aspects of the invention, the engineered signaling polypeptide is a Chimeric Antigen Receptor (CAR) or a polynucleotide encoding a CAR, which is referred to herein as a "CAR" for simplicity. The CAR of the present disclosure comprises: a) At least one Antigen Specific Targeting Region (ASTR); b) A transmembrane domain; and c) an intracellular activation domain. In an illustrative embodiment, the antigen-specific targeting region of the CAR is the scFv portion of the antibody against the antigen of interest. In an illustrative embodiment, the intracellular activation domain is from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70, and in some other illustrative embodiments, from CD3z. In an illustrative embodiment, the CAR further comprises a co-stimulatory domain, such as any of the co-stimulatory domains provided in the regulatory domain section above, and in other illustrative embodiments the co-stimulatory domain is an intracellular co-stimulatory domain of 4-1BB (CD 137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some embodiments, the CAR comprises any of the transmembrane domains listed above in the transmembrane domain section.

The CARs of the present disclosure can be present in the plasma membrane of eukaryotic cells (e.g., mammalian cells), where suitable mammalian cells include, but are not limited to, cytotoxic cells, T lymphocytes, stem cells, progeny of stem cells, progenitor cells, progeny of progenitor cells, and NK cells, NK-T cells, and macrophages. When present in the plasma membrane of eukaryotic cells, the CARs of the present disclosure are activated in the presence of one or more antigens of interest (under certain conditions, binding to astm). The antigen of interest is the second member of the specific binding pair. The antigen of interest of a specific binding pair may be a soluble (e.g., not bound to a cell) factor; factors present on the surface of cells such as target cells; factors present on the surface of the entity; factors present on lipid bilayers, and the like. When astm is an antibody and the second member of the specific binding pair is an antigen, the antigen may be a soluble (e.g., not bound to a cell) antigen; an antigen present on the surface of a cell such as a target cell; an antigen present on the surface of the entity; antigens present on lipid bilayers, and the like.

In some embodiments, the ASTR of the CAR is expressed as a polypeptide separate from the intracellular signaling domain. In such embodiments, one or both polypeptides can include any of the transmembrane domains disclosed herein. In some embodiments, one or both polypeptides may include a heterologous signal sequence and/or a heterologous membrane attachment sequence. In some embodiments, the heterologous membrane linker is a GPI anchor linker.

In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more antigens of interest, increases expression of at least one nucleic acid in the cell. For example, in some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by one or more antigens of interest, increases expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or greater than the level of transcription of the nucleic acid in the absence of the one or more antigens of interest.

As an example, a CAR of the present disclosure can include an intracellular signaling polypeptide comprising an immune receptor tyrosine-based activation motif (ITAM).

In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more antigens of interest, can cause the cell to produce one or more cytokines to increase. For example, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by one or more antigens of interest, can increase cytokine production by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or greater than the amount of cytokine produced by the cell in the absence of the one or more antigens of interest. Cytokines whose production may be increased include, but are not limited to, interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-a), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; growth factors, and the like.

In some embodiments, the CARs of the present disclosure can cause increased transcription of nucleic acids in cells and increased cytokine production by cells when present in the plasma membrane of eukaryotic cells and when activated by one or more antigens of interest.

In some cases, the CARs of the disclosure, when present in the plasma membrane of eukaryotic cells and when activated by one or more antigens of interest, produce cytotoxic activity of the cells towards target cells that express on their cell surfaces an antigen that binds to the antigen binding domain of the first polypeptide of the CAR. For example, when the eukaryotic cell is a cytotoxic cell (e.g., NK cell or cytotoxic T lymphocyte), a CAR of the present disclosure, when present in the plasma membrane of the eukaryotic cell and activated by one or more antigens of interest, increases the cytotoxic activity of the cell towards the target cell, which expresses the one or more antigens of interest on its cell surface. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, the CAR of the present disclosure, when present in the plasma membrane of the eukaryotic cell and activated by one or more antigens of interest, increases the cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or greater than the cytotoxic activity of the cell in the absence of the one or more antigens of interest.

In some embodiments, the CARs of the present disclosure, when present in the plasma membrane of eukaryotic cells and when activated by one or more antigens of interest, can cause other CAR activation related events, such as proliferation and expansion (due to increased cell division or anti-apoptotic response).

In some embodiments, the CARs of the present disclosure, when present in the plasma membrane of eukaryotic cells and when activated by one or more antigens of interest, can cause other CAR activation-related events, such as modulation of intracellular signaling, cell differentiation, or cell death.

In some embodiments, the CARs of the present disclosure are subject to microenvironment. This property is typically a result of the microenvironmentally constrained nature of the ASTR domain of the CAR. Thus, the CARs of the present disclosure may have lower binding affinity or, in an illustrative embodiment, may have higher binding affinity for one or more antigens of interest under conditions of the microenvironment than under conditions of the normal physiological environment.

In certain illustrative embodiments, a CAR provided herein comprises a co-stimulatory domain in addition to an intracellular activation domain, wherein the co-stimulatory domain is any one of the intracellular signaling domains provided herein for a Lymphoproliferative Element (LE), e.g., an intracellular domain of a CLE. In certain illustrative embodiments, the co-stimulatory domain of a CAR herein is the first intracellular domain (P3 domain) or P4 domain identified herein with respect to the CLE, which is displayed as the effective intracellular signaling domain of the CLE herein in the absence of the P3 domain. Furthermore, in certain illustrative embodiments, the co-stimulatory domain of the CAR may comprise P3 and P4 intracellular signaling domains identified herein with respect to CLE. Certain illustrative sub-embodiments include, inter alia, potent P3 and P4 partner intracellular signaling domains as identified herein with respect to CLE. In illustrative embodiments, the co-stimulatory domain is not an ITAM-containing intracellular domain of the CAR, either as part of the co-stimulatory domain or in other illustrative embodiments, as the sole co-stimulatory domain.

In these embodiments comprising a CAR, the CAR has the co-stimulatory domain identified herein as the effective intracellular domain of LE, the co-stimulatory domain of the CAR may be any of the intracellular signaling domains in table 1 provided herein. An active fragment of any of the intracellular domains in table 1 can be a co-stimulatory domain of a CAR. In an illustrative embodiment, the astm of the CAR comprises a scFV. In an illustrative embodiment, these CARs comprise, in addition to the C-stimulatory intracellular domain of CLE, an intracellular activation domain, which in an illustrative embodiment is CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C. DAP10/CD28, or ZAP70 intracellular activation domain, or in other illustrative embodiments, CD3z intracellular activation domain.

In these illustrative embodiments, the co-stimulatory domain of the CAR may comprise an intracellular domain or a functional signaling fragment thereof, including signaling domains from: CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL ST, IL7RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23R, IL RA, IL31RA, LEPR, LIFR, LMP1, MPL, myD88, OSMR or PRLR. In some embodiments, the co-stimulatory domain of the CAR may include an intracellular domain or a functional signaling fragment thereof, comprising a signaling domain from: CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL ST, IL9R, IL RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP, MPL, myD88, OSMR or PRLR. In some embodiments, the co-stimulatory domain of the CAR may include an intracellular domain or a functional fragment thereof, comprising a signaling domain from: CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL RB, IL11RA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL31RA, LEPR, MPL, myD or OSMR. In some embodiments, the co-stimulatory domain of the CAR may include an intracellular domain or fragment thereof comprising a signaling domain from: CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL RB, IL11RA, IL13RA2, IL17RD, IL31RA, LEPR, MPL, myD88 or OSMR. In some embodiments, the co-stimulatory domain of the CAR may include an intracellular domain or a functional signaling fragment thereof, comprising a signaling domain from: CSF2RB, CSF3R, IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RC, IL17RE, IL18R1, IL27RA, IL31RA, MPL, myD88, OSMR or PRLR. In some embodiments, the co-stimulatory domain of the CAR may include an intracellular domain or a functional signaling fragment thereof, comprising a signaling domain from: CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE, IL31RA, MPL or MyD88.

In some embodiments, the co-stimulatory domain of the CAR may include an intracellular domain or fragment thereof comprising a signaling domain from: CSF3R, IL ST, IL27RA, MPL and MyD88. In certain illustrative sub-embodiments, the intracellular activation domain of the CAR is derived from CD3z.

Recombinant T Cell Receptor (TCR)

T Cell Receptors (TCRs) recognize specific protein fragments derived from intracellular and extracellular proteins. When a protein breaks down into peptide fragments, it is presented on the cell surface along with another protein called the major histocompatibility complex or MHC, which in humans is called the HLA (human leukocyte antigen) complex. Three different T cell antigen receptor combinations in vertebrates are αβ TCR, γδ TCR, and pre-TCR. Such combinations are formed by dimerization between members of the dimerization subtype (e.g., TCR subunits and βtcr subunits, γtcr subunits and δtcr subunits, and for pre-TCRs, pta subunits and βtcr subunits). The collection of TCR subunits dimerizes and recognizes peptide fragments of interest presented in the context of MHC. The pre-TCR is expressed only on the surface of immature αβ T cells, while αβ TCR is expressed on the surface of mature αβ T cells and NKT cells, and γδ TCR is expressed on the surface of γδ T cells. The T cell surface of the alpha beta TCR recognition MHCI or MHCII presented peptides, and NK T cell surface of the alpha beta TCR recognition CD1 presented lipid antigen. γδ TCRs can recognize MHC and MHC-like molecules, as well as non-MHC molecules, such as viral glycoproteins. After ligand recognition, the αβ TCR and γδ TCR transmit activation signals through the cd3ζ chain, which stimulate T cell proliferation and cytokine secretion.

TCR molecules belong to the immunoglobulin superfamily, whose antigen specificity resides in the V region, with CDR3 having higher variability than CDR1 and CDR2, directly determining the antigen binding specificity of the TCR. When the MHC-antigen peptide complex is recognized by a TCR, CDRl and CDR2 recognize and bind to the side wall of the MHC molecule antigen binding channel, and CDR3 binds directly to the antigen peptide. Thus, recombinant TCRs can be engineered that recognize tumor-specific protein fragments presented on MHC.

Thus, recombinant TCRs with specificity for tumor-specific proteins can be produced, such as those derived from recognition of specific peptides with common HLA, human tcra and tcrp pairs (Schmitt, TM et al, 2009). The target of the recombinant TCR may be a peptide derived from any antigen target of CAR ASTR provided herein, but more typically is derived from an intracellular tumor specific protein, such as carcinoembryonic antigen, or a mutant variant of a normal intracellular protein or other cancer specific neoepitope. In some embodiments, the recombinant TCR binds an idiotype of the antibody. In such embodiments, the TCR is an anti-idiotype polypeptide as disclosed in more detail herein. Libraries of TCR subunits can be screened for their selectivity for the antigen of interest. Screening of native and/or recombinant TCR subunits may identify a collection of TCR subunits having high affinity and/or reactivity for the antigen of interest. Members of such a collection of TCR subunits can be selected and cloned to produce one or more polynucleotides encoding TCR subunits.

Polynucleotides encoding such a collection of TCR subunits may be included in the replication-defective recombinant retroviral particle to genetically modify lymphocytes or, in an illustrative embodiment, T cells or NK cells, such that the lymphocytes express the recombinant TCR. Thus, in any aspect or embodiment provided herein that includes a polynucleotide encoding a CAR or an engineered signal polypeptide that is a CAR, the CAR can be replaced with a γδ TCR chain or, in an illustrative embodiment, a collection of αβ TCR chains. The TCR chains forming the collection can be co-expressed using a variety of different techniques to co-express two TCR chains as disclosed herein for expression of two or more other engineered signaling polypeptides, such as CARs and lymphoproliferative elements. For example, protease cleavage epitopes (e.g., 2A protease), internal Ribosome Entry Sites (IRES) and separate promoters can be used.

Several strategies have been used to reduce the likelihood of mixed TCR dimer formation. Typically, this involves modification of the constant (C) domains of the TCR α and TCR β chains to facilitate preferential pairing of the introduced TCR chains with each other, while making it unlikely that they will pair successfully with the endogenous TCR chain. An in vitro method that demonstrates some promise involves replacing the C domain of the human tcra and tcrp chains with the mouse counterpart. Another approach involves mutations in the human tcra public domain and tcrp chain public regions to facilitate self pairing, or expression of endogenous tcra and tcrp mirnas within viral gene constructs. Thus, in some embodiments provided herein that include one or more sets of TCR chains as engineered signaling polypeptides, each member of the set of αβ TCR chains in the illustrative embodiments comprises a modified constant domain that facilitates preferential pairing with each other. In some sub-embodiments, the TCR chains, in illustrative embodiments, each member of the set of αβ TCR chains, comprises a mouse constant domain from the same TCR chain type, or a constant domain from the same TCR chain subtype that has sufficient sequence derived from the mouse constant domain, such that dimerization of the set of TCR chains with each other takes place in preference to or in a manner that precludes dimerization with a human TCR chain. In other sub-embodiments, the TCR chains, in illustrative embodiments, each member of the set of αβ TCR chains comprises a respective mutation in its constant domain such that dimerization of the set of TCR chains with each other takes place in preference to or in a manner that precludes dimerization with a TCR chain having a human constant domain. In illustrative embodiments, such preferential or exclusive dimerization is performed under physiological conditions.

In some embodiments provided herein that include one or more sets of TCR chains that are engineered signaling polypeptides, the constant regions of members of each of the one or more sets of TCR chains are exchanged. Thus, the collective αtcr subunits have βtcr constant regions, and the collective βtcr subunits have αtcr constant regions. Without being limited by theory, it is believed that such exchanges may prevent mismatches with endogenous counterparts.

Lymphoproliferative element

Many of the embodiments provided herein include lymphoproliferative elements, or nucleic acids encoding the same, typically as part of an engineered signaling polypeptide. Thus, in some aspects of the invention, for example for modified and/or genetically modified lymphocytes to be introduced or reintroduced by subcutaneous injection, the engineered signaling polypeptide is a Lymphoproliferative Element (LE), such as a Chimeric Lymphoproliferative Element (CLE). Typically, LE comprises an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain that drives proliferation, and in an illustrative embodiment, a second intracellular signaling domain.

The extracellular, transmembrane and intracellular domains of LE can vary in their respective amino acid lengths. For example, for embodiments that include replication defective recombinant retroviral particles (RIPs), there is a limit to the length of polynucleotides that can be packaged into retroviral particles so that LEs with shorter amino acid sequences can be advantageous in certain illustrative embodiments. In some embodiments, the total length of the LE may be between 3 and 4000 amino acids, for example between 10 and 3000 amino acids, 10 and 2000 amino acids, 50 and 2000 amino acids, 250 and 2000 amino acids, and in illustrative embodiments between 50 and 1000 amino acids, 100 and 1000 amino acids, or 250 and 1000 amino acids. When present to form the extracellular domain and the transmembrane domain, the extracellular domain may be between 1 and 1000 amino acids, and typically between 4 and 400 amino acids, between 4 and 200 amino acids, between 4 and 100 amino acids, between 4 and 50 amino acids, between 4 and 25 amino acids, or between 4 and 20 amino acids. In one embodiment, the extracellular region is a GGGS for the extracellular domain and transmembrane domain of this aspect of the invention. The transmembrane domain or extracellular domain and the transmembrane region of the transmembrane domain may be between 10 and 250 amino acids, and more typically at least 15 amino acids in length, and may for example be between 15 and 100 amino acids, 15 and 75 amino acids, 15 and 50 amino acids, 15 and 40 amino acids, 15 and 30 amino acids in length. The intracellular signaling domain may be, for example, between 10 and 1000 amino acids, 10 and 750 amino acids, 10 and 500 amino acids, 10 and 250 amino acids, or 10 and 100 amino acids. In illustrative embodiments, the intracellular signaling domain may be at least 30 amino acids, or between 30 and 500 amino acids, 30 and 250 amino acids, 30 and 150 amino acids, 30 and 100 amino acids, 50 and 500 amino acids, 50 and 250 amino acids, 50 and 150 amino acids, or 50 and 100 amino acids. In some embodiments, the intracellular signaling domain of a particular gene is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to at least 10, 25, 30, 40 or 50 or all amino acids (up to the size of the entire intracellular domain sequence) from the sequence of the intracellular signaling domain (the sequence of the intracellular domain as provided herein), and may include, for example, up to an additional 1, 2, 3, 4, 5, 10, 20 or 25 amino acids, provided such sequences still are capable of providing any of the properties of the LEs disclosed herein.

In some embodiments, the lymphoproliferative element may include a first and/or a second intracellular signaling domain. In some embodiments of the present invention, in some embodiments, the first and/or second intracellular signaling domain may comprise CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD B, CD27, mutant δLck CD28, CD40, CD79A, CD79B, CRLF, CSF2RB, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA IL4R, IL RA, IL6R, IL6ST, IL7RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23R, IL RA, IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, or a functional mutant and/or fragment thereof. In an illustrative embodiment, the first intracellular signaling domain may comprise MyD88 or a functional mutant and/or fragment thereof. In other illustrative embodiments, the first intracellular signaling domain may comprise MyD88 or a functional mutant and/or fragment thereof, and the second intracellular signaling domain may comprise ICOS, TNFRSF4 or tnffr 18 or a functional mutant and/or fragment thereof. In some embodiments, the first intracellular domain is MyD88 and the second intracellular domain is an ITAM-containing intracellular domain, e.g., an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C, DAP10/CD28 or ZAP 70. In some embodiments, the second intracellular signaling domain may comprise TNFRSF18 or a functional mutant and/or fragment thereof.

In some embodiments, the lymphoproliferative element may comprise a fusion of an extracellular domain and a transmembrane domain. In some embodiments, the fusion of the extracellular domain and the transmembrane domain may include eTAG IL7 rais PPCL (interleukin 7 receptor), myc LMP1, eTAG CRLF2, eTAG CSF2RB, eTAG CSF3R, eTAG EPOR, eTAG GHR, eTAG truncated after Fn F523C IL27RA, or eTAG truncated after Fn S505N MPL, or functional mutants and/or fragments thereof. In some embodiments, the lymphoproliferative element may include an extracellular domain. In some embodiments, the extracellular domain can include a cell tag having 0, 1, 2, 3, or 4 additional alanine at the carboxy terminus. In some embodiments, the extracellular domain may comprise Myc or eTAG or a functional mutant and/or fragment thereof having 0, 1, 2, 3 or 4 additional alanine at the carboxy terminus. For any of the embodiments of the lymphoproliferative elements disclosed herein that include a cell tag, there are corresponding embodiments that are identical but lack a cell tag and optionally lack any linker sequence that links the cell tag to the lymphoproliferative element.

In some embodiments, the lymphoproliferative element may include a transmembrane domain. In some embodiments, the transmembrane domain may include a transmembrane domain from: BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD, CD5, CD7, CD8A, CD8B, CD, CD11A, CD11A, CD11A, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49A, CD49A, CD64, CD79A, CD79A, CD80, CD84, CD86, CD96 (tactile), CD100 (SEMA 4D), CD103, C134 CD137, CD154, CD160 (BY 55), CD162 (SELPLG), CD226 (DNAM 1), CD229 (Ly 9), CD247, CRLF2, CRTAM, CSF2RA, CSF2RB, CSF 3A, CD 1A, CD 2A, CD2, GHR, HVEM (LIGHTR), IA4, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA IL 4A, CD RA, IL 6A, CD ST, IL7RA, IL7RAIns PPCL, IL 9A, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD RA, IL31RA, ITGA1, ITGA4, ITGA6, A, CD1, ITGB2, ITGB7, KIRDS2, LEPR, LFA-1 (CD 11a, CD 18), A, CD (KLRF 1), OSMR, PAG/A, CD1, SLAM (SLAMF 1, CD150, IPO-3), AMF4 (CD 244, 2B 4), SLF 6 (NTB-A, CD 108), SLAMF7, SLAMF8 (SLSF 8, TNSF 2, TNSF 6, or a mutant thereof.

CLE for any aspect or embodiment herein may include any CLE disclosed in WO2019/055946 (which is incorporated herein by reference in its entirety), which is designed and considered to have constitutive activity in large part, typically because they constitutively activate signaling pathways, typically through functional domains on their intracellular domains. In some embodiments, the constitutively active signaling pathway comprises activation of the Jak pathway, stat pathway, or Jak/Stat pathway, including Jak1, jak2, jak3, and Tyk2, and Stat, e.g., stat1, stat2, stat3, stat4, stat5, stat6, and in illustrative embodiments, stat3 and/or Stat5. Thus, in certain embodiments, provided herein are lymphoproliferative elements comprising a means for activating any one or more of these pathways, and typically an intracellular domain, which is a means for activating any one or more of these pathways. In certain embodiments, the lymphoproliferative element comprises a means, such as an intracellular domain that is part of the dimerized lymphoproliferative element in the illustrative embodiment, as a means for transmitting a signal that promotes proliferation of T cells and/or NK cells. In some embodiments, the CLE comprises one or more STAT activation domains. In some embodiments, the CLE comprises two or more, three or more, four or more, five or more, or six or more STAT activation domains. In some embodiments, at least one of the one or more STAT activation domains is or is derived from BLNK, IL2RG, EGFR, epoR, GHR, IFNAR1, IFNAR2, IFNAR1/2, IFNLR1, IL10R1, IL12Rb2, IL21R, IL2Rb, IL2small, IL7R, IL Ra, IL9R, IL R, and IL21R as known in the art. In some embodiments, two or more STAT activation domains are or are derived from two or more different receptors. In some embodiments, a constitutively active signaling pathway comprises activating a TRAF pathway by activating a TNF receptor related factor such as TRAF3, TRAF4, TRAF7, and in illustrative embodiments TRAF1, TRAF2, TRAF5, and/or TRAF 6. Thus, in certain embodiments, the lymphoproliferative elements used in any of the kits, methods, uses or compositions herein are constitutively active and comprise intracellular signaling domains that activate the Jak/Stat pathway and/or TRAF pathway. In some embodiments, the constitutive active signaling pathway comprises activation of the PI3K pathway. In some embodiments, the constitutive active signaling pathway comprises activation of a PLC pathway. Thus, in certain embodiments, the lymphoproliferative elements used in any of the kits, methods, uses, or compositions herein are constitutively active and include intracellular signaling domains that activate the Jak/Stat pathway, TRAF pathway, PI3K pathway, and/or PLC pathway. As shown therein, in the case where the first and second intracellular signaling domains of the CLE are present, the first intracellular signaling domain is located between the membrane association motif (e.g., transmembrane domain) and the second intracellular domain.

In some embodiments, the lymphoproliferative elements provided herein include one or more or all binding domains (including those disclosed herein) that are responsible for signaling found in the corresponding lymphoproliferative elements in nature. In some embodiments, lymphoproliferative elements provided herein include one or more JAK binding domains. In some embodiments, the JAK binding domain is or is derived from EPOR, GP130, PRLR, GHR, GCSFR, or TPOR/MPLR. JAK binding domains from these proteins are known in the art and the skilled person will understand how to use them. For example, residues 273-338 of EpoR and residues 478-582 of tpox are known to be JAK binding domains. Conserved motifs found in the intracellular domains of cytokine receptors responsible for this signaling are known and are present in certain exemplary lymphoproliferative elements provided herein (see, e.g., morris et al, molecular details of cytokine signaling via the JAK/STAT pathway (The molecular details of cytokine signaling via the JAK/STAT pathway), protein Science (2018) 27:1984-2009). Box1 and Box2 motifs are involved in binding to JAK and signal transduction, but proliferation signals do not always require the presence of Box2 motifs (Murakami et al, proc. Natl. Acad. Sci. USA, 1991, 12, 15; 88 (24): 11349-53; fukunaga et al, european journal of molecular biology (EMBO J.), 1991, 10 (10): 2855-65; and O' Neal and Lee, [ lymphokine cytokine study (Lymphokine Cytokine Res.) ] 1993, 10 months; 12 (5): 309-12). Thus, in some embodiments, the lymphoproliferative element herein is a cytokine receptor comprising a transgenic Box 1-comprising an intracellular domain of a cytokine receptor comprising a Box1 Janus kinase (JAK) binding motif, optionally a Box2 JAK binding motif, and a signal transducer comprising a tyrosine residue and a transcription activator (STAT) binding motif. In some embodiments, the lymphoproliferative element comprises two or more JAK binding motifs, e.g., three or more or four or more JAK binding motifs, which in the illustration are binding motifs found in the native form of the corresponding lymphoproliferative element. In some embodiments, the lymphoproliferative element comprises an intracellular domain that is a means of transmitting a signal that promotes proliferation of T cells and/or NK cells that is part of the dimerized lymphoproliferative element.

Intracellular domains from IFNAR1, IFNGR1, IFNLR1, IL2RB, IL4R, IL5RB, IL6R, IL ST, IL7RA, IL9R, IL10RA, IL21R, IL27R, IL31RA, LIFR and OSMR are known in the art for activating JAK1 signaling and thus include JAK1 binding motifs. The intracellular domains from CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNGR2, IL3RA, IL5RA, IL6ST, IL20RA, IL20RB, IL23R, IL27R, LEPR, MPL and PRLR are known in the art for activating JAK2 and thus include JAK2 binding motifs. The intracellular domain from IL2RG is known in the art for activating JAK3 and thus includes a JAK3 binding motif. Intracellular domains from GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL2RB, IL2RG, IL4R, IL RA, IL5RB, IL7RA, IL9R, IL R, IL RA1, IL31RA, LIFR, MPL and OSMR are known in the art for activating STAT1. Intracellular domains from IFNAR1 and IFNAR2 are known in the art for activating STAT2. Intracellular domains from GHR, IL2RB, IL2RG, IL6R, IL7RA, IL9R, IL RA, IL10RB, IL21R, IL RA1, IL23R, IL27R, IL31RA, LEPR, LIFR, MPL and OSMR are known in the art for activating STAT3. Intracellular domains from IL12RB1 are known in the art for activating STAT4. Intracellular domains from CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IL RB, IL2RG, IL3RA, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL15RA, IL20RB, IL21R, IL22RA1, IL31RA, LIFR, MPL, OSMR and PRLR are known in the art for activating STAT5. Intracellular domains from IL4R and OSMR are known in the art for activating STAT6. The gene found in the first intracellular domain and its intracellular domain are identical to the optional second intracellular domain, except that if the first intracellular domain and the second intracellular domain are identical, at least one and typically neither the transmembrane domain nor the extracellular domain is derived from the same gene.

In some embodiments, lymphoproliferative elements herein may include one or more intracellular signaling domains comprising one or more Box1 motifs. In some embodiments, the one or more intracellular signaling domains comprising one or more of the Box1 motifs may be IL7RA (Box 1 motif at residues 9-17 of SEQ ID NOs:248 and 249), IL12RB (Box 1 motif at residues 10-12 of SEQ ID NOs:254 and 255; and Box1 motif at residues 107-110 and 139-142 of SEQ ID NO: 256), IL31RA (Box 1 motif at residues 12-15 of SEQ ID NOs:275 and 276), CSF2RB (Box 1 motif at residues 14-22 of SEQ ID NO: 213), IL2RB (Box 1 motif at residues 13-21 of SEQ ID NO: 240), IL6ST (Box 1 motif at residues 10-18 of SEQ ID NO: 247), IL2 RA (Box 1 motif at residues 3-11 of SEQ ID NO: 241), IL27RA (Box 1 motif at residues 273-17 of SEQ ID NO: 273), RG 1 at residues 25 of SEQ ID NO: 25), EPOR 1 at residues 25-25 of SEQ ID NO:25, or (Box 1 at residues 25-25 of SEQ ID NO: 25), or (Box 1 motif at residues 16, SEQ ID NO:25, or 16).

In some embodiments, the lymphoproliferative elements herein may include one or more intracellular signaling domains comprising one or more Box2 motifs. In some embodiments, the one or more intracellular signaling domains comprising one or more Box2 motifs may be MPL (Box 2 motif at residues 46-64 in SEQ ID NO: 283), IFNAR2 (Box 1 motif at residues 37-46 in SEQ ID NO: 227), CSF3R, or EPOR (Box 2 motif at residues 303-313 of full length EPOR). EPOR also comprises an extended Box2 motif (residues 329-372 of full length EPOR) important for binding to the tyrosine kinase receptor KIT, and in some embodiments, lymphoproliferative elements may include this Box2 motif. CSF3R further comprises a Box3 motif, which in some embodiments may be included in the lymphoproliferative element.

Some intracellular signaling domains have hydrophobic residues at positions-1, -2 and-6 relative to the Box1 motif that form a "switch motif" required for cytokine-induced JAK2 activation but not for JAK2 binding (Constantinescu et al, molecular cells, 2 nd month 2001; 7 (2): 377-85; and Huang et al, molecular cells, 12 nd 2001; 8 (6): 1327-38). Thus, in certain embodiments, the lymphoproliferative element comprising the Box1 motif has a switch motif, which in the illustrative embodiments has one or more, and preferably all, hydrophobic residues at positions-1, -2 and-6 relative to the Box1 motif. In certain embodiments, the ICD of the lymphoproliferative element is located proximal to the Transmembrane (TM) domain (e.g., 5 to 15 or about 10 residues downstream of the TM domain) relative to the Box2 motif, which Box2 motif is located proximal to the transmembrane domain (e.g., 10 to 50 residues downstream of the TM domain) relative to the STAT binding motif. STAT binding motifs typically comprise tyrosine residues whose phosphorylation affects binding of STAT to STAT binding motifs of lymphoproliferative elements. In some embodiments, the ICD comprises a plurality of STAT binding motifs, wherein the plurality of STAT binding motifs are present in a native ICD (e.g., EPO receptor and IL-6 receptor signaling chain (gp 130)). In some embodiments, the switch motif comprising the intracellular signaling domain may be MPL (switch motifs at residues 11, 15 and 16 of SEQ ID NO: 283).

In some embodiments, lymphoproliferative elements herein may include one or more intracellular signaling domains including one or more phosphorylatable residues, e.g., phosphorylatable serine, threonine, or tyrosine. In some embodiments, the one or more intracellular signaling domains comprising one or more phosphorylable residues may be IL31RA (phosphorylable tyrosine at residues Y96, Y237 and Y165 of SEQ ID NO: 275; absent in SEQ ID NO: 276), CD27 (phosphorylable serine at residue S6 of SEQ ID NO: 205), CSF2RB (phosphorylable tyrosine at residue Y306 of SEQ ID NO: 213), IL6ST (phosphorylable serine at residues S20, S26, S141, S148, S188 and S198 of SEQ ID NO: 247), MPL (phosphorylable tyrosine at residues Y8, Y29, Y78, Y113 and Y118 of SEQ ID NO: 283), CD79B (phosphorylable tyrosine at residues Y16 and Y27 of SEQ ID NO: 211), OSMR (phosphorylable serine at residues S65 and S128 of SEQ ID NO: 294) or CD3G (phosphorylable serine at residues S123 and S126 of SEQ ID NO: 123). In some embodiments, the lymphoproliferative element comprising the intracellular domain of CSF3R may comprise one, two, three or all of the tyrosine residues corresponding to Y704, Y729, Y744 and Y764 of full-length CSF3R, various combinations of which have been shown to be important in binding Stat3, SOCS3, grb2 and p21 Ras. In some embodiments, lymphoproliferative elements herein may include one or more intracellular signaling domains having one or more of their phosphorylatable residues mutated to a phosphorus mimetic residue, such as aspartic acid or glutamic acid. In some embodiments, the lymphoproliferative elements herein may include one or more intracellular signaling domains having one or more of its phosphorylatable tyrosine mutated to a non-phosphorylatable residue, such as alanine, valine, or phenylalanine. In some embodiments, the lymphoproliferative element comprising the intracellular domain of CSF3R may comprise one or more mutations corresponding to T615A and T618I of full-length CSF3R, which have been shown to increase receptor dimerization and activity.

In some embodiments, lymphoproliferative elements herein may include one or more intracellular signaling domains comprising one or more ubiquitinated targeting motif residues. In some embodiments, the one or more intracellular signaling domains comprising one or more ubiquitinated targeting motif residues may be MPL (residues at K40 and K60 of SEQ ID NO: 283) or OX40 (residues at K17 and K41 of SEQ ID NO: 296). In some embodiments herein, the intracellular domain comprising a ubiquitinated targeting motif residue may have one or more lysines mutated to arginine or another amino acid.

In some embodiments, lymphoproliferative elements herein may include one or more intracellular signaling domains comprising one or more TRAF binding sites. Without being limited by theory, TRAF1, TRAF2 and TRAF3 binding sites comprise the amino acid sequence PXQXT (SEQ ID NO: 303), wherein each X may be any amino acid, the different TRAF2 binding sites comprise the consensus sequence SXXE (SEQ ID NO: 304), wherein each X may be any amino acid, and the TRAF6 binding site comprises the consensus sequence QXPXEX (SEQ ID NO: 305). In some embodiments, the one or more intracellular signaling domains comprising one or more TRAF binding sites may be CD40 (TRAF 1, TRAF2, and TRAF3 binding sites at residues 35-39 of SEQ ID NO: 208; TRAF2 binding sites at residues 57-60 of SEQ ID NO: 208; TRAF6 binding sites at residues 16-21 of SEQ ID NO: 208) or OX40 (TRAF 1, TRAF2, TRAF3, and TRAF5 binding motifs at residues 20-27 of SEQ ID NO: 296).

In some embodiments, the lymphoproliferative elements herein may include one or more intracellular signaling domains comprising a TIR domain. In some embodiments, the one or more intracellular signaling domains comprising a TIR domain may be IL17RE (TIR domain at residues 13-136 of SEQ ID NO: 265), IL18R1 (TIR domain at residues 28-170 of SEQ ID NO: 266), or MyD88 (TIR domain at residues 160-304 of SEQ ID NO: 284).

In some embodiments, lymphoproliferative elements herein may include one or more intracellular signaling domains comprising a PI3K binding motif domain. In some embodiments, one or more of the intracellular signaling domains comprising the PI3K binding motif may be CD28 (PI 3K binding motif at residues 12-15 of SEQ ID NOs:206 and 207, which also binds to Grb 2), ICOS (PI 3K binding motif at residues 19-22 of SEQ ID NO:225, which may mutate F21Q to increase IL-2 production and/or to bind to Grb 2), OX40 (p 85 PI3K binding motif at residues 34-57 of full length OX 40).

In some embodiments, the lymphoproliferative elements herein may include one or more intracellular signaling domains comprising a dilucium motif. In some embodiments, one or more intracellular signaling domains comprising a dual leucine motif may be IFNGR2 (dual leucine motifs at residues 8-9 of SEQ ID NO: 230) or CD3G (dual leucine motifs at residues 131-132 of full length CD 3G). In some embodiments, one or both residues in the dual leucine motif may be mutated.

In some embodiments, a lymphoproliferative element herein may include one or more intracellular signaling domains comprising one or more N-terminal death domains. In some embodiments, the one or more intracellular signaling domains comprising one or more N-terminal death domains may be MyD88 (the N-terminal death domain at residues 29-106 of SEQ ID NO: 284) or TNFR. The cytoplasmic domain of the TNF receptor (TNFR), which in an illustrative example may be TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14 or TNFRSF18, may recruit signaling molecules, including TRAF (TNF receptor-related factor) and/or "death domain" (DD) molecules. The domains, motifs and point mutations of TNFR that induce proliferation and/or survival of T cells and/or NK cells are known in the art and one of skill in the art can recognize the corresponding domains, motifs and point mutations in the TNFR polypeptide. Those skilled in the art will be able to identify TRAF and/or DD binding motifs in different TNFR families using, for example, sequence alignment with known binding motifs. In some embodiments, the lymphoproliferative element comprising the intracellular domain of TNFR may comprise one or more TRAF binding motifs. In some embodiments, the lymphoproliferative element comprising the intracellular domain of TNFR does not comprise a DD binding motif, or has one or more DD binding motifs deleted or mutated within the intracellular domain. In some embodiments, lymphoproliferative elements comprising an intracellular domain of TNFR can recruit TRADD and/or TRAF2.TNFR also includes Cysteine Rich Domains (CRD) which are important for ligand binding (Locksley RM et al, cell, 2001, 23/month; 104 (4): 487-501). In some embodiments, the lymphoproliferative element comprising an intracellular domain of TNFR does not comprise TNFR CRD.

In some embodiments, the lymphoproliferative elements herein may include one or more intracellular signaling domains comprising one or more intermediate domains that interact with an IL-1R-related kinase. In some embodiments, the one or more intracellular signaling domains comprising one or more intermediate domains may be MyD88 (intermediate domain at residues 107-156 of SEQ ID NO: 284).

In some embodiments, lymphoproliferative elements comprising an intracellular domain from IL7RA may include one or more of the S region or the T region (S region at residues 359-394 and T region at residues Y401, Y449, and Y456 of full length IL7 RA). In an illustrative embodiment of a lymphoproliferative element comprising a first intracellular domain derived from IL7RA, the second intracellular domain may be derived from TNFRSF8.

In an illustrative embodiment of a lymphoproliferative element comprising a first intracellular domain derived from CD40, the second intracellular domain may not be derived from the following intracellular domains: myD88, a CD28 family member (e.g., CD28, ICOS), a pattern recognition receptor, a C-reactive protein receptor (i.e., nodi, nod2, ptX 3-R), a TNF receptor, CD40, RANK/TRANCE-R, OX, 4-1BB, HSP receptors (Lox-1 and CD 91), or CD28. Pattern recognition receptors include, but are not limited to, endocytic pattern recognition receptors (i.e., mannose receptor, scavenger receptor (i.e., mac-1, LRP, peptidoglycan, creatine, toxin, CD11c/CR 4)); external signaling pattern recognition receptors (Toll-like receptors (TLR 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR 10), peptidoglycan recognition proteins (PGRP binds bacterial peptidoglycans, and CD 14), internal signaling pattern recognition receptors (i.e., NOD-receptors 1 and 2) and RIG1.

In some embodiments, the lymphoproliferative element comprising an intracellular domain from MyD88 may comprise one or more of mutations L93P, R193C and L265P in full-length MyD88 (mutations L93P, R C and L260P in SEQ ID NO: 284). In an illustrative embodiment comprising a lymphoproliferative element derived from a first intracellular domain of MyD88, the second intracellular domain may be derived from TNFRSF4 or TNFRSF8. In other illustrative embodiments comprising a lymphoproliferative element derived from a first intracellular domain of MyD88, the second intracellular domain may not be derived from an intracellular domain that: members of the CD28 family (e.g., CD28, ICOS), pattern recognition receptors, C-reactive protein receptors, TNF receptors, or HSP receptors.

In some embodiments, cells expressing lymphoproliferative elements comprising intracellular and transmembrane domains of MPL may be contacted with or exposed to eltrombopag, or a patient or subject into which such cells have been infused may be treated with eltrombopag. Without being limited by theory, eltrombopag binds to the transmembrane domain of MPL and induces activation of the intracellular domain of MPL.

The domains, motifs and point mutations of MPL that induce proliferation and/or survival of T cells and/or NK cells are known in the art and one of skill in the art can recognize the corresponding domains, motifs and point mutations in MPL polypeptides, some of which are discussed in this paragraph. The region deleted covering amino acids 70 to 95 in SEQ ID NO:283 proved to support viral transformation in the case of v-mpl (Benit et al J virol.) 8 month 1994, 68 (8): 5270-4), thus indicating that this region is not necessary for the function of mpl in this case. Morello et al Blood, 7, 1995; 86 (8) 557-71 use of the same deletion to demonstrate that this region is not required to stimulate transcription of the erythropoietin receptor-reactive CAT reporter gene construct and further find that this deletion results in slightly enhanced transcription as expected with respect to removal of non-essential and negative elements in this region, as suggested by Drachman and Kaushansky. Thus, in some embodiments, the MPL intracellular signaling domain does not contain the region comprising amino acids 70 to 95 of SEQ ID NO 283. Using computer modeling, lee et al found that clinically relevant mutations in the transmembrane domain of MPL should activate MPL in the following order of activation: W515K (amino acid substitution corresponding to SEQ ID NO: 283W 2K) > S505A (amino acid substitution corresponding to SEQ ID NO: 187S 14A) > W515I (amino acid substitution corresponding to SEQ ID NO: 283W 2I) > S505N (amino acid substitution corresponding to SEQ ID NO: 187S 14N, which was tested as part T075 (SEQ ID NO: 188)) (Lee et al, science public library complex (PLoS one.)), 2011;6 (8): e 23496). It is predicted that the simulation of these mutations may result in constitutive activation of JAK2 (a kinase partner of MPL). In some embodiments, the intracellular portion of MPL may include one or more or all of the domains and motifs described herein that are present in SEQ ID NO 283. In some embodiments, the transmembrane portion of MPL may include one or more or all of the domains and motifs described herein that are present in SEQ ID NO. 187. In an illustrative embodiment of a lymphoproliferative element comprising a first intracellular domain derived from MPL, a second intracellular domain may be derived from CD79B.

In an illustrative embodiment of a lymphoproliferative element comprising a second intracellular domain derived from CD79B, the first intracellular domain may be derived from CSF3R.

In some embodiments, lymphoproliferative elements comprising the intracellular domain of PRLR may include the growth hormone receptor binding domain of PRLR and any known mutations (growth hormone receptor binding domain at residues 28-104 of SEQ ID NO: 295).

In some embodiments, the lymphoproliferative element comprising an ICOS intracellular domain may comprise a calcium signaling motif (the calcium signaling motif at residues 5-8 of SEQ ID NO: 225). In some embodiments, the lymphoproliferative element comprising an ICOS intracellular domain may comprise at least one of a first conserved motif and a second conserved motif (the first conserved motif and the second conserved motif located at residues 9-18 and 24-30, respectively, of SEQ ID NO: 225). In some embodiments, the lymphoproliferative element comprising an ICOS intracellular domain does not include at least one of a first conserved motif or a second conserved motif.

EPOR also contains a short segment (residues 267-276 of full length EPOR) important for EPOR internalization. In some embodiments, the lymphoproliferative element comprising an EPOR intracellular domain does not comprise an internalizing segment.

The domains, motifs and point mutations of intracellular signaling domains that induce proliferation and/or survival of T cells and/or NK cells are known in the art and the skilled person can identify corresponding domains, motifs and point mutations in polypeptides, some of which are described above and the skilled person can identify corresponding domains, motifs and point mutations in other polypeptides. The skilled artisan will be able to identify these domains, motifs and point mutations in similar polypeptides using, for example, sequence alignment with known binding motifs. In some embodiments, the lymphoproliferative elements herein may include any one, e.g., one or more up to all, domains, motifs and mutations of the intracellular signaling domains disclosed herein or otherwise known to induce proliferation and/or survival of T cells and/or NK cells.

In another embodiment, the LE provides, is capable of providing and/or has the following characteristics (or a cell modified, genetically modified and/or transduced with LE can provide, be suitable for, possess and/or be modified for use in) in vivo driving T cell expansion.

In some embodiments, the lymphoproliferative element may comprise any of the sequences listed in Table 1 (SEQ ID NOS: 84-302). Table 1 shows the parts, names (including gene names) and amino acid sequences of the domains tested in CLE. In certain illustrative embodiments, a CLE may include an extracellular domain (denoted P1), a transmembrane domain (denoted P2), a first intracellular domain (denoted P3), and a second intracellular domain (denoted P4). Typically, the lymphoproliferative element comprises a first intracellular domain. In illustrative embodiments, the first intracellular domain can include any of the portions listed as S036 to S0216 in table 1 or functional mutants and/or fragments thereof. In some embodiments, the lymphoproliferative element may include a second intracellular domain. In illustrative embodiments, the second intracellular domain can include any of the portions listed as S036 to S0216 in table 1 or functional mutants and/or fragments thereof. In some embodiments, the lymphoproliferative element may include an extracellular domain. In illustrative embodiments, the extracellular domain may include any of the sequences listed in table 1 as part of M001 to M049 or E006 to E015, or functional mutants and/or fragments thereof. In some embodiments, the lymphoproliferative element may include a transmembrane domain. In illustrative embodiments, the transmembrane domain may include any of the moieties listed in table 1 as M001 to M049 or T001 to T082, or functional mutants and/or fragments thereof. In some embodiments, the lymphoproliferative element may be a fusion of the extracellular/transmembrane domain (M001 to M049 in table 1), the first intracellular domain (S036 to S0216 in table 1) and the second intracellular domain (S036 to S216 in table 1). In some embodiments, the lymphoproliferative element may be a fusion of an extracellular domain (E006 to E016 in table 1), a transmembrane domain (T001 to T082 in table 1), a first intracellular domain (S036 to S0216 in table 1), and a second intracellular domain (S036 to S0216 in table 1). For example, the lymphoproliferative element may be a fusion of E006, T001, S036 and S216, also written E006-T001-S036-S216. In illustrative embodiments, the lymphoproliferative element may be a fusion E010-T072-S192-S212, E007-T054-S197-S212, E006-T006-S194-S211, E009-T073-S062-S053, E008-T001-S121-S212, E006-T044-S186-S053, or E006-T016-S186-S050.

In an illustrative embodiment, the intracellular domain of LE or the first intracellular domain in an LE having two or more intracellular domains is not a functional intracellular activation domain from an intracellular domain containing ITAM, e.g., an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70 and in other illustrative embodiments CD3 z. In an illustrative embodiment, the LE extracellular domain does not comprise a single chain variable fragment (scFv). In other illustrative embodiments, the LE extracellular domain that activates LE upon binding to a binding partner does not comprise a single chain variable fragment (scFv). CLE does not contain an ASTR and an activation domain from: CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70. If the LE does include an ASTR (rather than the activation domains in the previous list), then in the illustrative embodiment the ASTR of the LE does not include an scFv. In some embodiments, the lymphoproliferative element does not include an extracellular domain.

In some embodiments, the lymphoproliferative element and in illustrative embodiments the CLE is not covalently linked to a cytokine. In some aspects, the lymphoproliferative element, and in illustrative embodiments the CLE, comprises a cytokine polypeptide covalently linked to its cognate receptor. In any of these embodiments, the CLE may be constitutively active and typically constitutively activates the same Jak/STAT and/or TRAF pathways as the corresponding activated wild type cytokine receptor. In some embodiments, the chimeric cytokine receptor is an interleukin. In some embodiments, the CLE is IL-7 covalently linked to IL-7 RA or IL-15 covalently linked to IL-15 RA. In other embodiments, the CLE is not IL-15 covalently linked to IL15 RA. In other aspects, the CLE comprises a cytokine polypeptide covalently linked to only a portion of its cognate receptor, the cognate receptor comprising a functional portion capable of binding to the extracellular domain of the cytokine polypeptide, the transmembrane domain and/or intracellular domain is from a heterologous polypeptide, and the CLE is constitutively active. In one embodiment, the CLE is IL-7 covalently linked to the extracellular and transmembrane domains of IL7RA and the intracellular domain from IL2 RB. In another embodiment, a CLE is a cytokine polypeptide covalently linked to a portion of its cognate receptor that includes a functional portion capable of binding to the extracellular domain of the cytokine polypeptide, the heterologous transmembrane domain, and the intracellular domain of the lymphoproliferative element provided herein. In some embodiments, the lymphoproliferative element is a cytokine receptor that does not bind to a cytokine.

In some aspects, the lymphoproliferative element is capable of binding to a soluble cytokine or growth factor, and such binding is necessary for activity. In certain illustrative embodiments, the lymphoproliferative element is constitutively active, and therefore does not need to bind to soluble growth factors or cytokines to obtain activity. Typically, the constitutively active lymphoproliferative element does not bind to soluble cytokines or growth factors. In some embodiments, the lymphoproliferative element is a chimeric comprising an extracellular binding domain from one receptor and an intracellular signaling domain from a different receptor. In some embodiments, the CLE is a counter-receptor that is activated when bound to a ligand that inhibits proliferation and/or survival when bound to its natural receptor, but instead results in proliferation and/or survival when the CLE is activated. In some embodiments, the counter receptor comprises a chimera comprising an extracellular ligand binding domain from IL4Ra and an intracellular domain from IL7Ra or IL 21. Other examples of inverse cytokine receptors include chimeras comprising an extracellular ligand binding domain from a receptor (e.g., a receptor for IL-4, IL-10, IL-13, or TGFb) that will inhibit proliferation and/or survival when bound to its natural ligand, as well as any of the lymphoproliferative element intracellular domains disclosed herein. In an illustrative aspect, the lymphoproliferative element does not bind a cytokine. In further illustrative aspects, the lymphoproliferative element does not bind any ligand. In illustrative embodiments, the lymphoproliferative element that does not bind any ligand is constitutively dimerized or otherwise multimerized, and is constitutively active. In any of the illustrative embodiments provided herein, including methods and compositions of lymphoproliferative elements, the intracellular domain may be derived from the intracellular portion of the transmembrane protein CD40 of the TNF receptor family. The domains, motifs and point mutations of CD40 that induce proliferation and/or survival of T cells and/or NK cells are known in the art and one of skill in the art can recognize the corresponding domains, motifs and point mutations in CD40 polypeptides, some of which are discussed in this paragraph. The CD40 protein contains several binding sites for TRAF proteins. Without being bound by theory, the binding sites for TRAF1, TRAF2 and TRAF3 are located in the membrane distal domain of the intracellular portion of CD40 and include the amino acid sequence PXQXT (SEQ ID NO: 303), where each X may be any amino acid (corresponding to amino acids 35-39 of SEQ ID NO: 208) (Elgueta et al, immunol review (Immunol Rev.)) (2009, month 5; 229 (1): 152-72). TRAF2 was also shown to bind to the common sequence SXXE (SEQ ID NO: 304), where each X may be any amino acid (amino acids 57-60 corresponding to SEQ ID NO: 208) (Elgueta et al, reviewed in immunology, 5 months 2009; 229 (1): 152-72). The different binding sites of TRAF6 are located in the membrane proximal domain of the intracellular portion of CD40 and include the common sequence QXPXEX (SEQ ID NO: 305), where each X may be any amino acid (corresponding to amino acid 16-21 of SEQ ID NO: 208) (Lu et al J biochemistry journal (J Biol chem.) 11, 14, 2003; 278 (46): 45414-8). In an illustrative embodiment, the intracellular portion of transmembrane protein CD40 may include all binding sites for TRAF protein. TRAF binding sites are known in the art, and one skilled in the art will be able to recognize the corresponding TRAF binding site in a CD 40-like polypeptide. In some embodiments, suitable intracellular domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:208 or SEQ ID NO: 209. In some embodiments, the intracellular domain derived from CD40 has a length of about 30 amino acids (aa) to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, or about 60aa to about 65 aa. In illustrative embodiments, the intracellular domain derived from CD40 has a length of about 30aa to about 66aa, e.g., 30aa to 65aa or 50aa to 66 aa. In an illustrative embodiment of a lymphoproliferative element comprising a first intracellular domain derived from CD40, the second intracellular domain may not be derived from the following intracellular domains: myD88, a CD28 family member (e.g., CD28, ICOS), a pattern recognition receptor, a C-reactive protein receptor (i.e., nodi, nod2, ptX 3-R), a TNF receptor, CD40, RANK/TRANCE-R, OX, 4-1BB, HSP receptors (Lox-1 and CD 91), or CD28. Pattern recognition receptors include, but are not limited to, endocytic pattern recognition receptors (i.e., mannose receptor, scavenger receptor (i.e., mac-1, LRP, peptidoglycan, creatine, toxin, CD11c/CR 4)); external signaling pattern recognition receptors (Toll-like receptors (TLR 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR 10), peptidoglycan recognition proteins (PGRP binds bacterial peptidoglycans, and CD 14), internal signaling pattern recognition receptors (i.e., NOD-receptors 1 and 2) and RIG1.

In any of the illustrative embodiments provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the transmembrane protein MPL. Thus, in some embodiments, the lymphoproliferative element comprises MPL or MPL, or a variant and/or fragment thereof, comprising a variant and/or fragment comprising at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the intracellular domain of MPL (with or without the transmembrane and/or extracellular domain of MPL), wherein the variant and/or fragment retains the ability to promote cell proliferation of PBMCs and in some embodiments T cells. In some embodiments, cells expressing lymphoproliferative elements comprising intracellular and transmembrane domains of MPL may be contacted with eltrombopag (eltrombopag), exposed Yu Aiqu baupa, or treated with eltrombopag. Without being limited by theory, eltrombopag binds to the transmembrane domain of MPL and induces activation of the intracellular domain of MPL. The domains, motifs and point mutations of MPL that induce proliferation and/or survival of T cells and/or NK cells are known in the art and one of skill in the art can recognize the corresponding domains, motifs and point mutations in MPL polypeptides, some of which are discussed in this paragraph. The transmembrane MPL protein contains the Box1 motif PXXP (SEQ ID NO: 306) and the Box2 motif, which are regions of increased serine and glutamate content (corresponding to amino acids 46-64 in SEQ ID NO: 283), in PXXP each X may be any amino acid (corresponding to amino acids 17-20 in SEQ ID NO: 283) (Drachman and Kaushansky, proc. Natl. Acad. Sci. USA, 1997, 3, 18; 94 (6): 2350-5). Box1 and Box2 motifs are involved in binding to JAK and signal transduction, but proliferation signals do not always require the presence of Box2 motifs (Murakami et al, proc. Natl. Acad. Sci. USA, 1991, 12, 15; 88 (24): 11349-53; fukunaga et al, european journal of molecular biology (EMBO J.), 1991, 10 (10): 2855-65; and O' Neal and Lee, [ lymphokine cytokine study (Lymphokine Cytokine Res.) ] 1993, 10 months; 12 (5): 309-12). Many cytokine receptors have hydrophobic residues at positions-1, -2 and-6 relative to the Box1 motif (corresponding to amino acids 16, 15 and 11 of SEQ ID NO:283, respectively) that form a "switch motif" required for cytokine-induced JAK2 activation but not for JAK2 binding (Constantinescu et al, molecular cells (Mol cell.), month 2 2001; 7 (2): 377-85; and Huang et al, molecular cells, month 12 2001; 8 (6): 1327-38). The region deleted covering amino acids 70 to 95 in SEQ ID NO:283 is shown to support viral transformation in the case of v-mpl (Benit et al J virol.) (1994, month 8; 68 (8): 5270-4), thus indicating that this region is not necessary for the function of mpl in this case. Morello et al Blood, 7, 1995; 86 (8) 557-71 use of the same deletion to demonstrate that this region is not required to stimulate transcription of the erythropoietin receptor-reactive CAT reporter gene construct and further find that this deletion results in slightly enhanced transcription as expected with respect to removal of non-essential and negative elements in this region, as suggested by Drachman and Kaushansky. Thus, in some embodiments, the MPL intracellular signaling domain does not contain the region comprising amino acids 70 to 95 of SEQ ID NO 283. In full-length MPL, lysines K553 (corresponding to K40 of SEQ ID NO: 283) and K573 (corresponding to K60 of SEQ ID NO: 283) are shown to serve as negative regulatory sites for portions of the ubiquitination targeting motif (Saur et al, blood, 11, 2010, 115 (6): 1254-63). Thus, in some embodiments herein, MPL intracellular signaling domains do not comprise these ubiquitinated targeting motif residues. In full-length MPL, tyrosine Y521 (corresponding to Y8 of SEQ ID NO: 283), Y542 (corresponding to Y29 of SEQ ID NO: 283), Y591 (corresponding to Y78 of SEQ ID NO: 283), Y626 (corresponding to Y113 of SEQ ID NO: 283) and Y631 (corresponding to Y118 of SEQ ID NO: 283) have been shown to be phosphorylated (Varghese et al, front endocrinology (Lausane) 2017, 3 months 31; 8:59). Y521 and Y591 of full-length MPL are negative regulatory sites that act as part of a lysosome targeting motif (Y521) or through interaction with the adapter protein AP2 (Y591) (Drachman and Kaushansky, proc. Natl. Acad. Sci. USA 1997, 18; 94 (6): 2350-5; and Hitchcock et al blood, 2008, 9, 15; 112 (6): 2222-31). Y626 and Y631 of full length MPL are positive regulatory sites (Drachman and Kaushansky, proc. Natl. Acad. Sci. USA, 1997, 3, 18; 94 (6): 2350-5) and murine homologs of Y626 are required for cell differentiation and phosphorylation of Shc (Alexander et al, J. Mol. Biol.1996, 2 nd. 1996; 15 (23): 6531-40) and Y626 is also required for constitutive signaling in MPL using the W515A mutation described below (pecque et al, blood, 4 th 2010; 115 (5): 1037-48). MPL contains the Shc phosphorylated tyrosine binding motif NXXY (SEQ ID NO: 307), where each X may be any amino acid (corresponding to amino acids 110-113 of SEQ ID NO: 283), and this tyrosine is phosphorylated and is important for TPO-dependent phosphorylation of Shc, SHIP and STAT3 (Laminet et al, J.Biochemical., 1996, 5; 271 (1): 264-9; and van der Geer et al, proc. Natl. Acad. Sci. USA, 1996, 2, 6; 93 (3): 963-8). MPL also contains the STAT3 consensus binding sequence YXXQ (SEQ ID NO: 308), where each X may be any amino acid (corresponding to amino acids 118 to 121 of SEQ ID NO: 283) (Stahl et al Science, 3 rd 1995; 267 (5202): 1349-53). Tyrosine of this sequence can be phosphorylated and MPL is capable of partial STAT3 recruitment (Drachman and Kaushansky, proc. Natl. Acad. Sci. USA, 1997, 3, 18; 94 (6): 2350-5). MPL also contains the sequence yl pl (SEQ ID NO: 309) (corresponding to amino acids 113-116 of SEQ ID NO: 283) which is similar to the consensus binding site for STAT5 recruitment of pyl xl (SEQ ID NO: 310), where pY is phosphotyrosine and X can be any amino acid (May et al, joint society of biochemistry (FEBS lett.), 30 th 1996, 394 (2): 221-6). Using computer modeling, lee et al found that clinically relevant mutations in the transmembrane domain of MPL should activate MPL in the following order of activation: W515K (amino acid substitution corresponding to SEQ ID NO: 283W 2K) > S505A (amino acid substitution corresponding to SEQ ID NO: 187S 14A) > W515I (amino acid substitution corresponding to SEQ ID NO: 283W 2I) > S505N (amino acid substitution corresponding to SEQ ID NO: 187S 14N, which was tested as T075 (SEQ ID NO: 188)) (Lee et al, science public library complex (PLoS One.)), 2011;6 (8): e 23396). It is predicted that the simulation of these mutations may result in constitutive activation of JAK2 (a kinase partner of MPL). In some embodiments, the intracellular portion of MPL may include one or more or all of the domains and motifs described herein that are present in SEQ ID NO 283. In some embodiments, the transmembrane portion of MPL may include one or more or all of the domains and motifs described herein that are present in SEQ ID NO. 187. The domains, motifs and point mutations of MPL provided herein are known in the art, and those of skill in the art will recognize that the MPL intracellular signaling domains herein will, in an illustrative embodiment, include the corresponding domains, motifs and point mutations that are shown to promote proliferative activity, and will not include those that are shown to inhibit MPL proliferative activity. Any or all of these domains, motifs and point mutations of MPL may be present in the intracellular signaling domain and may be included in any of the aspects and embodiments disclosed herein. In some embodiments, suitable intracellular domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO 283. In some embodiments, the intracellular domain derived from MPL has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to about 150aa, about 150aa to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, about 250aa to about 300aa, about 300aa to 350aa, about 350aa to about 400aa, about 400aa to about 450aa, about 450aa to about 500aa, about 500aa to about 550aa, about 550aa to about 600aa, or about 600aa to about 635 aa. In illustrative embodiments, the intracellular domain derived from MPL has a length of about 30aa to about 200aa, e.g., a length of 30aa to 150aa, 30aa to 119aa, 30aa to 121aa, 30aa to 122aa, or 50aa to 125 aa. In an illustrative embodiment of a lymphoproliferative element comprising a first intracellular domain derived from MPL, a second intracellular domain may be derived from CD79B.

The lymphoproliferative elements and CLE that may be included in any aspect disclosed herein may be any LE or CLE disclosed in WO 2019/055946. Therein, CLE is disclosed that promotes proliferation in cell culture from day 7 to day 21, 28, 35 and/or 42 after transduction with PBMCs transduced with CLE-encoding lentiviral particles. Furthermore, wherein CLE is identified that promotes in vivo proliferation in mice in the presence or absence of an antigen recognized by the CAR, wherein T cells expressing one of CLE and CAR are introduced into the mice. As exemplified therein, the examples provide that the test and/or criteria can be used to identify any test polypeptide, including an LE or a test domain of an LE, such as whether the first intracellular domain or the second intracellular domain or both the first and second intracellular domains are indeed valid intracellular domains of an LE or LE, or particularly valid intracellular domains of an LE or LE. Thus, in certain embodiments, any aspect or other embodiment provided herein that includes an LE or a polynucleotide or nucleic acid encoding an LE may demonstrate that an LE meets any one or more of the identified tests or criteria for identifying an LE provided herein, or provides characteristics of the test or criteria, or is capable of providing and/or having characteristics of the test or criteria, or a cell genetically modified, transduced and/or stably transfected with a recombinant nucleic acid vector (e.g., a cell transduced with lentiviral particles encoding an LE) is capable of providing, adapting, having and/or being modified to achieve the results of one or more of the tests. In one embodiment, the LE provides, is capable of providing and/or has the following properties (or a cell genetically modified and/or transduced with a retroviral particle encoding LE is capable of providing, is suitable for, has the following properties and/or is modified for use) compared to a control retroviral particle (e.g., a lentiviral particle under the same conditions): amplification of preactivated PBMCs transduced with lentiviruses comprising nucleic acid encoding LE and anti-CD 19 CAR comprising a cd3ζ intracellular activation domain (but not a co-stimulatory domain) in the absence of exogenously added cytokines at days 7 through 21, 28, 35 and/or 42 of in vitro post-transduction culture. In some embodiments, an improved or enhanced viability, expansion, and/or proliferation element test for cells transduced with a retroviral particle (e.g., a lentiviral particle) having a genome encoding a test construct encoding a hypothetical LE (test cell) or a cell transduced with a control retroviral (e.g., lentiviral) particle that is the same as a lentiviral particle comprising a nucleic acid encoding a lymphoproliferative element, but does not have a lymphoproliferative element, or does not have one or more intracellular domains of a test polypeptide construct, but comprises the same extracellular domain (if present), and the same transmembrane region or membrane targeting region of a corresponding test polypeptide construct, can be performed based on comparison to a control cell. In some embodiments, control cells are transduced with a retroviral particle (e.g., a lentiviral particle) having a genome encoding a lymphoproliferative element or an intracellular domain thereof as identified herein by the exemplified lymphoproliferative element. In such embodiments, the test criteria may include: when tested using retroviral particles (e.g., lentiviral particles) having a genome encoding a test construct relative to a control lymphoproliferative element, typically by analysis of cells transduced therewith, there is at least sufficient enrichment, survival and/or amplification, or no statistical difference in enrichment, survival and/or amplification. In some embodiments, the illustrative or explanatory embodiments of lymphoproliferative elements herein are explanatory embodiments of control lymphoproliferative elements for such tests.

In some embodiments, this test for improved properties of the putative or tested lymphoproliferative element is performed by performing replication and/or performing statistical tests. Those skilled in the art will recognize that many statistical tests may be used for such lymphoproliferative element tests. Such tests in the examples are contemplated to be any such test known in the art. In some embodiments, the statistical test may be a T-test or a Mann-Whitney-Wilcoxon test (Mann-Whitney-Wilcoxon test). In some embodiments, the normalized enrichment level of the test construct is significant at a p-value of less than 0.1 or less than 0.05 or less than 0.01.

In another embodiment, upon transduction with an anti-CD 19 CAR comprising a cd3ζ intracellular activation domain but no co-stimulatory domain, LE provides, is capable of providing and/or has the following properties (or a cell modified and/or transduced with an LE gene is capable of providing, is suitable for, has the following properties and/or is modified for use) on days 7 to 21, 28, 35 and/or 42 of in vitro culture in the absence of exogenously added cytokines: at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold amplification, or 1.5-fold to 25-fold amplification, or 2-fold to 20-fold amplification, or 2-fold to 15-fold amplification, or 5-fold to 25-fold amplification, or 5-fold to 20-fold amplification, or 5-fold to 15-fold amplification of preactivated PBMCs transduced with nucleic acids encoding LE. In some embodiments, the test is performed in the presence of PBMCs, e.g., at a 1:1 ratio of transduced cells to PBMCs (which may be, e.g., from matched donors), and in some embodiments, the test is performed in the absence of PBMCs. In some embodiments, the analysis of amplification of any of these tests is performed as described in WO 2019/055946. In some embodiments, the test may comprise other statistical tests and cut-offs, such as a P-value below 0.1, 0.05, or 0.01, wherein the test polypeptide or nucleic acid encoding the test polypeptide is required to meet one or two thresholds (i.e., fold amplification and statistical cut-off).

For any of the lymphoproliferative element tests provided herein, the number of test cells was compared to the number of control cells between day 7 and day 14, day 21, day 28, day 35, day 42, or day 60 after transduction. In some embodiments, the number of test and control cells can be determined by sequencing the DNA and counting the identifiers present in each construct. In some embodiments, the number of test and control cells can be counted directly, for example, with a cytometer or a cytometer. In some embodiments, all test cells and control cells can be grown in the same container, well or flask. In some embodiments, the test cells may be seeded in one or more wells, flasks, or containers, and the control cells may be seeded in one or more flasks or containers. In some embodiments, test and control cells may be individually seeded into wells or flasks, e.g., one cell per well. In some embodiments, the enrichment level can be used to compare the number of test cells to a control cell. In some embodiments, the level of enrichment of the test or control construct can be calculated by dividing the number of cells at a later time point (day 14, day 21, day 28, day 35, or day 45) by the number of cells on day 7 for each construct. In some embodiments, the cells can be isolated by administering the cell antigen to the non-transduced cells at one time point (day 14, day 21, day 28 Day 35 or day 45) divided by the number of cells at the time point to calculate the level of enrichment of the test or control construct. In some embodiments, the enrichment level of each test construct can be normalized to the enrichment level of the respective control construct to produce a normalized enrichment level. In some embodiments, the LE encoded in the test construct provides (or a cell genetically modified and/or transduced with a retroviral particle (e.g., lentiviral particle) having a genome encoding LE is capable of providing, suitable for, has the following characteristics and/or is modified for) at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, or 1.5-fold to 25-fold normalized enrichment level, or 3-fold to 20-fold normalized enrichment level, or 5-fold to 25-fold normalized enrichment level, or 5-fold to 20-fold normalized enrichment level, or 5-fold to 15-fold normalized enrichment level). Enrichment can be measured, for example, by direct cell counting. The cut-off value may be based on a single test, or two, three, four or five repetitions, or on a number of repetitions. The cut-off value may be met when the lymphoproliferative element meets one or more repeated tests, or meets or exceeds a cut-off value for all repetitions. In some embodiments, the enrichment is measured as log ₂ ((normalized count data on test day+1)/(normalized count data on day 7+1)).

Additional details regarding the test for identifying LE are described in WO2019/055946, including experimental conditions.

As illustrated in WO2019/055946, the test construct was identified as CLE because CLE induced proliferation/expansion in these fed or not fed cultures without the need to add cytokines, such as IL-2, between day 7 and day 21, day 28, day 35 and/or day 42. For example, as illustrated in WO2019/055946, between day 7 and 21, 28, 35 and/or 42 post transduction compared to a control construct that does not include any intracellular domains, effective CLEs are identified by identifying test CLEs that provide increased amplification of these in vitro cultures, whether fed or not fed with non-transduced PBMCs. WO2019/055946 discloses at least one and often more than one test CLE comprising an intracellular domain from a test gene providing more amplification than each control construct present on day 7 post transduction that does not comprise an intracellular domain. WO2019/055946 also provides statistical methods for identifying particularly effective genes with respect to the first intracellular domain and one or more exemplary intracellular domains from these genes. The method uses the Mannheim-Wilkek Kessen test with a wig appearance cutoff of less than 0.1 or less than 0.05. WO2019/055946 identifies particularly potent genes of the first or second intracellular domain, for example by analyzing the scores of genes calculated as combined scores of all constructs with the genes. Such assays can use a cutoff value of greater than 1, or greater than a negative control construct without any intracellular domains, or greater than 2, as demonstrated by some of the tests disclosed in WO 2019/055946.

In another embodiment, the LE provides, is capable of providing and/or has the following characteristics (or a cell modified and/or transduced with the LE gene is capable of providing, is suitable for, possesses, and/or is modified for use in): in vivo driving T cell expansion. For example, in vivo testing may utilize a mouse model and T cell expansion is measured in vivo on days 15 to 25, or on days 19 to 21, or about day 21, after T cells are contacted with a lentiviral vector encoding LE introduced into the mouse, as disclosed in WO 2019/055946.

In exemplary aspects and embodiments including LEs (which generally include CARs), the genetically modified cells are modified so as to have new properties that the cells do not have in advance prior to genetic modification and/or transduction, as provided herein, and uses thereof. Such properties may be provided by genetic modification using nucleic acids encoding either CAR or LE (and in an illustrative embodiment, both CAR and LE). For example, in certain embodiments, the genetically modified and/or transduced cells are capable of, suitable for, have the following characteristics and/or are modified for use in: survival and/or proliferation in an in vitro culture of at least 7, 14, 21, 28, 35, 42, or 60 days or 7 th to 14, 21, 28, 35, 42, or 60 days after transduction in the absence of added IL-2 or in the absence of added cytokines such as IL-2, IL-15, or IL-7 and in certain illustrative embodiments in the presence of antigen recognized by the CAR, wherein the method comprises modification with retroviral particles having pseudotyped elements and optionally separate or fused activation domains on the surface and generally without prior activation.

In certain embodiments, being able to enhance survival and/or proliferation refers to a cell that is genetically modified and/or transduced to exhibit, be able to, be suitable for, have the following characteristics and/or be modified for use: improved survival or expansion in culture in vitro or in vitro in the absence of one or more added cytokines (e.g., IL-2, IL-15, or IL-7) or added lymphomitogens, as compared to control cells (which are identical to genetically modified and/or transduced cells prior to genetic modification and/or transduction) or control cells transduced with the same retroviral particles in the test (which comprise LE or a putative LE, but do not comprise an intracellular domain of LE or LE), wherein said survival or proliferation of said control cells is facilitated by the addition of said one or more cytokines (e.g., IL-2, IL-15, or IL-7) or said lymphomitogens to the culture medium. By added cytokine or lymphocyte mitogen is meant that the cytokine or lymphocyte mitogen is added from an external source to the medium such that during culturing of the cells, the concentration of the cytokine or lymphocyte mitogen is increased in the medium as compared to the initial medium, and in some embodiments, there may be no initial medium prior to the addition. "adding" or "exogenously adding" refers to adding such cytokines or lymphocyte mitogens to a lymphocyte medium for culturing modified, genetically modified and/or transduced cells after modification, wherein the medium may or may not have cytokines or lymphocyte mitogens. In the absence of exogenously added cytokines or lymphocyte mitogens, all or part of the medium, including a mixture of various medium components, is typically stored and, in an illustrative embodiment, transported to the site where the culturing takes place. In some embodiments, the lymphocyte medium is purchased from a vendor and a user (e.g., a technician) not employed by the vendor and not within the vendor's facility adds exogenously added cytokines or lymphocyte mitogens to the lymphocyte medium and then cultures the genetically modified and/or transduced cells in the presence or absence of such exogenously added cytokines or lymphocyte mitogens.

In some embodiments, improved or enhanced survival, amplification and/or proliferation may be demonstrated by an increase in the number of cells determined by sequencing DNA from cells transduced with retroviral particles (e.g., lentiviral particles) having a genome encoding a CLE and counting the occurrence of sequences present in the unique identifier of each CLE. In some embodiments, increased survival and/or increased expansion may be determined by counting cells directly with a cytometer or a cell counter at each time point. In some embodiments, improved survival and/or improved expansion and/or enrichment can be calculated by dividing the number of cells at a later time point (day 21, day 28, day 35, and/or day 45) by the number of cells on day 7 for each construct. In some embodiments, the cells may be counted by a cytometer or a cell counter. In some embodiments, the level of enrichment determined using the nucleic acid count or cell count of each particular test construct can be normalized to the level of enrichment of the respective control construct (i.e., a construct having the same extracellular domain and transmembrane domain but lacking the intracellular domain present in the test construct). In these embodiments, the LE encoded in the test construct provides (or cells genetically modified and/or transduced with a retroviral particle (e.g., lentiviral particle) having a genome encoding LE can provide, be suitable for, have the following characteristics and/or be modified for) at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold standardized enrichment level, or 1.5-fold to 25-fold standardized enrichment level, or 3-fold to 20-fold standardized enrichment level, or 5-fold to 25-fold standardized enrichment level, or 5-fold to 20-fold standardized enrichment level, or 5-fold to 15-fold standardized enrichment level).

In some embodiments, the lymphoproliferative element may comprise a cytokine receptor or fragment comprising a signaling domain thereof. In some embodiments, the cytokine receptor may be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL RA, IL2RB, IL2RG, IL3RA, IL4R, IL RA, IL6R, IL6ST, IL7R, IL RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL13R, IL RA1, IL13RA2, IL15R, IL RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23 f R, IL27 f R, IL RA, IL31 β R, TGF β decoy, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14 or TNFRSF18.

In some embodiments, the lymphoproliferative element comprising CLE comprises an intracellular activation domain as disclosed above. In some illustrative embodiments, the lymphoproliferative element is a CLE comprising an intracellular activation domain comprising a domain comprising ITAM, thus, the CLE may comprise an intracellular activation domain having at least 80%, 90%, 95%, 98% or 100% sequence identity to a CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP, FCERlG, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70 domain provided herein, wherein the CLE does not comprise astm.

In some embodiments, one or more domains of the lymphoproliferative element are fused to a regulatory domain (e.g., a costimulatory domain) and/or an intracellular activation domain of the CAR. In some embodiments of the compositions and method aspects for transducing lymphocytes in whole blood, one or more intracellular domains of the lymphoproliferative element can be part of the same polypeptide as the CAR or can be fused and optionally functionally linked to some component of the CAR. In other embodiments, the engineered signaling polypeptide may include an ASTR, an intracellular activation domain (e.g., a CD3 zeta signaling domain), a costimulatory domain, and a lymphoproliferative domain. Additional details regarding co-stimulatory domains, intracellular activation domains, ASTR, and other CAR domains are disclosed elsewhere herein.

Lymphoproliferative elements provided herein generally include a transmembrane domain. For example, the transmembrane domain may have 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any of the transmembrane domains from the following genes and representative sequences disclosed in WO 2019/055946: CD8 beta, CD4, CD3 zeta, CD28, CD134, CD7, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A CD8B, CD27, CD28, CD40, CD79A, CD79B, CRLF2, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA IL6R, IL6ST, IL7RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23R, IL RA, IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, and TNFRSF18 or mutants thereof, they are known to promote signaling activity in certain cell types in these mutants. TM domains suitable for use in any engineered signaling polypeptide include, but are not limited to, constitutively active cytokine receptors, TM domains from LMP1, and TM domains from TM proteins of type 1 comprising dimerization motifs, as discussed in more detail herein. In any aspect disclosed herein that contains a transmembrane domain from a type I transmembrane protein, the transmembrane domain may be a type I growth factor receptor, hormone receptor, T cell receptor, or TNF family receptor.

In some embodiments, the CLE comprises an extracellular portion and a transmembrane portion from the same protein (in the illustrative embodiments, the same receptor), either of which is a mutant in the illustrative embodiments, thus forming an extracellular domain and a transmembrane domain. These domains may be from cytokine receptors or mutants thereof, or hormone receptors or mutants thereof, which in some embodiments are reported to be constitutively active when expressed in at least some cell types. In illustrative embodiments, such extracellular domains and transmembrane domains do not include a ligand binding region. It is believed that such domains do not bind to ligands when present in CLE and expressed in B cells, T cells and/or NK cells. Mutations in these receptor mutants may occur in the transmembrane region or in the extracellular juxtamembrane region. Without being bound by theory, mutations in at least some of the extracellular-transmembrane domains of the CLEs provided herein are responsible for signaling of the CLE in the absence of the ligand by bringing together the activation chains that are not normally together or by altering the confirmation of the linked transmembrane and/or intracellular domains.

Exemplary extracellular and transmembrane domains of CLEs comprising embodiments of these domains (in the illustrative embodiment, the extracellular domains) are typically less than the 30 amino acids from the membrane proximal extracellular domain together with the transmembrane domain of a mutant receptor reported to be constitutive, which does not require ligand binding for activation of the relevant intracellular domain. In an illustrative embodiment, these extracellular and transmembrane domains include IL7RA Ins PPCL, CRLF 2F 232C, CSF RB V449E, CSF R T N, EPOR L251C I C, GHR E260C I270C, IL RA F523C, and MPL S505N. In some embodiments, the extracellular domain and the transmembrane domain do not comprise more than 10, 20, 25, 30, or 50 constitutive amino acids in sequence that are identical to the extracellular domain and/or the portion of the transmembrane domain of IL7RA or a mutant thereof. In some embodiments, the extracellular domain and transmembrane domain are not IL7RA Ins PPCL. In some embodiments, the extracellular domain and the transmembrane domain do not comprise more than 10, 20, 25, 30, or 50 constitutive amino acids in sequence that are identical to portions of the extracellular domain and/or the transmembrane domain of IL 15R.

In embodiments in which the transmembrane domain is any of these aspects of a type I transmembrane protein, the transmembrane domain may be a type I growth factor receptor, hormone receptor, T cell receptor or TNF family receptor. In embodiments of any of the aspects and embodiments wherein the chimeric polypeptide comprises an extracellular domain and wherein the extracellular domain comprises a dimerization motif, the transmembrane domain may be a type I cytokine receptor, a hormone receptor, a T cell receptor, or a TNF family receptor.

In some embodiments, the ectodomain and transmembrane domain are viral protein LMP1 or mutants and/or fragments thereof. LMP1 is a multi-transmembrane protein known to activate cell signaling independent of ligand either when targeting lipid rafts or when fusing to CD40 (Kaykas et al, journal of european molecular biology, 20:2641 (2001)). Fragments of LMP1 are typically long enough to span the plasma membrane and activate the attached intracellular domains. For example, LMP1 may be between 15 and 386 amino acids, between 15 and 200 amino acids, between 15 and 150 amino acids, between 15 and 100 amino acids, between 18 and 50 amino acids, between 18 and 30 amino acids, between 20 and 200 amino acids, between 20 and 150 amino acids, between 20 and 50 amino acids, between 20 and 30 amino acids, between 20 and 100 amino acids, between 20 and 40 amino acids, or between 20 and 25 amino acids. Mutants and/or fragments of LMP1 retain their ability to activate intracellular domains when included in the CLE provided herein. Furthermore, if present, the extracellular domain comprises at least 1, but typically at least 4 amino acids and it is typically linked to another functional polypeptide, such as a gap domain, e.g. eTag. In some embodiments, the lymphoproliferative element comprises an LMP1 transmembrane domain. In an illustrative embodiment, the lymphoproliferative element comprises an LMP1 transmembrane domain and one or more intracellular domains do not comprise an intracellular domain from: TNFRSF proteins (i.e., CD40, 4-IBB, RANK, TACI, OX, CD27, GITR, LTR, and BAFFR), TLR 1-TLR 13, integrin, fcyRIII, dectinl, dectin2, NOD1, NOD2, CD16, IL-2R, I type II interferon receptor, chemokine receptor (e.g., CCR5 and CCR 7), G protein-coupled receptor, TREM1, CD79A, CD79B, ig- α, IPS-1, myD88, RIG-1, MDA5, CD3Z, myD88 Δ TIR, TRIF, TRAM, TIRAP, MAL, BTK, RTK, RAC1, SYK, NALP3 (NLRP 3), NALP3 Δlrr, NALP1, CARD9, DAI, IPAG, STING, zap70, or LAT.

In other embodiments of the CLE provided herein, the extracellular domain comprises a dimerization moiety. Many different dimerization moieties disclosed herein may be used in these embodiments. In an illustrative embodiment, the dimerizing moiety is capable of homodimerization. Without being bound by theory, the dimerization moiety may provide an activation function to an intracellular domain connected thereto via a transmembrane domain. In some embodiments, the dimerizing moiety of a CLE may be an anti-idiotype extracellular recognition domain of any of the anti-idiotype polypeptides herein. Thus, an anti-idiotype polypeptide comprising an anti-idiotype extracellular domain may be a CLE. For example, extracellular recognition domains attached to such CLEs may dimerize upon binding of a target antibody or antibody mimetic, as disclosed elsewhere herein. In other words, in some embodiments, the CLE is part of a fusion polypeptide comprising an anti-idiotype polypeptide, and the fusion polypeptide dimerizes by binding of a target antibody or antibody mimetic to an anti-idiotype extracellular recognition domain. In an illustrative embodiment, CLE is not constitutively active, but is activated after dimerization induced by binding of the target antibody to 2 anti-idiotype polypeptides that bind to the idiotype of the target antibody. In these embodiments, the target antibody generally does not induce cytotoxicity. In some embodiments, the lymphoproliferative elements provided herein comprise an extracellular domain and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In an illustrative embodiment of this aspect, the extracellular domain comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is the c-jun polypeptide region of ECD-11.

The extracellular domain with the dimerizing moiety may also serve the function of linking a cell tag polypeptide (e.g., an anti-idiotype extracellular recognition domain of an anti-idiotype polypeptide) to a cell expressing CLE. Thus, in such embodiments, the dimerization motif may function to link the anti-idiotype extracellular recognition domain to the handle of the membrane association domain, which is typically a transmembrane domain in LE and CLE. Such embodiments provide the advantage of having a transmembrane domain and a dimerization motif that anchor the anti-idiotype domain to the cell while retaining their function in LE. In some embodiments, such embodiments provide the following advantages: constitutive dimerization and inducible dimerization by LE dimerization motifs provides tetramerization for intracellular domains that form tetramers upon binding of target antibodies having an idiotype recognized by an anti-idiotype extracellular recognition domain. In these embodiments, the target antibody is generally not cytotoxic, but rather is used to tetramerize dimeric ICD. These may be used in some of the apoptosis-inducing embodiments as described in other sections herein.

In some embodiments, the dimerizing agent may be located intracellular rather than extracellular. In some embodiments, more than one or more dimerization domains may be used. In any aspect or embodiment wherein the extracellular domain of CLE comprises a dimerization motif, the dimerization motif may be selected from the group consisting of: leucine zipper motif-containing polypeptides, CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271 and CD324, and mutants and/or active fragments thereof that retain dimerization capacity. In any aspect or embodiment herein wherein the extracellular domain of the CLE comprises a dimerization motif, the dimerization motif may require a dimerization agent, and the dimerization motif and associated dimerization agent may be selected from the group consisting of: FKBP and rapamycin (rapamycin) or an analogue thereof, gyrB and coumaramycin (coumermycin) or an analogue thereof, DHFR and methotrexate or an analogue thereof, or DmrB and AP20187 or an analogue thereof, and mutants and/or active fragments of said dimerized proteins that retain dimerization ability. In some aspects and illustrative embodiments, the lymphoproliferative element is constitutively active and is not a lymphoproliferative element requiring a dimerizer for activation.

The internal dimerization and/or multimerization lymphoproliferative element in one embodiment is an integral part of a system using an analogue of the lipid permeable dimeric immunosuppressant drug FK506, which loses its normal biological activity while gaining the ability to crosslink molecules genetically fused to the FK506 binding protein FKBP 12. By fusing one or more FKBP and myristoylation sequences to the cytoplasmic signaling domain of the target receptor, signaling can be stimulated in a dimeric drug dependent but ligand and extracellular domain independent manner. This provides time control for the system, reversibility of use of monomeric drug analogs, and enhanced specificity. The high affinity of the third generation AP20187/AP1903 dimer drug for its binding domain FKBP12 allows specific activation of recombinant receptors in vivo without inducing non-specific side effects via endogenous FKBP 12. FKBP12 variants (e.g., FKBP12V 36) having amino acid substitutions and deletions which bind to dimeric drugs may also be used. Furthermore, synthetic ligands are resistant to proteolytic cleavage, making them more effective at activating receptors in vivo than most delivered protein agents.

The extracellular domain of the embodiment in which the extracellular domain has a dimerization motif is sufficiently long to form dimers, such as leucine zipper dimers. Thus, the extracellular domain comprising the dimerization moiety may be 15 amino acids to 100 amino acids, 20 amino acids to 50 amino acids, 30 amino acids to 45 amino acids, or 35 amino acids to 40 amino acids, in the illustrative example the c-Jun portion of the c-Jun extracellular domain. The extracellular domain of the polypeptide comprising the dimerizing moiety may not retain other functionality. For example, for leucine zipper dimers, these leucine zippers are capable of forming dimers, as they retain motifs of leucine separated by 7 residues along α. However, the leucine zipper portion of certain embodiments of CLEs provided herein may or may not retain their DNA binding function.

A spacer between 1 alanine residue and 4 alanine residues can be included in the CLE between the extracellular domain with the dimerization moiety and the transmembrane domain. Without being bound by theory, it is believed that the alanine spacer affects signaling of the intracellular domain linked to the leucine zipper extracellular region via the transmembrane domain by altering the orientation of the intracellular domain.

In an illustrative embodiment, the CLE comprises a cell tag domain. Details regarding cell tags are provided in other sections herein. Any of the cell tags provided herein may be part of a CLE. Typically, the cell tag is attached to the N-terminus of the extracellular domain. Without being bound by theory, in some embodiments, the extracellular domain includes a function that provides a linker (in the illustrative embodiment, a flexible linker) to attach the cell tag domain to a cell expressing CLE.

In addition, polynucleotides comprising a nucleic acid sequence encoding a CLE provided herein typically also comprise a signal sequence to directly express the plasma membrane. Exemplary signal sequences are generally provided herein in other sections. In certain embodiments, a component can be provided on a transcript such that both CAR and CLE are expressed from the same transcript.

Binding and fusogenic elements

Many of the methods, compositions, and kits provided herein include a RIP having an envelope protein on its surface, such as multiple copies of a T cell and/or NK cell binding polypeptide and multiple copies of a fusogenic polypeptide (also known as a fusogenic). "binding polypeptides" include one or more polypeptides, typically glycoproteins, that recognize and bind to a target host cell. The "fusogenic polypeptide" mediates fusion of the retroviral and target host cell membranes, thereby allowing entry of the retroviral genome into the target host cell. In certain embodiments, the binding polypeptide and the fusogenic polypeptide are located on the same envelope protein, e.g., a heterologous glycoprotein. In other embodiments, the binding polypeptide and the fusogenic polypeptide are located on two or more different heterologous glycoproteins.

One or both of these binding and fusogenic polypeptide functions may be provided by a pseudotyped element. In some embodiments, the pseudotyped element can be one or more viral envelope proteins. In some embodiments, the binding polypeptide function of the viral envelope protein may be altered, reduced, or eliminated (e.g., amino acids corresponding to the binding polypeptide function may be mutated or deleted). In some embodiments, viral envelope proteins with reduced or eliminated binding polypeptide function may be re-targeted with new binding polypeptide function or by mutating the original binding polypeptide function.

In some embodiments, the binding polypeptide function may be provided by any polypeptide that binds to a cell surface marker on the target cell. In some embodiments, the binding polypeptide function may be provided by an activating element, as disclosed elsewhere herein. Pseudotyping of replication-defective recombinant retroviral particles with heterologous envelope glycoproteins typically alters viral tropism and facilitates transduction of host cells. In some embodiments provided herein, the pseudotyped element is provided as a polypeptide/protein, or as a nucleic acid sequence encoding a polypeptide/protein.

In some embodiments, the pseudotyped element comprises envelope proteins from different viruses. In some embodiments, the pseudotyped element is a feline endogenous virus (RD 114) envelope protein, a tumor retrovirus amphotropic envelope protein, a vesicular stomatitis virus envelope protein (VSV-G) (SEQ ID NO: 336), a baboon retrovirus envelope glycoprotein (BaEV) (SEQ ID NO: 337), a murine leukemia envelope protein (MuLV) (SEQ ID NO: 338), an influenza glycoprotein HA surface glycoprotein (HA), an influenza glycoprotein Neuraminidase (NA), a paramyxovirus measles envelope protein H, a paramyxovirus measles envelope protein F, a tree shrew paramyxovirus (TPMV) envelope protein H, TPMV envelope protein F, glycoproteins G and F from the genus Hennopavirus, a NiV envelope protein F, niV envelope protein G, a Sindbis virus (SINV) protein E1, a SINV protein E2, and/or functional variants or fragments of any of these envelope proteins (see, e.g., ank and Bucholz molecular therapy, clinical methods Mol Ther Methods Clin-2016:17, 17-17.17, 17-17.8).

In some embodiments, the pseudotyped element may be a wild-type BaEV. Without being bound by theory, baEV contains R peptide that was demonstrated to inhibit transduction. In some embodiments, baEV may contain a deletion of the R peptide. In some embodiments, after the nucleotide encodes the amino acid sequence HA (referred to herein as BaEV ΔR (HA)) (SEQ ID NO: 339), the BaEV may contain a deletion of the inhibitory R peptide. In some embodiments, after the nucleotide encodes the amino acid sequence HAM (referred to herein as BaEV ΔR (HAM)) (SEQ ID NO: 340), baEV may contain a deletion of the inhibitory R peptide.

In some embodiments, the pseudotyped element can be wild-type MuLV. In some embodiments, muLV may contain one or more mutations to remove furin-mediated cleavage sites located between the Transmembrane (TM) and Surface (SU) subunits of the envelope glycoprotein. In some embodiments, muLV contains a SUx mutation (MuLVSUx) (SEQ ID NO: 372) that inhibits furin-mediated cleavage of the MuLV envelope protein in packaging cells. In certain embodiments, the C-terminus of the cytoplasmic tail of the MuLV or MuLVSUx protein is truncated by 4 to 31 amino acids. In certain embodiments, the C-terminus of the cytoplasmic tail of the MuLV or MuLVSUx protein is truncated by 4, 8, 12, 16, 20, 24, 28, or 31 amino acids.

In some embodiments, the pseudotyped element comprises a binding polypeptide and a fusogenic polypeptide derived from a different protein. In one aspect, the pseudotyped element can include the influenza proteins hemagglutinin HA and/or Neuraminidase (NA). In certain embodiments, the HA is from influenza a subtype H1N1. In an illustrative embodiment, the HA is from H1N1 PR8 1934, in which the trypsin-monosultap-dependent cleavage site HAs been mutated to a more promiscuous multiplex sequence (SEQ ID NO: 311). In certain embodiments, the NA is from influenza a virus subtype H10N 7. In an illustrative embodiment, NA is from H10N7-HKWF446C-07 (SEQ ID NO: 312). In some embodiments, the binding polypeptide may be a functional variant or fragment of VSV-G, baEV, baEV Δr (HA), baEV Δr (HAM), muLV, muLVSUx, influenza HA, influenza NA, or measles envelope protein H that retains the ability to bind to a target cell, and the fusogenic polypeptide may be a functional variant or fragment of VSV-G, baEV, baEV Δr (HA), baEV Δr (HAM), muLV, muLVSUx, influenza HA, influenza NA, or measles envelope protein F that retains the ability to mediate fusion of a retrovirus and a target host cell membrane.

In another aspect, replication-defective recombinant retroviral particles in the methods and compositions disclosed herein can be pseudotyped by fusion (F) and/or lectin (H) polypeptides of Measles Virus (MV), clinical wild-type strains of MV, and vaccine strains including Edmonston strain (MV-Edm) (GenBank; AF 266288.2) or fragments thereof, as non-limiting examples. Without being bound by theory, it is believed that both the lectin (H) and fusion (F) polypeptides may play a role in entry into host cells, wherein the H protein binds MV to the receptors CD46, SLAM and Nectin-4 on target cells, and F mediates fusion of the retroviral and host cell membranes. In an illustrative embodiment, particularly where the target cell is a T cell and/or NK cell, the binding polypeptide is a measles virus H polypeptide and the fusion polypeptide is a measles virus F polypeptide.

In some studies, lentiviral particles pseudotyped with truncated F and H polypeptides have a significant increase in titer and transduction efficiency (Funke et al, 2008. Molecular therapy 16 (8): 1427-1436), (Frecha et al, 2008. Blood 112 (13): 4843-4852). The highest titers were obtained when the F cytoplasmic tail was truncated by 30 residues, also known as MV (Ed) -FΔ30 (SEQ ID NO: 313). For H variants, optimal truncations occur when 18 or 19 residues (MV (Ed) -H2 18 (SEQ ID NO: 314) or (MV (Ed) -H2 19)) are deleted, but truncated variants with 24 residues also give optimal titers with and without alanine substitutions of the deleted residues (MV (Ed) -H2 24 (SEQ ID NO: 315) and MV (Ed) -H2+A). In some embodiments, including those directed against transduced T cells and/or NK cells, replication defective recombinant retroviral particles in the methods and compositions disclosed herein are pseudotyped with mutant or variant versions of measles virus fusion (F) polypeptide and lectin (H) polypeptide (in the illustrative examples, cytoplasmic domain deleted variants of measles virus F and H polypeptides). In some embodiments, the mutated F and H polypeptides are "truncated H" or "truncated F" polypeptides, the cytoplasmic portion of which has been truncated, i.e., the amino acid residues (or the nucleic acid encoding the corresponding nucleic acid molecule encoding the protein) have been deleted. "hΔy" and "fΔx" represent such truncated H and F polypeptides, respectively, wherein "Y" refers to 1 to 34 residues that have been deleted from the amino terminus, and "X" refers to 1 to 35 residues that have been deleted from the carboxy terminus of the cytoplasmic domain. In another embodiment, the "truncated F polypeptide" is fΔ24 or fΔ30 and/or the "truncated H protein" is selected from the group consisting of: hΔ14, hΔ15, hΔ16, hΔ17, hΔ18, hΔ19, hΔ20, hΔ21+a, hΔ24, and hΔ24+4a, and more preferably, hΔ18 or hΔ24. In an illustrative embodiment, the truncated F polypeptide is MV (Ed) -fΔ30, and the truncated H polypeptide is MV (Ed) -hΔ18.

In some embodiments, the pseudotyped element may be an envelope protein from the henipavirus genus (e.g., nipah virus, hendra virus, pine bay virus, meyer's patch virus, or keamax virus) and includes envelope glycoprotein G (henipav-G protein) and their fusion partner envelope glycoprotein F (henipav-F protein). In some embodiments, the henipav-F protein comprises the sequence of SEQ ID No. 374 and the henipav-G protein comprises the sequence of SEQ ID No. 375. In some embodiments, the henipav virus-F protein comprises a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID No. 374. In some embodiments, the henipav virus-G protein comprises a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of SEQ ID No. 375.

In some embodiments, the henipav-G protein may contain one or more mutations to modify (e.g., truncate) the cytoplasmic tail and thereby improve pseudotyping and particle incorporation efficiency (Palomares et al 2013 journal of virology 87 (8): 4794-4794; witting et al 2013: gene therapy 20 (10): 997-1005; bender et al 2016.Plos Pathog.12 (6): e 1005641). In certain embodiments, the N-terminus of the cytoplasmic tail of any henipav-G protein may be truncated by 1 amino acid to all of its amino acids. In some embodiments, residues of henipav-G protein involved in receptor binding are mutated to alter, and in illustrative embodiments remove, their natural interactions with their natural receptors. In certain embodiments, the henipav-G protein is mutated (giving amino acids for Nipah-G, also known as NiV-G) at one or more of Y389, E501, W504, E505, V507, Q530, E533, or I588 of SEQ ID No. 375, for example, and the skilled artisan will be able to identify the corresponding glutamine of other henipav-G proteins (Guillaume et al 2006 journal of virology 80 (15): 7546-7554; negrete et al 2007 journal of virology 81 (19): 10804-10814; xu et al, national academy of sciences of america 2008; 105 (29): 9953-9958; xu et al 2012, public library complex volume of sciences; 7 (11E 48742): bender et al 2016.Plos Pathog.12 (6)). In some embodiments, the henipav virus-G protein is SEQ ID No. 375 having mutations E533A and/or Q530A. In some embodiments, one or more N-glycosylation sites or O-glycosylation sites are mutated to improve pseudotyping and fusion (Biering et al 2012 J.Virol.86 (22): 11991-12002; stone et al 2016.Plos Pathog.12 (2): e 1005445). In some embodiments, one or more N-glycosylation sites are mutated to another amino acid, such as glutamine, for example, but not limited to, at one or more of N72, N159, N306, N378, N417, N481, or N529 of SEQ ID NO:375, or at the corresponding glutamine of other Huntington's virus-G protein. In some embodiments, one or more O-glycosylation sites are mutated from serine or threonine to another amino acid, such as alanine. In some embodiments, one or more serine or threonine residues in the highly O-glycosylated stem domain of amino acids 103 to 137 from SEQ ID NO. 375 are mutated to, for example, alanine. In other embodiments, the C-terminus of the henipav virus-G protein may be modified and fused to a binding polypeptide and in illustrative embodiments an activating element, such as an antibody or antibody mimetic, which in illustrative embodiments may be an anti-CD 3 antibody or antibody mimetic (Bender et al 2016.Plos Pathog.12 (6): e1005641; jamali et al 2019 methods of molecular therapy and clinical development (Mol. Ter-Meth Clin D.)) 13:371-379; frank et al 2020 Blood progression (Blood adv.)) 4 (22): 5702-5715).

In some embodiments, the F protein may contain one or more mutations to modify (e.g., truncate) the cytoplasmic tail, and thus improve pseudotyping, particle incorporation efficiency, and/or cleavage of the F protein from inactive F0 to cleavage active F1 form (Khetawat et al 2010 journal of virology 7:312; palomales et al 2013 journal of virology 87 (8): 4794-4794; witting et al 2013: gene therapy 20 (10): 997-1005; bender et al 2016.Plos Pathog.12 (6): e1005641; johnston et al 2017 journal of virology 91 (10): e 02150-16). In some embodiments, one or more N-glycosylation sites are mutated to another amino acid, such as glutamine, for example, but not limited to, at one or more of N64, N67, N99, N414, or N464. In certain embodiments, the C-terminus of the cytoplasmic tail of envelope glycoprotein F from henipavirus (henipavirus-F protein) is truncated 1 to all its amino acids. In some embodiments, the F protein may contain one or more mutations to make it more fusogenic (Aguilar et al 2007 J.Virol.81 (9): 4520-4532; weis et al 2015J Cell biol.) 94 (7-9): 316-322).

In some embodiments, the pseudotyped element may include henipav-F protein and henipav-G protein (i.e., homologous proteins) from an homologous virus of the henipav genus. In some embodiments, the pseudotyped element may include henipav-F protein and henipav-G protein (i.e., heterologous proteins) from different viruses of the henipav genus. In some embodiments, the pseudotyped element may comprise henipav virus-F protein, and the henipav virus-G protein may be a chimera consisting of domains of heterologous proteins (Bradel-trethesway et al 2019 journal of virology 93 (13): e 00577-19).

In some embodiments, any pseudotyped element can comprise one or more mutations to modify (e.g., truncate) the cytoplasmic tail, and thus improve pseudotyping and particle incorporation efficiency. In certain embodiments, the N-terminus of the cytoplasmic tail is truncated 1 to all its amino acids. In some embodiments, residues involved in receptor binding are mutated to alter, and in illustrative embodiments remove, their natural interactions with their natural receptors. Similar to the mutation of the Nipah-G protein, in some embodiments, the VSV-G protein is mutated, for example, but not limited to, in residues K47 or R354, such as K47A or K47Q and/or R354A or R354Q. In some embodiments, these pseudotyped elements are fused to a heterologous binding polypeptide that serves to direct or redirect the pseudotyped element to a new target protein on the same or a different cellular target.

In some embodiments, the isolated binding and/or fusogenic polypeptide comprises one or more non-virally derived proteins. In some embodiments, the binding polypeptide comprises an antibody, ligand, or receptor that binds to a polypeptide on a target cell. In some embodiments, the binding polypeptide comprises an alternative non-antibody scaffold (also referred to herein as an antibody mimetic). In any aspect or embodiment provided herein comprising a binding polypeptide, the binding polypeptide may be an antibody mimetic. In any aspect or embodiment provided herein that comprises a binding polypeptide as an antibody, a suitable antibody mimetic may be used in place of the antibody. In some embodiments, the antibody mimetic can be an affibody, avimer, affidine, alpha body, alphamab, anti-carrier protein, peptide aptamer, armadillo repeat protein, trimer, affimer (also known as avidity multimer), C-type lectin domain, cysteine knot microgrotein, cyclic peptide, cytotoxic T lymphocyte-associated protein-4, DARPin (designed ankyrin repeat protein), fibrinogen domain, fibronectin binding domain (FN 3 domain) (e.g., an attachment protein or monoclonal antibody), fynomer, kink bacteria, kunitz domain peptide, nanofilin, leucine-rich repeat domain, lipocalin domain, mAb 2, or Fcab ^TM Nanobodies, nanomembers, OBody, pronectin, single chain TCRs, triangular tetrapeptide repeat domains, VHH or V-like domains. In some embodiments, the binding polypeptide recognizes a protein on the surface of NK cells such as CD16, CD56, and CD 57. In some embodiments, the binding polypeptide recognizes a protein on the surface of a T cell, such as CD3, CD4, CD8, CD25, CD28, CD62L, CCR7, TCRa, and TCRb. In some embodiments, the binding polypeptide is also an activating element. In some embodiments, the binding polypeptide is a membrane polypeptide that binds CD 3. In some embodiments, the fusion source is derived from sindbis virus glycoprotein SV1 modified to remove its binding activity, and the binding polypeptide is a membrane-bound anti-CD 3 antibody (Yang et al 2009, pharmaceutical research (Pharm Res) 26 (6): 1432-1445).

In some embodiments, the viral particles are co-pseudotyped with envelope glycoproteins from 2 or more heterologous viruses. In some embodiments, the viral particles are co-pseudotyped with VSV-G or a functional variant or fragment thereof, and envelope proteins from RD114, baEV, muLV, influenza virus, measles virus, and/or a functional variant or fragment thereof. In some embodiments, the viral particles are co-pseudotyped with VSV-G and MV (Ed) -H glycoprotein or MV (Ed) -H glycoprotein and truncated cytoplasmic domain. In an illustrative embodiment, viral particles are co-pseudotyped with VSV-G and MV (Ed) -H2. In certain embodiments, VSV-G is co-pseudotyped by MuLV or MuLV with a truncated cytoplasmic domain. In other embodiments, VSV-G is co-pseudotyped by MuLVSUx or MuLVSUx with a truncated cytoplasmic domain. In other illustrative embodiments, VSV-G is co-pseudotyped with a fusion of an anti-CD 3scFv with MuLV.

In some embodiments, the fusogenic polypeptide is derived from a class I fusion partner. In some embodiments, the fusogenic polypeptide is derived from a class II fusion partner. In some embodiments, the binding polypeptide and the isolated fusogenic polypeptide are both virus-derived. In some embodiments, the fusion polypeptide includes multiple elements that are expressed as one polypeptide. In some embodiments, the binding polypeptide and fusion polypeptide are translated from the same transcript but from separate ribosome binding sites; in other embodiments, the binding polypeptides and fusion polypeptides are separated by a cleavage peptide site (which is not bound by theory, is cleaved after translation, as is common in the literature) or a ribosome jump sequence. In some embodiments, translation of the binding polypeptide and fusion polypeptide from the self-isolated ribosome binding site produces a higher amount of fusion polypeptide than the binding polypeptide. In some embodiments, the ratio of fusion polypeptide to binding polypeptide is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1. In some embodiments, the ratio of fusion polypeptide to binding polypeptide is 1.5:1, 2:1, or 3:1 as the low end of the range to 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 as the high end of the range.

In embodiments disclosed herein that include short contact times, many modified lymphocytes in the cell preparation have pseudotyped elements on their surface by associating with replication defective recombinant retroviral particles or by fusing the retroviral envelope with the plasma membrane of the modified lymphocytes during reintroduction of the modified lymphocytes into the subject. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the modified lymphocytes in the cell preparation may comprise pseudotyped elements on their surface. In some embodiments, the pseudotyped element can be bound to the surface of the modified lymphocyte and/or the pseudotyped element can be present in the plasma membrane of the modified lymphocyte.

In some embodiments, the RIP herein includes means on its surface for binding T cells and/or NK cells. In some embodiments, the RIP herein includes means on its surface for mediating fusion of the RIP and T cell and/or NK cell membranes. In some embodiments, the RIP herein includes means on its surface for binding to T cells and/or NK cells, as well as means for mediating fusion of RIP and T cells and/or NK cell membranes. In some embodiments, this is the same tool.

Activating element

Many of the methods and compositions of the present disclosure comprising replication defective recombinant retroviral particles further comprise an activating element (also referred to herein as a T cell activating element), or a nucleic acid encoding an activating element. The activating element is an envelope protein of a replication defective recombinant retroviral particle. Cells of the immune system (e.g., T lymphocytes) recognize and interact with specific antigens via receptors or receptor complexes, which, when recognized or interacted with such antigens, activate the cells and expand in vivo. An example of such a receptor is the antigen-specific T lymphocyte receptor complex (TCR/CD 3) expressed on the surface of T lymphocytes. TCRs recognize antigenic peptides that are presented by proteins of the Major Histocompatibility Complex (MHC) on the surface of antigen presenting cells and other T lymphocyte targets. Stimulation of the TCR/CD3 complex results in activation of T lymphocytes and subsequent antigen-specific immune responses. Thus, the activating assemblies provided herein activate T cells by binding to one or more components of a T cell receptor-related complex, for example by binding to CD 3. In some embodiments, the activation elements may be activated individually. In other cases, activation requires activation via the TCR receptor complex in order to further activate the cell. T lymphocytes also require a secondary, co-stimulatory signal to become fully active in vivo. Without this signal, T lymphocytes do not respond to antigens that bind to the TCR, or become non-allergic. However, the second co-stimulatory signal is not necessary for transduction and expansion of the T cell, and may be provided by a later co-stimulatory signal from the CAR or LE, e.g., after transduction, as provided elsewhere herein. In some embodiments, the co-stimulatory signal may be provided during transduction by, for example, CD28 (a T lymphocyte protein), CD28 interacting with CD80 and CD86 on antigen producing cells.

Activation of the T Cell Receptor (TCR) CD3 complex and co-stimulation by CD28 can occur by ex vivo exposure to solid surfaces (e.g., beads) coated with anti-CD 3 and anti-CD 28. In some embodiments of the methods and compositions disclosed herein, resting T cells are activated by exposure to solid surfaces coated ex vivo with anti-CD 3 and anti-CD 28. In other embodiments, resting T cells or NK cells (in the illustrative embodiment, T cells) are activated by exposure to soluble anti-CD 3 antibodies (e.g., at 50ng/ml to 150ng/ml, or 75ng/ml to 125ng/ml, or 100 ng/ml). In such embodiments, which may be part of a method for modification, genetic modification, or transduction, in illustrative embodiments where no pre-activation is performed, such activation and/or contacting may be performed by including anti-CD 3 in the transduction reaction mixture and performing the contacting and optionally incubating for any time provided herein. Furthermore, such activation by soluble anti-CD 3 may be performed by incubating lymphocytes, such as PBMCs and in the illustrative embodiments NK cells and in the illustrative higher embodiments T cells, after contact with the retroviral particles in an anti-CD 3 containing medium. Such incubation may be, for example, for 5, 10, 15, 30, 45, 60 or 120 minutes as the low end of the range to 15, 30, 45, 60, 120, 180 or 240 minutes as the high end of the range, for example 15 minutes to 1 hour or 2 hours.

In certain illustrative embodiments of the methods, kits and compositions provided herein, e.g., methods, kits and compositions for modifying, genetically modifying and/or transducing lymphocytes (particularly T cells and/or NK cells), polypeptides capable of binding to an activated T cell surface protein are presented as "activating elements" on the surface of replication defective recombinant retroviral particles. Thus, in some embodiments, the activating element may perform a binding polypeptide function. In some embodiments, the activating element is an envelope protein. Such T cell and/or NK cell activating elements on the surface of a retroviral particle are present in the examples herein for modifying, genetically modifying and/or transducing lymphocytes, for example, wherein the retroviral particle has a genome encoding a CAR, an autonomous CAR or an LE. In some embodiments, such retroviral particles having an activating element on their surface are used in methods and uses that include administration by subcutaneous administration, as well as in kit components for subcutaneous administration. The activating element functions discussed herein in this section, as well as the binding polypeptides and fusogenic polypeptides disclosed elsewhere herein, are found in certain illustrative embodiments to associate with the surface of a retroviral particle as part of one, two, or three proteins, in illustrative embodiments glycoproteins, and in further illustrative embodiments heterologous glycoproteins. For example, some activating element polypeptides, such as those capable of binding to CD3, may also provide T cell binding polypeptide function.

In some embodiments, the activating element is a polypeptide capable of binding to a polypeptide on the surface of a lymphocyte and in illustrative embodiments a T cell and/or NK cell. In an illustrative embodiment, the activating element is capable of binding to a TCR complex polypeptide. In some embodiments, the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCR a or tcrp. In some embodiments, the activating element capable of binding to a TCR complex polypeptide is a polypeptide capable of binding to one or more of CD3D, CD3E, CD3G, CD3Z, TCR a or TCR β. In an illustrative embodiment, the activation element activates ZAP-70.In some embodiments, the activating element comprises a polypeptide capable of binding to CD16A, NKG2C, NKG2D, NKG2E, NKG F or NKG 2H. In some embodiments, the polypeptide capable of binding to NKG2D is MIC-A, MIC-B or ULBP, e.g., ULBP1 or ULBP2. In further embodiments, polypeptides capable of binding to CD16A include polypeptides capable of binding to one or more of NKp46, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG2F, or NKG 2H. In some embodiments, the activating element is a polypeptide capable of binding to one or more of the following combinations: NKp46 and 2B4, NKp46 and CD2, NKp46 and DNAM, NKp46 and NKG2D, 2B4 and DNAM, or 2B4 and NKG2D. In some embodiments, the activating element may be two or more polypeptides capable of binding to polypeptides on the surface of lymphocytes. In some embodiments, the activating element may be one or more polypeptides capable of binding to at least one of the following combinations: NKp46 and 2B4, NKp46 and CD2, NKp46 and DNAM, NKp46 and NKG2D, 2B4 and DNAM, or 2B4 and NKG2D. In an illustrative embodiment, the activating element is a polypeptide capable of binding to CD 3E. In some embodiments, the polypeptide capable of binding to CD3 is an anti-CD 3 antibody or fragment thereof that retains the ability to bind to CD 3. In an illustrative embodiment, the anti-CD 3 antibody or fragment thereof is a single chain anti-CD 3 antibody, such as (but not limited to) an anti-CD 3 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD3 is an anti-CD 3scFvFc. In some embodiments, the activating element is an antibody. In some embodiments, the activating element comprises an alternative non-antibody scaffold, also referred to herein as an antibody mimetic. In any aspect or embodiment provided herein that comprises an activating element capable of binding to a polypeptide on the surface of a lymphocyte (and in the illustrative embodiment a T cell), the binding polypeptide may be an antibody mimetic. In some embodiments, the antibody mimetic can be an affibody, an affinity mer, an affinity block, an alpha body, an alphamab, an anti-carrier protein, a peptide aptamer, an armadillo repeat protein, a trimer, an affinity multimer (also known as an avidity multimer), a C-lectin domain, a cysteine knot microgrotein, a cyclic peptide, a cytotoxic T lymphocyte-associated protein-4, DARPin (engineered ankyrin repeat protein), a fibrinogen domain, fibronectin binding Domain (FN 3 domain) (e.g., an attachment protein or monoclonal antibody), fynomer, kink bacteria, kunitz domain peptide, nanofitin, leucine rich repeat domain, lipocalin domain, mAb 2 or Fcab ^TM Nanobodies, nanomembers, OBody, pronectin, single chain TCRs, triangular tetrapeptide repeat domains, VHH or V-like domains. In any aspect or embodiment provided herein that comprises an activating element as an antibody, a suitable antibody mimetic may be used in place of the antibody. In some embodiments, the activating element (e.g., tcrp) capable of binding to a polypeptide on the surface of a lymphocyte is a superantigen polypeptide.

A number of anti-human CD3 monoclonal antibodies and antibody fragments thereof are useful and can be used as T cell activating elements in the present invention, including, but not limited to UCHT1, OKT-3, HIT3A, TRX, X35-3, VIT3, BMA030 (BW 264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, TR66.opt, huM291, WT31, WT32, SPv-T3B, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01. Thus, the RIP herein includes means on its surface for activating T cells.

In other embodiments, the activating element on the surface of the replication defective recombinant retroviral particle may comprise one or more polypeptides capable of binding CD2, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81 and/or CD82 and optionally one or more polypeptides capable of binding CD 3. In an illustrative embodiment, the activating element is a polypeptide capable of binding to mitogenic four-transmembrane protein (mitogenic tetraspanin), e.g., a polypeptide capable of binding to CD81, CD9, CD53, CD63, or CD 82. In some embodiments, the activating element is a four transmembrane protein. Four transmembrane proteins are known in the art. In some embodiments, the four transmembrane protein may be TSPAN1 (TSP-1), TSPAN2 (TSPAN-2), TSPAN3 (TSPAN-3), TSPAN4 (TSPAN-4, NAG-2), TSPAN5 (TSPAN-5), TSPAN6 (TSPAN-6), TSPAN7 (CD 231/TALLA-1/A15), TSPAN8 (CO-029), TSPAN9 (NET-5), TSPAN10 (tetraspanin)), TSPAN11 (CD 151-like), TSPAN12 (NET-2), TSPAN13 (NET-6), TSPAN14, TSPAN15 (NET-7), TSPAN16 (TM 4-B), TSPAN17, TSPAN18, TSPAN19, TSPAN20 (UP 1B, UPK1A, UPK 1A), TSPAN22 (RDS, PRPH 2), TSPAN23 (ROM 1), TSPAN24 (CD 25), TSPAN10 (TSPAN 31 (CD 31), TSPAN32 (CD 33) or TSPAN32 (CD 31). In some embodiments, the four transmembrane protein may be TSPAN1 (TSP-1), TSPAN2 (TSPAN-2), TSPAN3 (TSPAN-3), TSPAN4 (TSPAN-4, NAG-2), TSPAN5 (TSPAN-5), TSPAN6 (TSPAN-6), TSPAN7 (CD 231/TALLA-1/A15), TSPAN8 (CO-029), TSPAN9 (NET-5), TSPAN10 (tetralin), TSPAN11 (CD 151-like), TSPAN12 (NET-2), TSPAN13 (NET-6), TSPAN14, TSPAN15 (NET-7), TSPAN16 (TM 4-B), TSPAN17, TSPAN18, TSPAN19, TSPAN20 (UP 1B, UPK 1B), AN21 (UP 1A, UP 22 (RDS, PRPH 2), TSPAN23 (ROM 1), TSPAN24 (CD 151), TSPAN26 (CD 37), TSPAN31 (TSPAN 33) or TSPAN33 (TSPAN 6). In illustrative embodiments, the four transmembrane protein is TSPAN7 (CD 231/TALLA-1/A15), TSPAN9 (NET-5), TSPAN24 (CD 151), TSPAN27 (CD 82), TSPAN28 (CD 81), TSPAN29 (CD 9) or TSPAN30 (CD 63). In some embodiments, the activating element is a four-transmembrane protein, and the four-transmembrane protein is TSPAN25 (CD 53), TSPAN27 (CD 82), TSPAN28 (CD 81), TSPAN29 (CD 9), or TSPAN30 (CD 63). In some embodiments, the four transmembrane protein is the only envelope protein. In some embodiments, the four transmembrane protein is a pseudotyped element comprising a binding polypeptide and a fusogenic element. In some embodiments, the four transmembrane protein is an activating element and a pseudotyped element. In an illustrative embodiment, the four transmembrane protein that is the activating and pseudotyped element is TSPAN29 (CD 9).

In some embodiments, one or more copies of these activating elements may be expressed as a polypeptide separate and distinct from the pseudotyped element on the surface of the replication defective recombinant retroviral particle. In some embodiments, the activating element can be expressed as a fusion polypeptide on a replication defective recombinant retroviral particle. In illustrative embodiments, the fusion polypeptide includes one or more activating elements and one or more pseudotyped elements or one or more binding and/or fusion promoting elements. In other illustrative embodiments, the fusion polypeptide includes an anti-CD 3, e.g., an anti-CD 3scFv, or an anti-CD 3scFvFc, and a viral envelope protein. In one example, the fusion polypeptide is an OKT-3scFv fused to the amino terminus of a viral envelope protein (e.g., muLV envelope protein), as shown in Maurie et al (2002). In some embodiments, the fusion polypeptide is UCHT1scFv fused to a viral envelope protein, such as a MuLV envelope protein (SEQ ID NO: 341), a MuLV SUx envelope protein (SEQ ID NO: 366), a VSV-G (SEQ ID NO: 367), or a functional variant or fragment thereof, including any of the membrane protein truncations provided herein. In illustrative embodiments, particularly for the compositions and methods used herein to transduce lymphocytes in whole blood, the fusion polypeptide does not include any blood protein (e.g., blood factor (e.g., factor X)) cleavage sites in the portion of the fusion protein that is located outside of the retroviral particle. In some embodiments, the fusion construct does not include any furin cleavage sites. Furin is a membrane-bound protease expressed in all mammalian cells examined, some of which are secreted and active in plasma (see, e.g., C.Fernandez et al, international journal of pharmacy (J.International. Medicine) (2018) 284; 377-387). The fusion construct may be mutated using known methods to remove such protease cleavage sites.

Because of its ability to activate resting T cells, a polypeptide that binds CD3, CD28, OX40, 4-1BB or ICOS is called an activating element. In certain embodiments, the nucleic acid encoding such activating elements is found in the genome of a replication defective recombinant retroviral particle containing activating elements on its surface. In an illustrative embodiment, the nucleic acid encoding the activating element is not found in the replication defective recombinant retroviral particle genome. In some embodiments, the nucleic acid encoding the activating element is found in the genome of the viral packaging cell.

In some embodiments, the activating element is a polypeptide capable of binding to CD28, such as an anti-CD 28 antibody or an anti-CD 28 scFv antibody, or a fragment thereof that retains the ability to bind to CD 28. In other embodiments, the polypeptide capable of binding to CD28 is CD80, CD86, or a fragment thereof capable of binding to CD28 and inducing CD 28-mediated activation of Akt, such as an external fragment of CD 80. In some aspects herein, an external fragment of a CD80 means a fragment that is normally present extracellular in a standard cellular location of a CD80 that retains the ability to bind to CD 28.

anti-CD 28 antibodies are known in the art and may include, as non-limiting examples, monoclonal antibodies 9.3 (IgG 2a antibody), KOLT-2 (IgG 1 antibody), 15E8 (IgG 1 antibody), 248.23.2 (IgM antibody), and EX5.3D10 (IgG 2a antibody).

In an illustrative embodiment, the activating element comprises two polypeptides, a polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD 28.

In certain embodiments, the polypeptide capable of binding to CD3 or CD28 is an antibody (single chain monoclonal antibody) or an antibody fragment (e.g., a single chain antibody fragment). Thus, an antibody fragment may be, for example, a single chain fragment variable region (scFv), an antibody binding (Fab) fragment of an antibody, a single chain antigen binding fragment (scFab), a cysteine-free single chain antigen binding fragment (scFab ac), a fragment variable region (Fv), a structure specific for an adjacent epitope of an antigen (CRAb) or a single domain antibody (VH or VL).

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the modified lymphocytes in the cell preparation may comprise T cell activating elements on their surface. In some embodiments, the T cell activating element may be bound to the surface of the modified lymphocyte by, for example, a T cell receptor, and/or the pseudotyped element may be present in the plasma membrane of the modified lymphocyte.

In any of the embodiments disclosed herein, the activating element or nucleic acid encoding the same may comprise a dimerizing or higher order multimerizing motif. Dimerization or multimerization motifs are well known in the art and those skilled in the art will understand how to incorporate them into polypeptides for efficient dimerization or multimerization. In illustrative embodiments, the polypeptide capable of binding to CD3 is an anti-CD 3scFvFc, which in some embodiments is considered an anti-CD 3 with a dimerization motif but without any additional dimerization motif, since anti-CD 3scFvFc constructs are known to dimerize without the need for a separate dimerization motif.

In some embodiments, the activating element comprising a dimerization motif may be active in the absence of a dimerization agent when present on the surface of the replication defective recombinant retroviral particle. In some embodiments, the dimerization or multimerization motif or nucleic acid sequence encoding the same may be an amino acid sequence from a transmembrane polypeptide that naturally occurs as a homodimer or multimer. In some embodiments, the dimerization or multimerization motif or nucleic acid sequence encoding the same may be an amino acid sequence from a fragment of a native protein or an engineered protein. In one embodiment, the homodimeric polypeptide is a leucine zipper motif-containing polypeptide (leucine zipper polypeptide). For example, leucine zippers are derived from c-JUN, non-limiting examples of which are disclosed in connection with Chimeric Lymphoproliferative Elements (CLE) herein. In some embodiments, these transmembrane homodimeric polypeptides may include CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271, CD324, or active fragments thereof.

In some embodiments, the activating element comprising a dimerization motif may be active in the presence of a dimerization agent when present on the surface of the replication defective recombinant retroviral particle. In some embodiments, the dimerization motif and nucleic acid encoding the same may include an amino acid sequence of a transmembrane protein that dimerizes upon binding of a ligand (also referred to herein as a dimer or dimerizer). In some embodiments, the dimerization motif and dimer may include (where the dimer is in parentheses after the dimer binding pair): FKBP (rapamycin or analogue thereof); gyrB and GyrB (coumarone or analog thereof); DHFR and DHFR (methotrexate); or DmrB and DmrB (AP 20187). As mentioned above, rapamycin may be used as a dimer. Alternatively, rapamycin derivatives or analogues may be used (see, e.g., WO96/41865, WO 99/36553, WO 01/14387; and Ye et al (1999) Science 283:88-91). Coumarone analogues may be used (see, e.g., farrrar et al (1996) Nature 383:178-181; and U.S. Pat. No. 6,916,846). Although some embodiments of the lymphoproliferative element include a dimerizing agent, in some aspects and illustrative embodiments, the lymphoproliferative element is constitutively active and does not require a dimerizing agent for activation.

In some embodiments, the activating element is fused to a heterologous signal sequence and/or a heterologous membrane-attachment sequence or membrane-binding protein, all of which help direct the activating element onto the membrane. In some embodiments, post-translational lipid modification may occur via myristoylation, palmitoylation, or GPI anchoring. In some embodiments, the heterologous membrane linker is a GPI anchor linker. The heterologous GPI anchor linkage sequence may be derived from any known GPI anchor protein. In some embodiments, the heterologous GPI anchor linkage sequence is a GPI anchor linkage sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD 87. In some embodiments, the heterologous GPI anchor linkage sequence is derived from CD16. In an illustrative embodiment, the heterologous GPI anchor linkage sequence is derived from the Fc receptor fcγriiib (CD 16 b) or Decay Accelerating Factor (DAF), otherwise known as complement decay accelerating factor or CD55. In some embodiments, the activating element is attached to the membrane via an endogenous transmembrane protein.

In some embodiments, one or more of the activating elements comprises a heterologous signal sequence that facilitates direct expression of the activating element to the cell membrane. Any signal sequence active in a packaging cell line may be used. In some embodiments, the signal sequence is a DAF signal sequence. In an illustrative embodiment, the activating element is fused to the DAF signal sequence at its N-terminus and the GPI-anchored linking sequence at its C-terminus.

In an illustrative embodiment, the activating element comprises an anti-CD 3 scFvFc fused to a GPI-anchored linking sequence derived from CD14, and a CD80 fused to a GPI-anchored linking sequence derived from CD16 b; and both expressed on the surface of the replication defective recombinant retroviral particles provided herein. In some embodiments, the anti-CD 3 scFvFc is fused to its N-terminal DAF signal sequence and its C-terminal GPI-anchored linking sequence derived from CD14, and CD80 is fused to its N-terminal DAF signal sequence and its C-terminal GPI-anchored linking sequence derived from CD16 b; and both expressed on the surface of the replication defective recombinant retroviral particles provided herein. In some embodiments, the DAF signal sequence comprises amino acid residues 1-30 of the DAF protein.

In some embodiments, the activating element may be separate from the replication defective recombinant retroviral particle. Thus, in some embodiments, the replication defective recombinant retroviral particle does not comprise an activating element on its surface.

The nucleic acid aptamer may serve as a surrogate molecule for antibodies that bind and/or elicit biological functions, including use as immunotherapy (Freage 2020). In any aspect or embodiment provided herein that includes an activating element (including an activating element on the RIP surface), a suitable DNA aptamer may be used in place of the polypeptide. Thus, in certain embodiments, a nucleic acid aptamer capable of binding to CD2, CD28, OX40, 4-1BB, ICOS, CD, CD53, CD63, CD81, and/or CD82 is used in place of the polypeptide. In some embodiments, a DNA aptamer capable of binding CD3 is used in place of a polypeptide capable of binding CD 3. In some embodiments, ZUCH-1 or variants thereof is used to bind CD3 and activate T cells.

In some embodiments, more than one activation element is used. In some embodiments, the activating element may be a superantigen, such as lipopolysaccharide, SEC3, and staphylococcal enterotoxin B. In some embodiments, the activating element may be a cytokine. In some embodiments, the activating element may be Phorbol Myristate Acetate (PMA), ionomycin, or Phytohemagglutination (PHA). In some embodiments, the concentration of PMA in the cell preparation or to be administered separately from the replication defective recombinant retroviral particles may be 10ng/ml, 25ng/ml, 50ng/ml, 75ng/ml or 100ng/ml or 10 to 100ng/ml or 25 to 75ng/ml. In some embodiments, the concentration of ionomycin in the cell preparation or to be administered separately from the replication defective recombinant retroviral particles may be at least or about 100ng/ml, 250ng/ml, 500ng/ml or 750ng/ml or 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml or 5 μg/ml or 100ng/ml to 5 μg/ml or 500ng/ml to 2 μg/ml. In some embodiments, the concentration of PHA in the cell preparation or to be administered separately from the replication defective recombinant retroviral particles can be at least or about 0.1 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml, 7.5 μg/ml, or 10 μg/ml or 0.1 μg/ml to 10 μg/ml, 1 μg/ml to 10 μg/ml, or 2.5 μg/ml to 7.5 μg/ml. In some embodiments, the activating element is administered within 5, 10, 15, 20, 30, 45, or 60 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, or 24 hours or 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days of administration of the cell preparation. In some embodiments, one or more activating elements are administered multiple times, e.g., on different days after administration of the cell preparation.

Membrane-bound cytokines

Some embodiments of the methods and composition aspects provided herein include a membrane-bound cytokine, or a polynucleotide encoding a membrane-bound cytokine. Cytokines are usually, but not always, secreted proteins. Naturally secreted cytokines can be engineered into membrane-bound fusion proteins. Membrane-bound cytokine fusion polypeptides are included in the methods and compositions disclosed herein and are also aspects of the invention. In some embodiments, the replication defective recombinant retroviral particle has a membrane bound cytokine fusion polypeptide on its surface capable of binding T cells and/or NK cells and promoting proliferation and/or survival thereof. Typically, the membrane-binding polypeptide is incorporated into the membrane of a replication-defective recombinant retroviral particle, and upon transduction of a cell by the replication-defective recombinant retroviral particle, fusion of the retroviral and host cell membranes produces a polypeptide that binds to the membrane of the transduced cell.

In some embodiments, the cytokine fusion polypeptide includes one or more of IL-2, IL-7, IL-15, or active fragments thereof.

The membrane-bound cytokine fusion polypeptide is typically a cytokine fused to a heterologous signal sequence and/or a heterologous membrane-linked sequence. In some embodiments, the heterologous membrane linker is a GPI anchor linker. The heterologous GPI anchor linkage sequence may be derived from any known GPI anchor protein (reviewed in Ferguson MAJ, kinoshita T, hart GW., glycosyl phosphatidylinositol anchors, varki A, cummings RD, esko JD et al, essential for glycobiology, 2 nd edition Cold Spring Harbor (NY): cold Spring Harbor Laboratory Press;2009, chapter 11). In some embodiments, the heterologous GPI anchor linkage sequence is a GPI anchor linkage sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD 87. In some embodiments, the heterologous GPI anchor linkage sequence is derived from CD16. In one illustrative embodiment, the heterologous GPI anchor linkage sequence is derived from the Fc receptor fcyriiib (CD 16 b). In some embodiments, the GPI anchor is a GPI anchor of a DAF.

In an illustrative embodiment, the membrane-bound cytokine is a fusion polypeptide of a cytokine fused to DAF. DAF is known to accumulate in lipid rafts incorporated into the membrane of replication-defective recombinant retroviral particles sprouting from packaging cells. Thus, without being bound by theory, it is believed that the DAF fusion protein preferentially targets portions of the membrane of the packaging cell that will become part of the recombinant retroviral membrane.

In non-limiting illustrative embodiments, the cytokine fusion polypeptide is IL-7, or an active fragment thereof fused to DAF. In a particular non-limiting illustrative embodiment, the fusion cytokine polypeptide comprises, in order: DAF signal sequence (residues 1-31 of DAF), IL-7 without its signal sequence, and residues 36-525 of DAF.

In some embodiments, the membrane bound cytokine fusion polypeptide comprises a cleavage site. In some embodiments, the cleavage site may be within the sequence of the cytokine. In some embodiments, the cleavage site may be within the sequence of the heterologous signal sequence. In some embodiments, the cleavage site may be within the sequence of the heterologous membrane attachment sequence. In some embodiments, the cleavage site may be between the cytokine and the heterologous signal sequence or the heterologous membrane attachment sequence.

In some embodiments, the membrane-bound cytokine fusion polypeptide can include a linker as disclosed elsewhere herein.

Packaging cell lines/methods for preparing recombinant retroviral particles

The present disclosure provides mammalian packaging cells and packaging cell lines that produce replication defective recombinant retroviral particles. The cell line that produces replication defective recombinant retroviral particles is also referred to herein as a packaging cell line. Non-limiting examples of such methods are described in WO 2019/055946. Other exemplary methods for preparing retroviral particles are provided herein, e.g., in the examples section herein. Such methods include, for example, 4 plasmid systems or 5 plasmid systems when nucleic acids encoding other membrane-bound proteins (e.g., T cell activating elements that are not fused to the viral envelope, such as GPI linked anti-CD 3) are included (see WO 2019/05546). In an illustrative embodiment, provided herein is a 4 plasmid system wherein a T cell activating element, such as GPI-linked anti-CD 3, is encoded on one packaging plasmid (e.g., a plasmid encoding a viral envelope or a plasmid encoding REV) and optionally, a second viral membrane-associated transgene, such as a membrane-bound cytokine, may be encoded on another packaging plasmid. In each case, the nucleic acid encoding the viral protein is isolated from the transgene by an IRES or ribosome spanning sequence (e.g., P2A or T2A). Such 4 plasmid systems and related polynucleotides are described in the examples, providing increased titers in transient transfection compared to 5 vector systems, and thus providing illustrative embodiments herein. The present disclosure provides packaging cells and mammalian cell lines that are packaging cell lines that produce replication defective recombinant retroviral particles that genetically modify a mammalian cell of interest and the mammalian cell line of interest itself. In an illustrative embodiment, the packaging cell comprises a nucleic acid sequence encoding a packagable RNA genome of a replication-defective recombinant retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyped element.

The cells of the packaging cell line may be adherent cells or suspension cells. Exemplary cell types are provided below. In an illustrative embodiment, the packaging cell line may be a suspension cell line, i.e., a cell line that does not adhere to a surface during growth. The cells may be grown in a chemically defined medium and/or serum-free medium. In some embodiments, the packaging cell line may be a suspension cell line derived from an adherent cell line, e.g., HEK293 may be grown under conditions that produce a suspension adapted HEK293 cell line according to methods known in the art. Packaging cell lines are typically grown in a chemically defined medium. In some embodiments, the packaging cell line medium can include serum. In some embodiments, the packaging cell line medium may include serum substitutes, as known in the art. In an illustrative embodiment, the packaging cell line medium can be a serum-free medium. This medium may be a chemically defined serum-free formulation manufactured according to current pharmaceutical good manufacturing practices (Current Good Manufacturing Practice; CGMP) regulations of the U.S. food and drug administration (US Food and Drug Administration; FDA). The packaging cell line medium may be xeno-free and intact. In some embodiments, the packaging cell line media is purged by regulatory authorities for ex vivo cell processing, such as FDA 510 (k) purge devices.

Accordingly, in one aspect, provided herein is a method for preparing a replication defective recombinant retroviral particle comprising: A. culturing packaging cells in suspension in a serum-free medium, wherein the packaging cells comprise a nucleic acid sequence encoding a packagable RNA genome of a replication-defective retroviral particle, REV protein, gag polypeptide, pol polypeptide and pseudotyped elements; replication defective recombinant retroviral particles are collected from serum-free medium. In another aspect, provided herein is a method for transducing lymphocytes with replication-defective recombinant retroviral particles, comprising: A. culturing packaging cells in suspension in a serum-free medium, wherein the packaging cells comprise a nucleic acid sequence encoding a packagable RNA genome of a replication-defective retroviral particle, REV protein, gag polypeptide, pol polypeptide and pseudotyped elements; B. collecting replication-defective recombinant retroviral particles from the serum-free medium; contacting lymphocytes with replication defective recombinant retroviral particles, wherein the contacting is performed for less than 24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 30 minutes or 15 minutes (or between contacting as the low end of the range and not incubating or incubating for 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours or 4 hours and incubating for 1, 2, 3, 4, 6, 8, 12, 18, 20 or 24 hours as the high end of the range), thereby transducing lymphocytes.

In some illustrative embodiments, the packagable RNA genome is designed to express one or more polypeptides of interest, including, as non-limiting examples, inhibitory RNA molecules in which any one of the engineered signaling polypeptides disclosed herein and/or one or more (e.g., two or more) are directed against (e.g., encoded on opposite strands and in opposite directions) retroviral components such as gag and pol. For example, from 5 'to 3', the packagable RNA genome may include: a 5' long terminal repeat or an active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; nucleic acid sequences encoding a first and optionally a second polypeptide of interest, such as, but not limited to, an engineered signal polypeptide of opposite orientation that can be driven off in this opposite orientation relative to the 5' long terminal repeat and the cis-acting RNA packaging element by a promoter, which in some embodiments is referred to as a "fourth" promoter (and sometimes referred to herein as a promoter active in T cells and/or NK cells) for convenience only, which is active in target cells (such as T cells and/or NK cells), but not in the packaging cells in the illustrative example, or is only inducible or minimally active in packaging cells; and a 3' long terminal repeat or an active truncated fragment thereof. In some embodiments, the packagable RNA genome may include a central polypurine region (cPPT)/Central Termination Sequence (CTS) element. In some embodiments, the retroviral cis-acting RNA packaging element can be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging element can be a Rev response element. In exemplary embodiments, the engineered signaling polypeptide driven by a promoter that is oppositely directed to the 5' long terminal repeat is one or more engineered signaling polypeptides disclosed herein, and may optionally express one or more inhibitory RNA molecules as disclosed herein and in more detail in WO2017/165245A2, WO2018/009923A1, and WO2018/161064 A1. In some aspects, provided herein is a packagable RNA genome designed to express a self-driven CAR. Details regarding such replication defective recombinant retroviral particles, as well as compositions and method aspects including the self-driven CAR, are disclosed in more detail herein, e.g., in the self-driven CAR methods and compositions section and in the illustrative embodiments section. In an illustrative embodiment, a first one or more transcriptional units encoding a lymphoproliferative element are encoded in the reverse direction and a second one or more transcriptional units encoding a CAR are encoded in the forward direction.

It is understood that the numbering of promoters such as the first promoter, second promoter, third promoter, fourth promoter, etc. is for convenience only. A promoter referred to as a "fourth" promoter should not be taken to imply that any other promoter is present, such as the first, second or third promoter, unless other promoters are explicitly recited. It should be noted that each of the promoters is capable of driving expression of the transcript in the appropriate cell type, and that such transcripts form transcriptional units.

In some embodiments, the engineered signaling polypeptide may include a first lymphoproliferative element. Suitable lymphoproliferative elements are disclosed elsewhere herein. As a non-limiting example, the lymphoproliferative element can be expressed as a fusion with a cell tag, such as eTag, as disclosed herein. In some embodiments, the packagable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide, including a chimeric antigen receptor encoding any of the CAR embodiments provided herein. For example, the second engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere herein. In some embodiments in which the target cell is a T cell, the promoter active in the target cell is active in the T cell, as disclosed elsewhere herein.

In some embodiments, the engineered signaling polypeptide can include a CAR, and the nucleic acid sequence can encode any CAR embodiment provided herein. For example, an engineered polypeptide can include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere herein. In some embodiments, the packagable RNA genome can further include a nucleic acid sequence encoding a second engineered polypeptide. In some embodiments, the second engineered polypeptide may be a lymphoproliferative element. In some embodiments wherein the target cell is a T cell or NK cell, the promoter active in the target cell is active in the T cell or NK cell, as disclosed elsewhere herein.

In some embodiments, a packagable RNA genome included in any of the aspects provided herein can further include a riboswitch, as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064 A1. In some embodiments, the nucleic acid sequence encoding the engineered signaling polypeptide may be reverse oriented relative to the 5 'to 3' orientation established by the 5'ltr and the 3' ltr. In other embodiments, the packagable RNA genome can further comprise a riboswitch, and optionally the riboswitch can be in a reverse orientation. In any of the embodiments disclosed herein, the polynucleotide comprising any of the elements may comprise a primer binding site. In illustrative embodiments, an isolator and/or polyadenylation sequence may be located before, after, between or near the gene to prevent or reduce unregulated transcription. In some embodiments, the isolator may be a chicken HS4 isolator, a Kaiso isolator, a SAR/MAR element, a chimeric chicken isolator-SAR element, a CTCF isolator, a gypsy isolator, or a β -globin isolator, or a fragment thereof, as known in the art. In some embodiments, the spacer and/or polyadenylation sequence may be hGH polyA (SEQ ID NO: 316), SPA1 (SEQ ID NO: 317), SPA2 (SEQ ID NO: 318), B-globin polyA spacer B (SEQ ID NO: 319), B-globin polyA spacer A (SEQ ID NO: 320), 250cHS4 spacer v1 (SEQ ID NO: 321), 250cHS4 spacer v2 (SEQ ID NO: 322), 650cHS4 spacer (SEQ ID NO: 323), 400cHS4 spacer (SEQ ID NO: 324), 650cHS4 spacer and B-globin polyA spacer B (SEQ ID NO: 325) or B-globin polyA spacers B and 650cHS4 spacers (SEQ ID NO: 326).

In any of the embodiments disclosed herein, the nucleic acid sequence encoding Vpx can be located on the second transcriptional unit or on an optionally present third transcriptional unit, or on an additional transcriptional unit operably linked to the first inducible promoter.

Some aspects of the disclosure include or are cells, in an illustrative example mammalian cells that are used as packaging cells to make replication defective recombinant retroviral particles, such as lentiviral particles for transduction of T cells and/or NK cells. In some aspects, provided herein are packaging cells to make replication defective recombinant retroviral particles comprising a polynucleotide encoding a self-driven CAR. Details regarding such replication defective recombinant retroviral particles, as well as compositions and method aspects including the self-driven CAR, are disclosed in more detail herein, e.g., in the self-driven CAR methods and compositions section and in the illustrative embodiments section.

Any of a wide variety of cells is optionally selected to produce a virus or viral particle in vitro, such as a reset-to-recombinant retroviral particle according to the present invention. Eukaryotic cells, particularly mammalian cells, including human cells, simian cells, canine cells, feline cells, equine cells, and rodent cells are typically used. In an illustrative example, the cell is a human cell. In other illustrative embodiments, the cells proliferate indefinitely, and thus immortalize. Examples of cells that may be advantageously used in the present invention include NIH 3T3 cells, COS cells, madin-Darby canine kidney cells, human embryonic 293T cells, and any cells derived from such cells, such as gpnlslacZ

Cells derived from 293T cells. Highly transfectable cells such as human embryonic kidney 293T cells can be used. By "highly transfectable" is meant that at least about 50%, preferably at least about 70%, optimally at least about 80% of the cells express the gene of the introduced DNA.

Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, heLa cells (e.g., american type culture Collection (American Type Culture Collection; ATCC) accession number CCL-2), CHO cells (e.g., ATCC accession number CRL9618, CCL61, CRL 9096), 293 cells (e.g., ATCC accession number CRL-1573), vero cells, NIH 3T3 cells (e.g., ATCC accession number CRL-1658), huh-7 cells, BHK cells (e.g., ATCC accession number CCLO), PC12 cells (ATCC accession number CRL 1721), COS cells, COS-7 cells (ATCC accession number CRL 1651), RATl cells, mouse L cells (ATCC accession number CCL.3), human Embryonic Kidney (HEK) cells (ATCC accession number CRL 1573), HLHepG2 cells, hut-78, jurkat, HL-60, and the like.

Nucleic acid

The present disclosure provides polynucleotides having nucleic acids encoding the polypeptides of the present disclosure, and nucleic acids for use in the various methods herein are disclosed. In some embodiments, the nucleic acid will be DNA, including, for example, a recombinant expression construct, or as all or part of the genome of, for example, a T cell or NK cell. In some embodiments, the nucleic acid will be RNA, such as a retrovirus genome or expressed transcript within a packaging cell line, T cell, or NK. In some embodiments, the nucleic acid will be RNA, e.g., RNA synthesized in vitro. In some embodiments, the nucleic acid may be isolated. As used herein, the term "isolated" refers to the removal of a material from its original environment (e.g., the natural environment when it is naturally occurring). For example, a naturally occurring polynucleotide present in a living animal, or in other embodiments a polypeptide, is not isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector, and/or such polynucleotides or polypeptides may be part of a composition, and still be isolated, as such vector or composition is not part of its natural environment. For example, the isolated nucleic acid may be part of a recombinant nucleic acid vector, such as an expression vector, which in an illustrative embodiment may be a replication defective recombinant retroviral particle. In some embodiments, the nucleic acid is produced in accordance with cGMP, as discussed herein for kit components.

In some embodiments, nucleic acids for producing the polypeptides of the disclosure (e.g., in mammalian cells) are provided. In other cases, the subject nucleic acids provide for amplification of nucleic acids encoding the polypeptides of the disclosure.

Some polynucleotides provided herein include a promoter. Promoters suitable for use may be constitutive or inducible. For expression of viral particle RNA, an LTR promoter or hybrid LTR promoter may be used. For example, separate RSV/LTR, TRE/LTR or LTR may be used to transcribe the nucleic acid to be packaged. Examples of LTRs include, but are not limited to MSCV, GALV, HIV-1, HIV-2 and MuLV. For example, in packaging lines containing large T antigens, the incorporation of the SV40 origin of replication may be contained in one or more packaging vectors to amplify the circular plasmid DNA during transcription and/or translation. The use of multiple promoters may be used to prevent competition for transcription factors. For example, CMV, SV40, RSV, HSVTK, TRE and other promoters may be used to express different components of LV particles. In some cases, the viral particle component may be expressed from an integrated expression vector. In other cases, one or more of the nucleic acids may be introduced via transient expression. In some embodiments, inducible promoters are used to minimize cytotoxicity prior to viral particle packaging.

For expression of a transgene (e.g., a CAR or an anti-idiotype polypeptide) in a genetically modified cell (e.g., a lymphocyte, macrophage or dendritic cell), suitable promoters include any constitutive promoter known in the art. In some embodiments, the constitutive promoter may be an EF1-a promoter, a PGK promoter, a CMV promoter, an MSCV-U3 promoter (see, e.g., jones et al, (2009) Gene therapy 20:630-40), an SV40hCD43 promoter, a VAV promoter, a TCR beta promoter, a UBC promoter, a cytomegalovirus immediate early promoter, a herpes simplex virus thymidine kinase promoter, early and late SV40 promoters, promoters found in long terminal repeats from retroviruses, a mouse metallothionein-I promoter, and various tissue-specific promoters known in the art. In some embodiments, the constitutive promoter may include the EF1-a promoter nucleotide sequence (SEQ ID NO: 350), the PGK promoter nucleotide sequence (SEQ ID NO: 351), or a functional portion or variant thereof. In some embodiments, the constitutive promoter may include a promoter other than the EF1-a promoter. In some embodiments, the promoter comprises light chain and/or heavy chain immunoglobulin gene promoters and enhancer elements.

In some embodiments, the promoter is inactive in the packaging line or minimally active in the packaging line. Such embodiments have the advantage that they will reduce, minimize or in the illustrative embodiments substantially eliminate or even eliminate the expression of the engineered T cell receptor or CAR in an encapsulated nucleic acid vector, such as a RIR retroviral particle or a virus-like particle, because the expression of the engineered T cell receptor or CAR is reduced, low, negligible, substantially none or none in the packaging cell line used to prepare the encapsulated nucleic acid vector. In illustrative embodiments, such expression is reduced, substantially eliminated, or eliminated on the surface of an encapsulated nucleic acid vector (e.g., a RIR particle or a virus-like particle). In some embodiments, the promoter may be a T cell specific promoter, a CD8 cell specific promoter, a CD4 cell specific promoter, a NKT cell specific promoter, or an NK cell specific promoter. In some embodiments, the T cell specific promoter may be a CD3 zeta promoter or a CD3 delta promoter (see, e.g., ji et al, J. Biochem. 12, 6, 2002; 277 (49): 47898-906). In an illustrative embodiment, the T cell specific promoter may be a cd3ζ promoter. In some embodiments, the T cell specific promoter may be a CD8 gene promoter. In some embodiments, the T cell specific promoter may be a CD4 gene promoter (see, e.g., salmon et al (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al (2003) blood 101:3416). In some embodiments, the NK cell specific promoter may be the Neri (p 46) promoter (see, e.g., eckelhart et al (2011) blood 117:1565). In some embodiments, the specific protein encoded by the recombinant polynucleotide vector is not expressed in, displayed on, and/or incorporated into the surface of a polynucleotide vector (e.g., RIP). In some embodiments, this is accomplished by a T cell specific promoter that drives transgene expression in the polynucleotide vector. In some embodiments, the promoter is a promoter from the CD3 family. In other embodiments, it is a hybrid CD3 promoter. In other embodiments, the packaging cell line encodes a repressor protein capable of substantially inhibiting expression of a lentiviral transgene in the packaging cell line. In some embodiments, the inhibitor may be a TET repressor protein. In other embodiments, the transcription factor activated against the protein in the packaging line has been inhibited or inactivated. In some embodiments, inactivation may be achieved by DNA editing nucleases. In other embodiments, inactivation is achieved by shRNA or miRNA. In other embodiments, inhibition of the transcription factor is achieved by a dominant negative protein or degradation determinant of the transcription factor. In other embodiments, the viral nucleic acid is controlled via a ligand-inducible or repressible promoter that is not activated in the packaging cell line.

In other embodiments, the promoter may be a reversible promoter. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from a variety of organisms, such as eukaryotes and prokaryotes. Modifications to reversible promoters derived from first organisms (e.g., first and second prokaryotes, etc.) for use in second organisms are known in the art. Such reversible promoters and systems based on such reversible promoters but also including other control proteins include, but are not limited to, alcohol dehydrogenase I (alcA) gene promoters, promoters responsive to alcohol transactivator protein (AlcR), and the like, tetracycline-regulated promoters (e.g., promoters including TetActivators, tetON, tetOFF, and the like), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone (mifepriston) promoter systems, and the like), metal-regulated promoters (e.g., metallothionein promoter systems, and the like), related pathogenesis-regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, and the like), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, and the like), light-regulated promoters, synthetically-inducible promoters, and the like, as suitable promoters for use in various aspects and as discussed herein.

In some embodiments, the promoter is inducible in the cell to be genetically modified (e.g., CAR-T cell). In some embodiments, the inducible promoter may include a T cell specific response element or an NFAT response element. In other embodiments, the promoter may be regulated by environmental conditions, such as hypoxia, temperature, glucose, pH or light. In other embodiments, the promoter may be responsive to the concentration of extracellular molecules. In some cases, a locus or construct or transgene containing a suitable promoter is irreversibly transformed by induction of an inducible system. Suitable systems for inducing irreversible transformations are well known in the art, e.g., induction of irreversible transformations may use Cre-lox mediated recombination (see, e.g., fuhrmann-Benzakey et al, proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art may be used to create an irreversibly converted promoter. Methods, mechanisms and requirements for performing site-specific recombination described elsewhere herein find use in promoters that produce irreversible exchanges, and are well known in the art, see, e.g., grindley et al, (2006) annual review of biochemistry (AnnualRehewaniew biochemistry), 567-605 and trop (2012), molecular biology (Jones & Bartlett publication, sudbury, mass.), the disclosures of which are incorporated herein by reference.

In some aspects, provided herein are polynucleotides comprising promoters that are particularly useful for self-driven CARs. Details regarding such promoters, as well as compositions and method aspects including self-driven CARs comprising such promoters, are disclosed in more detail herein, e.g., in the self-driven CAR methods and compositions section and exemplary embodiments section. In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a macrophage-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter may be used.

The isolated nucleotide sequence encoding a polypeptide of the present disclosure may be present in a eukaryotic expression vector and/or a cloning vector. The nucleotide sequences encoding the two separate polypeptides may be cloned in the same or different vectors. Expression vectors can include selectable markers, origins of replication, and other features that provide for replication and/or maintenance of the vector and expression of the transgene. For example, expression vectors typically include a promoter operably linked to a transgene. Suitable expression vectors are known in the art and include, for example, plasmid and viral vectors. In some embodiments, the expression vector is a recombinant retroviral particle, as disclosed in detail herein.

Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for insertion of nucleic acid sequences encoding heterologous proteins. Optional markers for manipulation in the expression host may be present.

As described above, in some embodiments, the nucleic acid encoding a polypeptide of the disclosure will be RNA, e.g., RNA synthesized in vitro, in some embodiments. Methods for in vitro synthesis of RNA are known in the art; any known method may be used to synthesize RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, zhao et al (2010) cancer research 15:9053. Introduction of RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure into a host cell may be performed in vitro or ex vivo or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo by RNA comprising a nucleotide sequence encoding a polypeptide of the disclosure.

Various aspects and embodiments including polynucleotides, nucleic acid sequences, and/or transcription units and/or vectors including the same further include one or more of the following: a Kozak-like sequence (also referred to herein as a Kozak-related sequence), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a dual or triple stop codon, wherein one or more of the dual or triple stop codons define a termination of a read by at least one of the one or more transcriptional units. In certain embodiments, the polynucleotide, nucleic acid sequence, and/or transcription unit, and/or vector comprising the same, further comprises a Kozak-type sequence having a 5' nucleotide within 10 nucleotides upstream of the start codon of at least one of the one or more transcription units. Kozak determined the Kozak consensus sequence (GCC) GCCRCCATG (SEQ ID NO: 327) for 699 vertebrate mRNAs, where R is a purine (A or G) (Kozak, nucleic acids research, 10 months, 26 days 1987; 15 (20): 8125-48). In one embodiment, the Kozak-type sequence is or includes CCACCAT/UG (G) (SEQ ID NO: 328), CCGCCAT/UG (G) (SEQ ID NO: 329), GCCGCCGCCAT/UG (G) (SEQ ID NO: 330) or GCCGCCACCAT/UG (G) (SEQ ID NO: 331) (wherein the nucleotides in brackets represent optional nucleotides and the nucleotides separated by a diagonal line represent possible nucleotides that differ in position, e.g., depending on whether the nucleic acid is DNA or rna. In these embodiments comprising an AU/TG start codon, a may be considered as position 0. In certain illustrative embodiments, the nucleotides at-3 and +4 are identical, e.g., the-3 and +4 nucleotides may be G. In another embodiment, kozak type sequence comprises a or G in position 3 upstream of the ATG, wherein the ATG is the start codon, in another embodiment, kozak type sequence comprises a or G in position 3 upstream of the AUG, wherein the AUG is the start codon, in an illustrative embodiment, kozak sequence is (GCC) GCCRCCATG (SEQ ID NO: 327), wherein R is purine (a or G). In an illustrative embodiment, kozak type sequence is (Kozak) 62 is a nucleotide in another embodiment, e.g. in a nucleotide sequence in a sequence or a sequence in a sequence, or a sequence in a sequence, a sheet, or a sheet of a sheet, or easily, or sheet, or made has n made has been made has n made has been made has or has or has, the WPRE element is located 3' to the stop codon of one or more transcription units and 5' to the 3' LTR of the polynucleotide. In another embodiment, which can be combined with any or both of the preceding embodiments (i.e., an embodiment in which the polynucleotide comprises a Kozak sequence and/or an embodiment in which the polynucleotide comprises a WPRE sequence), the one or more transcriptional units terminate with one or more stop codons that are dual stop codons or triple stop codons, wherein the dual stop codons comprise a first stop codon in a first reading frame and a second stop codon in a second reading frame, or a first stop codon in a frame with a second stop codon, and wherein the triple stop codons comprise a first stop codon in a first reading frame, a second stop codon in a second reading frame, and a third stop codon in a third reading frame, or a first stop codon in a frame with a second stop codon and a third stop codon.

The triple stop codons herein include three stop codons, one within 10 nucleotides of each other in each reading frame, and preferably having overlapping sequences, or three stop codons in the same reading frame, preferably at consecutive codons. A double stop codon means two stop codons, each in a different reading frame, within 10 nucleotides of each other, and preferably having overlapping sequences, or two stop codons in the same reading frame, preferably at consecutive codons.

In some of the methods and compositions disclosed herein, the introduction of DNA into PBMCs, B cells, T cells and/or NK cells and optionally the incorporation of DNA into the host cell genome is performed using methods that utilize recombinant nucleic acid vectors rather than replication-defective recombinant retroviral particles. For example, other viral vectors, such as those derived from adenovirus, adeno-associated virus, or herpes simplex virus type 1, may be utilized as non-limiting examples.

In some embodiments, the methods provided herein, as well as related uses, reaction mixtures, kits, and cell preparations, can include transfecting a cell with a polynucleotide that is not encoded in a viral vector. Such polynucleotides may be referred to as non-viral vectors. In any of the embodiments disclosed herein that utilize non-viral vector genetic modification or transfection of cells, non-viral vectors (including, for example, plasmids or naked DNA) can be introduced into cells (such as PBMC, B cells, T cells, and/or NK cells) using methods that include electroporation, nuclear transfection, lipid formulations, lipids, dendrimers, cationic polymers (such as poly (ethyleneimine) (PEI) and poly (l-lysine) (PLL)), nanoparticles, cell penetrating peptides, microinjection, and/or non-integration of lentiviral vectors. In some embodiments, the lipid preparation, lipid, dendrimer, PEI, PLL, nanoparticle, and cell penetrating peptide may be modified to include lymphocyte targeting ligands, such as anti-CD 3 antibodies. PEI conjugated to anti-CD 3 antibodies has been shown to be effective in transfecting PBMC with exogenous nucleic acids (O' Neill et al, gene therapy 3 month 2001; 8 (5): 362-8). Similarly, T lymphocytes were transfected with nanoparticles made of polyglutamic acid molecules conjugated to anti-CD 3e f (ab') 2 fragments (Smith et al, nature nanotechnology, 8 months 2017; 12 (8): 813-820). In some embodiments, the DNA may be introduced into cells (e.g., PBMC, B cells, T cells, and/or NK cells) having a complex of a lipid and a protamine. Other methods for ex vivo transfection of T cells and/or NK cells that may be used in embodiments of the methods provided herein are known in the art (see, e.g., morgan and Boyerinas, biomedicine (biomedicines.) month 4, 20, 2016; 4 (2) pii: E9, incorporated herein by reference in its entirety).

In some embodiments of the methods provided herein, transposon-based vector systems may be used to integrate DNA into the genome by co-transfection, co-nuclear transfection or co-electroporation of the target DNA (as a plasmid containing transposon ITR fragments in the 5 'and 3' ends of the relevant gene) and a transposase vector system (as DNA or mRNA or protein or site-specific serine recombinases, such as phiC31 integrating the relevant gene in the pseudo attP site in the human genome), in which case the DNA vector contains 34 to 40bp attB sites, which are recognition sequences for the recombinases (Bhaskar Thyagarajan et al, by phage

Integrase-mediated mammalian cellsSite-specific genomic integration (Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage->

Integrase), molecular Cell biology (Mol Cell biol.), 6 months 2001; 21 3926-3934) and co-transfected with a recombinase. Regarding T cells and/or NK cells, transposon-based systems that may be used in certain methods provided herein utilize SleepingBeauty DNA vector systems (see, e.g., U.S. Pat. No. 6,489,458 and U.S. patent application Ser. No. 15/434,595, which are incorporated herein by reference in their entirety), piggyBac DNA vector systems (see, e.g., manuri et al, human Gene therapy, month 2010 4; 21 (4): 427-37, which are incorporated herein by reference in their entirety), or ToLCDR2 transposon systems (see, e.g., tsukahara et al, gene therapy, month 2015; 22 (2): 209-215, which are incorporated herein by reference in their entirety), in the form of DNA, mRNA, or protein. In some embodiments, prior to introduction into T cells and/or NK cells, transposons and/or transposases of the transposon-based vector system may be generated in the form of mini-circular DNA vectors (see, e.g., hudecek et al, current cancer research results (Recent Results Cancer Res.)) 2016;209:37-50 and Monjezi et al, leukemia (Leukemia.)), 2017, month 1, 31 (1): 186-194, which are incorporated herein by reference in their entirety). However, in some cases, transposase-based vector systems are not the preferred method of introducing exogenous nucleic acids. Thus, in some embodiments, the polynucleotide of any aspect or embodiment disclosed herein does not include a transposon ITR fragment. In some embodiments, the modified, genetically modified, and/or transduced cells of any aspects or embodiments disclosed herein do not include a transposase vector system that is DNA or mRNA or protein.

The integration of an anti-idiotype polypeptide, CAR or lymphoproliferative element into defined and specific sites in the genome using CRISPR or TALEN mediated integration can also be accomplished by adding 50-1000bp homology arms 5 'and 3' homologous to the target site (Jae Seong Lee et al science report (Scientific Reports) 5, article number 8572 (2015), site specific integration in CHO cells mediated by CRISPR/Cas9 and homology directed DNA repair pathways). CRISPR or TALEN provide specific and genome-targeted cleavage and the construct will integrate by homology-mediated end-joining (Yao X et al, cell research (Cell res.) "6 months 2017; 27 (6): 801-814.Doi:10.1038/cr.2017.76.Epub 2017, 5 months 19). CRISPR or TALEN can be co-transfected with a plasmid of interest as DNA, mRNA or protein.

For any method for modifying, genetically modifying and/or transducing T cells and/or NK cells (e.g., in whole blood or in a whole blood fraction such as TNF or PBMC), or use including such methods, or modified cells produced using such methods, and any other method or definition product provided herein, one of skill in the art will appreciate that an exogenous nucleic acid may be introduced into a cell using a method that does not include replication-defective recombinant retroviral particles, e.g., using another type of recombinant vector (e.g., a plasmid associated with a lipofection agent).

Inhibitory RNA molecules

Embodiments of any aspect provided herein may include recombinant retroviral particles whose genomes are configured to induce expression of one or more, and in illustrative embodiments, two or more, inhibitory RNA molecules (e.g., miRNA or shRNA) upon integration into a host cell (e.g., a lymphocyte (e.g., a T cell and/or NK cell)). Such inhibitory RNA molecules may be encoded within introns, including, for example, the EF1-a intron. This uses the method teachings of the present invention to maximize functional elements that may be included in the packagable retroviral genome to overcome the shortcomings of the previous teachings and maximize the effectiveness of such recombinant retroviral particles in adoptive T cell therapies.

In some embodiments, the inhibitory RNA molecule includes a 5 'strand and a 3' strand (in some examples, a sense strand and an antisense strand) that are partially or fully complementary to each other, such that the two strands are capable of forming an RNA duplex of 18 to 25 nucleotides within the cellular environment. The 5 'strand may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, and the 3' strand may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The 5 'strand and the 3' strand may be the same or different lengths, and the RNA duplex may include one or more mismatches. Alternatively, the RNA duplex has no mismatches. In some illustrative embodiments, a vector or genome herein comprises 2 or more of the inhibitory RNAs provided herein.

The inhibitory RNA molecules included in the compositions and methods provided herein are in certain illustrative embodiments absent and/or not naturally expressed in T cells into which they are inserted into their genome. In some embodiments, the inhibitory RNA molecule is a miRNA or shRNA. In some embodiments, the inhibitory molecules in embodiments of the present disclosure may be precursors to mirnas (e.g., pri-miRNA or Pre-miRNA), or precursors to shRNA. In some embodiments, the miRNA or shRNA is artificially derived (i.e., an artificial miRNA or siRNA). In other embodiments, the inhibitory RNA molecule is dsRNA treated to siRNA (transcribed or artificially introduced) or the siRNA itself. In some embodiments, the miRNA or shRNA has a sequence not found in nature, or has at least one functional segment not found in nature, or has a combination of functional segments not found in nature.

In some embodiments, the inhibitory RNA molecules are placed in a first nucleic acid molecule in a serial or multiplex arrangement such that multiple miRNA sequences are simultaneously expressed from a single polycistronic miRNA transcript. In some embodiments, inhibitory RNA molecules may be directly or indirectly contiguous with each other using a nonfunctional linker sequence. In some embodiments, the linker sequence may be between 5 and 120 nucleotides in length, and in some embodiments, between 10 and 40 nucleotides in length, as non-limiting examples. In some embodiments, the functional sequence may be expressed from the same transcript as the inhibitory RNA molecule, such as any of the lymphoproliferative elements provided herein. In some embodiments, the inhibitory RNA molecules are naturally occurring miRNAs such as, but not limited to, miR-155, miR-30, miR-17-92, miR-122 and miR-21. Thus, in some embodiments, the 5 'to 3' orientation of the inhibitory RNA molecule comprises: a 5' microRNA flanking sequence, a 5' handle, a loop, a 3' handle partially or fully complementary to the 5' handle, and a 3' microRNA flanking sequence. In some embodiments, the 5 'handle (also referred to herein as a 5' arm) may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3 'handle (also referred to herein as a 3' arm) may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop is 3 to 40, 10 to 40, 20 to 40, or 20 to 30 nucleotides in length, and in illustrative embodiments, the loop may be 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, one handle is two nucleotides longer than the other handle. The longer handle may be a 5 'handle or a 3' handle. The inhibitory RNA molecule can be any of the inhibitory RNA molecules in the inhibitory RNA molecule section herein.

In some embodiments, the 5 'microRNA flanking sequence, the 3' microRNA flanking sequence, or both are derived from naturally-occurring miRNAs such as, but not limited to, miR-155, miR-30, miR-17-92, miR-122 and miR-21. In certain embodiments, the 5 'microRNA flanking sequence, the 3' microRNA flanking sequence, or both are derived from miR-155, e.g., miR-155 from a mouse or homo sapiens. The insertion of synthetic miRNA stem-loops into the miR-155 framework (i.e., the 5 'microrna flanking sequences, the 3' microrna flanking sequences, and the loops between the miRNA 5 'and 3' stems) is known to those of ordinary skill in the art (Chung, k., et al 2006 nucleic acids research 34 (7): e53; US 7,387,896), such as SIBR and eSIBR sequences. In some embodiments of the disclosure, the mirnas can be placed in the SIBR or eSIBR miR-155 framework. In the illustrative embodiments herein, the miRNA is placed in a miR-155 framework, which comprises the 5 'microRNA flanking sequence of miR-155 shown by SEQ ID NO. 333, or a functional variant thereof, the 3' microRNA flanking sequence shown by SEQ ID NO. 334 (nucleotides 221 to 265 of mouse BIC non-coding mRNA), or a functional variant thereof; and a modified miR-155 loop (SEQ ID NO: 335) or a functional variant thereof. However, any known microrna framework that functions to provide suitable processing within cells of the miRNA inserted therein to form a mature miRNA capable of inhibiting expression of the target mRNA to which it binds is encompassed by the present disclosure.

In some embodiments, when two or more inhibitory RNA molecules (in some examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 inhibitory RNA molecules) are included, these inhibitory RNA molecules are directed against the same or different RNA targets (e.g., mRNA transcribed from an autocorrelation gene).

In some embodiments, the one or more inhibitory RNA molecules are one or more lymphoproliferative elements, and thus, in any aspect or embodiment provided herein that includes a lymphoproliferative element, unless incompatible therewith or stated therein. In an illustrative embodiment, the mirnas inserted into the T cell genome in the methods provided herein are directed against a target such that proliferation and/or enhancement and/or apoptosis of the T cells are inhibited. In some embodiments, the RNA target is mRNA transcribed from a gene that is a miR-155 target.

In some embodiments, the inhibitory RNAs, e.g., mirnas, encode target mRNA for ABCG1, cbl protooncogene (RNF 55) (also known as Cbl and RNF 55) (HGNC: 1541 Entrez Gene: 867, OMIM: 165360), T cell receptor T3 ζ chain (CD 3 z) (HGNC: 1677, entrez gene 919, OMIM: 186780), T cell receptor alpha locus (TCRA) (also known as TCRaα) (HGNC: 12027, entrez gene 6955, OMIM: 186880), T cell receptor beta locus (TCRB) (also known as TCRβ) (HGNC: 12155, entrez gene 6957, OMIM: 186930), PD1, CTLA4, IFN gamma, T cell immunoglobulin mucin 3 (TIM 3) (also known as hepatitis A virus cell receptor 2) (HGNC: 184337 Entrez gene: 84868, OMIM: 606652), lymphocyte activation 3 (LAG 3) (HGNC: 6476, entrez gene: 3902, OMIM: 153337), SMAD2, TNF receptor superfamily member 10B (TNFRSF 10B) (HGNC: 12105, entrez gene: 8795, OMIM: 603612), protease 2 catalytic subunit alpha (HGCA 2) (HGNC: 99, IFN gamma, T cell immunoglobulin mucin 3 (TIMA 3) (also known as "light" G ") gene (HGNC: 1837 Entrez gene: 8468, OMIM: 606652), lymphocyte activation 3 (LAG 3) (HGNC: 6476, entrez gene (light) and human tumor cell death) (also known as" light ", light", light receptor "light) (including light receptor" light gene "light receptor" light) (HGNC "light gene well as light receptor well) 35) (HGNC: light gene, 35, entrez gene, 35, tumor gene, entrez Gene 135, OMIM 102776), aromatic Hydrocarbon Receptor (AHR) (HGNC 348, entrez Gene 196, OMIM 600253), degerming protein (EOMES) (HGNC 3372, entrez Gene 8320, OMIM 604615), SMAD family Member 3 (SMAD 3) (HGNC 6769, entrez Gene 4088, OMIM 603109), SMAD family Member 4 (SMAD 4) (GNC 6770, entrez Gene 4089, OMIM 600993), TGR 2, TRAIL2, PP2A, protein phosphatase 2 regulatory subunit B delta (PPP 2R 2D) (HGNC 23732, entrez Gene 55844, OMIM 613992), tumor necrosis factor ligand superfamily Member 6 (TNFSF 6) (also referred to as FASL) (HGNC 11936, entrez Gene 356, OMIM 638), cysteine protein 3 (CASP 3, SOCS1, SOCS 2), and signal transducer 2 (HGIM 1936, SOCS1, SOCS 2A, SOCS 2D) (HGNC 23732, entrez 2D): 8835, OMIM:605117), kruppel-like factor 10 (KLF 10) (also known as TGPB-inducible early growth reaction protein 1 (TIEG 1)) (HGNC:11810, entrez gene: 7071, OMIM:601878), junB protooncogene, AP-1 transcription factor subunit (JunB) (HGNC:6205, entrez gene: 3726, OMIM:165161), chromosome cassette 1 (Cbx) (HGNC:1551, entrez gene: 10951, OMIM:604511), cbx3, tet methylcytosine dioxygenase 2 (Tet 2) (HGNC:25941, entrez Gene: 54790, OMIM: 612839), hexokinase 2 (HK 2) (HGNC: 4923, entrez gene: 3099, OMIM: 60125) fv, phosphatase-1 (SHP 1) containing the Src homology region 2 domain (HGNC: 9658, entrez gene: 5777, OMIM: 176883), phosphatase-2 (SHP 2) containing the Src homology region 2 domain (HGNC: 9644, entrez gene: 5781, OMIM: 176876), colony stimulating factor 2 (CSF 2; GMCSF) (Entrez Gene: 1437). In some embodiments, the inhibitory RNA (e.g., miRNA) targets an antigen to which the aster of the CAR binds.

In some aspects, provided herein is a polynucleotide designed to express a self-driven CAR. Details regarding such replication defective recombinant retroviral particles, as well as compositions and method aspects including the self-driven CAR, are disclosed in more detail herein, e.g., in the self-driven CAR methods and compositions section and in the illustrative embodiments section. In some embodiments, a polynucleotide designed to express a self-driven CAR can include any inhibitory RNA molecule disclosed herein. Such polynucleotides may also have inhibitory RNA molecules that target inhibitors of the NFAT pathway, with or without other inhibitory RNA molecules disclosed herein. In some embodiments, the inhibitory RNA molecules may target CABIN, homer2, AKAP5, LRRK2, and/or DSCR1/MCIP (knockout of RNA molecules encoding these proteins may reduce inhibition of calcineurin or calmodulin); and/or Dyrk1A, CK1 and/or GSK3 (knockdown of RNA molecules encoding these proteins can prevent phosphorylation and nuclear export of NFAT). In some other illustrative embodiments, the vector or genome herein includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 of the inhibitory RNAs (e.g., mirnas) identified herein, e.g., in the paragraphs above.

In some embodiments provided herein, two or more inhibitory RNA molecules can be delivered in a single intron, such as (but not limited to) EF1-a intron a. Intronic sequences useful for carrying the mirnas of the disclosure include any intron that is processed within a T cell. The sequence requirements of introns are known in the art. In some embodiments, such intron processing is operably linked to a riboswitch, such as any riboswitch disclosed herein. Thus, the illustrative embodiments provided herein are combinations of mirnas directed to endogenous T cell receptor subunits, wherein expression of the mirnas is regulated by riboswitches, which may be any of the riboswitches discussed herein.

In some embodiments, the inhibitory RNA molecules may be provided on multiple nucleic acid sequences that may be included on the same or different transcriptional units. For example, a first nucleic acid sequence may encode one or more inhibitory RNA molecules and be expressed from a first promoter, and a second nucleic acid sequence may encode one or more inhibitory RNA molecules and be expressed from a second promoter. In illustrative embodiments, two or more inhibitory RNA molecules are located on a first nucleic acid sequence expressed from a single promoter. Promoters used to express such mirnas are typically promoters that are inactive in packaging cells used to express retroviral particles that deliver the mirnas in their genomes to the T cell of interest, but such promoters are constitutively active or active in an inducible manner within the T cell. The promoter may be a Pol I, pol II or Pol III promoter. In some illustrative embodiments, the promoter is a Pol II promoter.

Feature and commercial production method

The present disclosure provides various methods and compositions that can be used as research reagents in scientific experiments and for commercial production. This scientific experiment may include a method of characterizing lymphocytes (e.g., NK cells, and in an illustrative embodiment, T cells) using methods for modifying, e.g., genetically modifying and/or transducing, lymphocytes provided herein. These methods are useful, for example, for studying the activation of lymphocytes and detailed molecular mechanisms by which these cells are rendered transduced. In addition, provided herein are lymphocytes that have been modified, and in the illustrative examples, genetically modified, to be used, for example, as research tools to better understand factors affecting T cell proliferation and survival. In some embodiments, the modified cells may express an anti-idiotype polypeptide herein, which may, for example, serve as a cell tag for identifying transduced cells. These modified lymphocytes (e.g., NK cells, and in the illustrative embodiment, T cells) can be used in addition for commercial production, for example, for the production of certain factors, such as growth factors and immunomodulators, which can be harvested or tested or for the production of commercial products.

Scientific experimentation and/or characterization of lymphocytes may include any of the aspects, embodiments, or sub-embodiments provided herein for analyzing or comparing lymphocytes. In some embodiments, T cells and/or NK cells can be transduced with the replication-defective recombinant retroviral particles provided herein, including polynucleotides. In some embodiments, the transduced T cells and/or NK cells can include polynucleotides including polynucleotides encoding polypeptides of the present disclosure, e.g., anti-idiotype polypeptides, CARs, lymphoproliferative elements, and/or activating elements. In some embodiments, the polynucleotide may include an inhibitory RNA molecule as discussed elsewhere herein. In some embodiments, the lymphoproliferative element may be a chimeric lymphoproliferative element.

Illustrative embodiments

This illustrative embodiment section provides non-limiting illustrative aspects and embodiments provided herein and is discussed further throughout this specification. For the sake of brevity and convenience, all of the aspects and embodiments disclosed herein and all of the possible combinations of the disclosed aspects and embodiments are not listed in this section. Additional embodiments and aspects are provided in other sections herein. Moreover, it should be understood that the embodiments provided are specific to many aspects and as such are discussed throughout this disclosure. It is intended that any individual embodiment described below or in this complete disclosure may be combined with any aspect described below or in this complete disclosure in view of the complete disclosure herein, wherein it is an additional element that may be added to an aspect or because it is a narrower element than the element already presented in the aspect. This combination is specifically discussed in other sections of this detailed description. Thus, for example, any of the embodiments provided herein can be used with any of the polynucleotides, polynucleotide vectors, methods, uses, reaction mixtures, cell preparations, kits, cell processing components, filter assemblies, modified, genetically modified, and transduced cells such as T cells or NK cells, cell mixtures, or cell populations provided herein, unless incompatible or otherwise indicated.

Thus, in some aspects, provided herein are polynucleotides comprising a nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain (anti-id ERD) that recognizes the idiotype of a target antibody or target antibody mimetic. Polynucleotides may be referred to herein as anti-idiotype polynucleotides or polynucleotides encoding anti-idiotypes or anti-id polynucleotides, including nucleic acids encoding anti-idiotype extracellular recognition domains (anti-id ERDs) that recognize the idiotype of a target antibody or target antibody mimetic. The anti-id ERD of the anti-id polynucleotide may be any anti-id ERD disclosed herein, and the anti-idiotype polypeptide encoded by such anti-idiotype polynucleotide may be any anti-idiotype polypeptide disclosed herein, or encoded by any anti-idiotype polynucleotide herein. Such anti-idiotype polypeptides encoded by any of the anti-idiotype polypeptides herein are themselves separate embodiments herein. In some embodiments, the anti-idiotype polynucleotide further comprises a nucleic acid encoding a Membrane Associated Domain (MAD), and in some embodiments further comprises a nucleic acid encoding a handle. In some illustrative embodiments, the polynucleotide encoding the anti-idiotype polypeptide may further comprise a nucleic acid encoding one or more inhibitory RNA molecules and/or the first engineered signaling polypeptide.

In one aspect, provided herein is a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter, wherein the one or more transcriptional units comprise:

a) Nucleic acid encoding one or more inhibitory RNA molecules and/or a first engineered signaling polypeptide, and

b) A nucleic acid encoding an anti-idiotype polypeptide comprising an extracellular recognition domain and a membrane associated domain, wherein the extracellular recognition domain comprises an anti-idiotype domain that recognizes the idiotype of a target antibody or a target antibody mimetic.

In one aspect, provided herein is a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units comprise:

a) Nucleic acids encoding one or more inhibitory RNA molecules, lymphoproliferative elements, cytokines, chimeric Antigen Receptors (CARs), and recombinant T Cell Receptors (TCRs), and

b) A nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain that recognizes an idiotype of a target antibody or target antibody mimetic.

In one aspect, provided herein is a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units comprise:

a) Nucleic acid encoding one or more inhibitory RNA molecules and/or one or more of lymphoproliferative elements, cytokines, chimeric Antigen Receptor (CAR), and recombinant T Cell Receptor (TCR), and

b) A nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain that recognizes an idiotype of a target antibody or target antibody mimetic, a membrane association domain, and a handle that links the anti-idiotype extracellular recognition domain to the membrane association domain.

In one aspect, provided herein is a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter, wherein the one or more transcriptional units comprise:

a) Nucleic acid encoding one or more inhibitory RNA molecules and/or one or more engineered signaling polypeptides selected from lymphoproliferative elements, cytokines, chimeric Antigen Receptors (CARs) and recombinant T Cell Receptors (TCRs), and

b) A nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain, a membrane association domain, and a handle connecting the anti-idiotype extracellular recognition domain to the membrane association domain, wherein the anti-idiotype extracellular recognition domain comprises an anti-idiotype antibody or antibody mimetic idiotype binding variable region that recognizes the idiotype of a target antibody or target antibody mimetic.

In one aspect, provided herein is a polynucleotide comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units comprise:

a) A nucleic acid encoding one or more engineered signaling polypeptides, wherein a first engineered signaling polypeptide of the one or more engineered signaling polypeptides is a Chimeric Antigen Receptor (CAR), and

b) A nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain, a membrane association domain, and a handle connecting the anti-idiotype extracellular recognition domain to the membrane association domain, wherein the anti-idiotype extracellular recognition domain comprises an anti-idiotype antibody or antibody mimetic idiotype binding variable region that recognizes the idiotype of a target antibody or target antibody mimetic.

In some aspects, provided herein are modified cells, which may be isolated cells, and in illustrative embodiments mammalian cells, e.g., human cells, which, for example, comprise any of the polynucleotides disclosed herein, and in illustrative embodiments, express the polynucleotides. In an illustrative embodiment, the polynucleotide comprises a nucleic acid encoding an anti-idiotype polypeptide. Such anti-idiotype polypeptides may be according to any of the anti-idiotype polypeptide embodiments provided herein. In some embodiments, the cell is a primary cell. In some embodiments, the cells are not from a cell line. In some embodiments, the cell is not an immortalized cell. The cells of some embodiments are not immortalized cells. In some embodiments, the cell is an ex vivo cultured cell. In some embodiments, the cell is a cell type or lymphocyte type that does not produce antibodies in its native form. In some embodiments, the cell is a TIL. In some embodiments, the cell is a T cell or NK cell, a CAR-T cell or CAR-NK cell or a population of T cells and/or NK cells or a population of CAR-T cells and/or CAR-NK cells, or a population of any of the foregoing cells in this paragraph and throughout the present disclosure. In some embodiments, the cells, such as a population of human cells, are in a container for gene therapy or cell therapy, such as an infusion bag. In some embodiments, at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75% of the modified cells in the population comprise the polynucleotide, and in illustrative embodiments the anti-idiotype polypeptide is expressed. In some embodiments, from 1%, 2%, 5%, 10%, 20%, or 25% of the modified cells in the population that are the low end of the range to 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% of the modified cells that are the high end of the range comprise the anti-idiotype polynucleotide, and in illustrative embodiments the anti-idiotype polypeptide is expressed.

In some aspects, provided herein are methods for delivering a modified cell, typically a mammalian cell, such as a lymphocyte, a primary cell, a primary lymphocyte, a lymphocyte with an unnatural expressed antibody type, and in illustrative embodiments a T cell and/or NK cell, to a subject, wherein the modified mammalian cell is modified to include any of the polynucleotides disclosed herein. In an illustrative embodiment, the polynucleotide includes a nucleic acid encoding an anti-idiotype polypeptide, and thus generally encodes an anti-id ERD. The method generally comprises administering a modified cell, such as a T cell and/or NK cell, to a subject. In some embodiments, the method is a method for treating a disorder, such as a hyperproliferative disorder, e.g., cancer. In some embodiments, such methods further comprise instructing the user to deliver the target antibody or antibody mimetic to the subject if the adverse event of the subject is desired to be treated. For example, such instructions may indicate that treatment is needed if the subject experiences adverse events of grade 3 or grade 4 of cytokine release syndrome or IEC-related neurotoxicity syndrome. In some embodiments, such methods further comprise administering the target antibody to the subject, e.g., after an adverse event has occurred in the subject, as provided herein. In some embodiments, the target antibody or antibody mimetic is delivered in an amount sufficient to selectively kill at least 1%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the genetically modified T cells and/or NK cells, thereby selectively killing the genetically modified T cells and/or NK cells. Such methods herein can be considered methods for selectively killing target modified cells.

In some aspects, provided herein is a method of treating a subject in need thereof, comprising administering to the subject a modified cell, wherein the method comprises administering to the subject a modified cell. Particularly contemplated for such uses are any of the methods disclosed herein that involve delivery or administration of the modified cells to a subject.

In some aspects, provided herein are polynucleotide vectors comprising an anti-idiotype polynucleotide according to any aspect or embodiment herein. In some aspects, polynucleotides comprising nucleic acids encoding anti-idiotype polypeptides herein comprise a promoter to drive expression of the nucleic acids encoding the anti-idiotype polypeptides. The Tis promoter or another promoter may be used to drive expression from other nucleic acids on a polynucleotide. In any aspect herein relating to or including a promoter, the promoter may be a promoter active in mammalian cells, e.g., a promoter active in lymphocytes or specifically active in lymphocytes. As another example, the promoter may be a promoter active or specifically active in T cells and/or NK cells.

In some embodiments of any of the polynucleotide or vector embodiments herein, the vector is a viral vector, and in further embodiments is a replication defective viral vector. In some embodiments, the viral vector is a retroviral particle. In some embodiments, the surface of the retroviral particle comprises a membrane bound cytokine. In some embodiments, the membrane-bound cytokine is a chemokine. In some sub-embodiments, the vector comprises a cleavage signal effective to cleave the membrane-bound cytokine from the membrane. In some embodiments, the retroviral particle is a lentiviral vector. In some embodiments, the viral vector further comprises an activating element on the surface of the retroviral particle, wherein the activating element is fused to a heterologous membrane attachment sequence, and wherein the activating element is a polypeptide capable of binding to CD3 or a nucleic acid (e.g., an aptamer) capable of binding to CD3 on the surface of resting T cells and activating resting T cells, and wherein the activating element is not encoded by a polynucleotide in a retroviral particle. In some embodiments, the activating element comprises an anti-CD 3 antibody or antibody mimetic. In some embodiments, the viral particle comprises means on its surface for binding to CD3 on the surface of T cells.

In one aspect, provided herein are anti-idiotype polypeptides encoded by any of the polynucleotides provided herein comprising a nucleic acid encoding an anti-id ERD. In one aspect, provided herein are polypeptides comprising an extracellular recognition domain, a membrane associated domain, and a handle that connects the extracellular recognition domain to the membrane associated domain, and further comprising an intracellular domain (ICD) of a first engineered signaling polypeptide, wherein the first engineered signaling polypeptide is a Chimeric Antigen Receptor (CAR), a recombinant T Cell Receptor (TCR), or a Lymphoproliferative Element (LE), and wherein the extracellular recognition domain is an anti-idiotype extracellular recognition domain that recognizes an idiotype of a target antibody.

In some non-limiting embodiments, the anti-idiotype polypeptide comprises an extracellular recognition domain, a membrane association domain, and a handle connecting the extracellular recognition domain to the membrane association domain, and further comprises an intracellular domain of the first engineered signaling polypeptide, wherein the extracellular recognition domain comprises a domain that recognizes the idiotype of the target antibody. In some embodiments, the first engineered signaling polypeptide is a Lymphoproliferative Element (LE), a cytokine, a Chimeric Antigen Receptor (CAR), and a recombinant T Cell Receptor (TCR).

In some non-limiting embodiments, an anti-idiotype polypeptide comprises an extracellular recognition domain, an intracellular domain, a membrane association domain, and a handle connecting the extracellular recognition domain to the membrane association domain, wherein the extracellular recognition domain comprises a domain that recognizes an idiotype of a target antibody.

In illustrative embodiments herein comprising any aspect of an anti-idiotype extracellular recognition domain, the anti-idiotype extracellular recognition domain comprises an idiotype binding variable region of an anti-idiotype antibody or an idiotype binding region of an anti-idiotype antibody mimetic. In some embodiments, the anti-idiotype extracellular recognition domain is an antibody selected from the group consisting of a single chain antibody, a Fab fragment, a Fab 'fragment, a (Fab') 2 fragment, a Fv fragment, a scFv, a bivalent single chain antibody, a bifunctional antibody, a scFv-Fc, a scF-CH, a scFab, or a scFv-zipper. In certain illustrative embodiments, the anti-idiotype extracellular recognition domain is an antibody comprising a scFV. In some embodiments, the idiotype-binding variable region comprises a framework region, and wherein the framework region is a human framework region.

In some embodiments of any aspect and embodiment herein including polynucleotides comprising polynucleotide vectors (including nucleic acids encoding anti-idiotype polypeptides comprising ICD) or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the polynucleotides further encode a second engineered signaling polypeptide, which in certain illustrative embodiments is a lymphoproliferative element. In some embodiments, the lymphoproliferative element comprises an intracellular domain, which is a means for transmitting a signal that promotes proliferation or survival of T cells and/or NK cells. In some embodiments, the lymphoproliferative element is constitutively active. In some embodiments, the polynucleotide comprises an Internal Ribosome Entry Site (IRES), ribosome jump sequence and/or cleavage signal between the nucleic acid encoding the first or second engineered signaling polypeptide and the nucleic acid encoding the anti-idiotype polypeptide. In some embodiments, the polynucleotide comprises an Internal Ribosome Entry Site (IRES), ribosome jump sequence and/or cleavage signal between the nucleic acid encoding the second engineered signaling polypeptide and the nucleic acid encoding the anti-idiotype polypeptide.

In some embodiments including polynucleotides comprising nucleic acids encoding anti-idiotype polypeptides, CARs, and LEs, the anti-idiotype polypeptides, CARs, and LEs may be expressed as one, two, or three separate polypeptides.

In some embodiments herein including any aspect of the anti-Id ERD, the target antibody or antibody mimetic recognized by the anti-Id ERD is a clinical antibody or clinical antibody mimetic that is the subject of FDA approved new drug research application (IND) or regulatory application equivalent approved for human initial clinical testing in another country or jurisdiction. In some embodiments of any aspect herein, the target antibody or antibody mimetic recognized by the anti-id ERD is a therapeutic antibody or therapeutic antibody mimetic, respectively. In some of any of these embodiments, the agent is a separate product according to IND or equivalent, wherein no other active therapeutic agent or ingredient is tested as part of IND. In some embodiments of any aspect herein, the target antibody or antibody mimetic recognized by the anti-id ERD has been shown to have an acceptable safety (i.e., adverse event) profile in one or more clinical trials. In some embodiments of any aspect herein, the target antibody or antibody mimetic that is recognized by the anti-id ERD is a clinical antibody or clinical antibody mimetic that has passed a human clinical safety test in an independent clinical trial of the clinical antibody or antibody mimetic. In some embodiments of any aspect herein, the target antibody or antibody mimetic recognized by the anti-id ERD is a clinical antibody or antibody mimetic, which has been filed in its independent regulatory approval application to the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), chinese National Medicines Administration (NMAN) (chinese FDA), or japanese medicines and food security administration (PFSB). In some embodiments of any aspect herein, the target antibody or antibody mimetic recognized by the anti-id ERD is a clinical antibody or antibody mimetic, which has been submitted to the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), chinese National Medicines Administration (NMAN) (chinese FDA), or japan medicine and food security administration (PFSB) for approval applications (e.g., biological product approval application (BLA)). In some illustrative embodiments, the target antibody or target antibody mimetic is an approved biological antibody or antibody mimetic approved by the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), national Medicines Administration (NMAN) (chinese FDA), or japanese medicine and food safety administration (PFSB). In some embodiments, the approved biological antibody or antibody mimetic is an approved biological antibody, and wherein the approved biologic antibody is cetuximab, moroxyab-CD 3, efalizumab, tositumomab-i 131, nebulomab, ibritumomab, cetuximab, daclizumab, olaparimab, aciumab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, timox, oxmazuzumab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, cetuzumab, you-tec mab, kanadumab, golimumab, ofatuzumab, tolizumab, denomumab, belimumab, ipilimumab, bentuximab, pertuzumab, enmetrastuzumab, risperidol, oxuzumab, setuximab, ramucirumab, vedolizumab, nivolumab, pembrolizumab, bolamiab, alemtuzumab, elkumab, idasemuzumab, valdecozumab, rituximab, dituzumab, threuzumab, meperimumab, alikumab, darumab, ai Luozhu mab, ethanomab, rayleimumab, bei Luotuo Shu Shan antibody, atuzumab, ottomab, buddamab, dulcamab, oxybutyramiab, guluzumab, sha Lilu mab, abamerab, eimerimumab, orebanab, benralizumab, de valvacizumab, getuzumab, erenumumab-aooe, ganezumab-gnlm), bromoxyzumab (buduzumab-xazeranab), fluzab-fluzab, mo Geli group antibody (mogamulizumab-kpkc), tizomib (tildrakizumab-asmn), remiranavizumab (fremanizumab-vfram), epothilone (ravulizumab-cvz), cimipramimab Li Shan (cemiimab-rwlc), ibalizumab (ibalizumab-uiyk), epavacizumab (emaprabeab-lzsg), praziram antibody (tildrakizumab-moxetumomab pasudotox-tdfk), calicheamab (cazamab-ydp), rapuzumab (ribankizumab-rka), vitamin-poisuogzub antibody (polatuzumab vedotin-piiq), luo Moshan antibody (romaqqg), ibuzumab (brizumab-dbibuzumab-dbab), ibuzumab (ibuzumab-vislizumab-Uzk), eprab (tizomib-35), tizomib (tizomib-37-35), tizomib (tizomib-35), tizomib-35, tizomib-95-45, tizomib-35, tizomib-95-anguzab (moxetumomab pasudotox), tizomib-35, tizomib-95-tizomib-anguzab-95, tizomet-95-tizomet (moxetumomab pasudotox), tizobezumab-95-tizobe, tizobeab-95-tizobezeab-95, tizobezeab-95-tizobeab (tizobe, tizobezeb-95-35, tizobezeb-tizob-tizobezeb, tizobeab-95-tizobezeb-95, tizobeab-tizobeab, tizobezeb-tizobezeb, tizobezeb-b-tsab, tizobezeb-tsab-tizobezeb, bezeb-stib, bezeb, the anti-cancer drugs include, but are not limited to, electric Mo Tuo mab (amivantamab-vmjw), aviumab-avwa, qu Luolu mab, aniumab (anifloumab-fnia), motuximab, statin-Tixostat mab, bimmetagezumab, naso Li Shan mab, terzerumab, meldi Li Shan mab, inomomab, baterimumab, rituximab, terlipp Li Shan mab, obrituximab, pie An Puli mab, tanitumumab, farnesimab, su Timo mab, telitumumab and remifex Li Shan-resistance.

In some embodiments herein including any aspect of the anti-Id ERD, the anti-idiotype polypeptide comprises means for binding to the idiotype of cetuximab. In some embodiments, the approved biological antibody is cetuximab. In some embodiments, the anti-idiotype polypeptide is capable of binding to cetuximab, and the anti-id ERD comprises any sequence provided herein for such cetuximab anti-idiotype ERD. In some embodiments, the anti-idiotype polypeptide is capable of blocking binding of cetuximab to an epidermal growth factor receptor. In some embodiments, the target antibody comprises an antigen binding site, and the anti-idiotype polypeptide is capable of binding to the antigen binding site of the target antibody.

In some embodiments of any aspect and embodiment herein including polynucleotides (including nucleic acids encoding anti-idiotype polypeptides) comprising polynucleotide vectors or embodiments directed against polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the membrane associated domain is a Gpi anchor.

In some embodiments of any aspect and embodiment herein including polynucleotides (including nucleic acids encoding anti-idiotype polypeptides) comprising a polynucleotide vector or embodiments directed to a polypeptide encoded by such nucleic acid or a cell comprising and optionally expressing a polynucleotide, the anti-idiotype polypeptide further comprises one or more intracellular domains (ICDs). In some embodiments, the ICD is 1 to 35 amino acids in length. In some embodiments, the ICD is 10 to 250 amino acids in length.

In some exemplary embodiments, the ICD has primarily or exclusively structural functions, and/or in related embodiments does not include a signaling domain, or does not include a proliferation, survival and/or apoptosis signaling domain. In some exemplary embodiments, the ICD comprises a signaling domain, which in illustrative embodiments is a proliferation, survival and/or apoptosis signaling domain.

In some embodiments and sub-embodiments, wherein the anti-idiotype polypeptide comprises ICD, the membrane associating domain is a transmembrane domain. The combination of the handle (sometimes referred to as a hinge), transmembrane domain, and ICD may be referred to herein as smicd. In some embodiments, the STMICD of the anti-idiotype polypeptide comprises the handle of PDGFR beta, the 8 amino acids of TM and ICD, and comprises the sequence as provided in SEQ ID NO. 676. In some embodiments, the STMICD of the anti-idiotype polypeptide comprises the 9 amino acids of the CD28 handle, TM, and ICD, and comprises the sequence as provided in SEQ ID NO: 677. In some embodiments, the anti-idiotype polypeptide of STMICD comprises CD28 hinge, TM and all ICDs, and comprises the sequence as provided in SEQ ID NO. 678. In some embodiments, the STMICD of the anti-idiotype polypeptide comprises the hinge, TM, and all ICDs of CD28 fused to all ICDs of CD80, and comprises the sequence as provided in SEQ ID NO. 679.

In some embodiments, in illustrative embodiments in which the membrane associating domain is a transmembrane domain, the ICD is an ICD of LE, and in further illustrative embodiments of these embodiments, the transmembrane domain is from LE. In some embodiments, the ICD comprises all or part of an intracellular signaling domain of one or more cytokine receptors, and wherein the ICD is capable of activating a signaling pathway. In some embodiments, the inducible lymphoproliferative element is capable of activating proliferative or survival signaling after binding of the anti-idiotype polypeptide to the target antibody or target antibody mimetic. In some embodiments, the ICD is capable of activating the Jak/Stat pathway, TRAF pathway, PI3K pathway, and/or PLC pathway after dimerization. In some embodiments, the one or more cytokine receptors are selected from the group consisting of CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL RA, IL2RB, IL2RG, IL3RA, IL4R, IL RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL13R, IL RA1, IL13RA2, IL15R, IL RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL RA1, IL23 6327R, IL RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TGF beta R, TGF beta decoy, TNFRSF4, frsf9, TNFRSF14, or TNFRSF18.

In some embodiments of any aspect and embodiment of polynucleotides (including nucleic acids encoding anti-idiotype polypeptides) including polynucleotide vectors herein or embodiments directed to polypeptides encoded by such nucleic acids or cells including and optionally expressing polynucleotides, the membrane associated domain is a transmembrane domain of a lymphoproliferative element, CAR, or recombinant TCR. In some embodiments of any aspect herein, the transmembrane domain is from: BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD, CD5, CD7, CD8A, CD8B, CD, CD11A, CD11A, CD11A, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49A, CD49A, CD64, CD79A, CD79A, CD80, CD84, CD86, CD96 (tactile), CD100 (SEMA 4D), CD103, C134 CD137, CD154, CD160 (BY 55), CD162 (SELPLG), CD226 (DNAM 1), CD229 (Ly 9), CD247, CRLF2, CRTAM, CSF2RA, CSF2RB, CSF 3A, CD 1A, CD 2A, CD2, GHR, HVEM (LIGHTR), IA4, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA IL 4A, CD RA, IL 6A, CD ST, IL7RA Ins PPCL, IL 9A, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD RA, IL31RA, ITGA1, ITGA4, ITGA6, A, CD1, ITGB2, ITGB7, KIRDS2, LEPR, LFA-1 (CD 11a, CD 18), A, CD (KLRF 1), OSMR, PAG/A, CD1, SLAM (SLAMF 1, CD150, IPO-3), AMF4 (CD 244, 2B 4), SLF 6 (NTB-A, CD 108), SLAMF7, SLAMF8 (SLSF 8, TNSF 2, TNSF 6, or a mutant thereof. In some embodiments of any aspect herein, the transmembrane domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids of one or more of: CD 8. Alpha. TM (SEQ ID NO: 17); CD 8. Beta. TM (SEQ ID NO: 18); CD4 handle (SEQ ID NO: 19); CD3Z TM (SEQ ID NO: 20); CD28 TM (SEQ ID NO: 21); CD134 (OX 40) TM: (SEQ ID NO: 22); CD7 TM (SEQ ID NO: 23); CD8 handle and TM (SEQ ID NO: 24); and CD28 handle and TM (SEQ ID NO: 25). In some embodiments of any aspect herein, the transmembrane domain is derived from an antibody. In some embodiments of any aspect herein, the transmembrane domain is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the transmembrane domain of IgD. In some embodiments of this embodiment, the polynucleotide further comprises a stretch of at least 24, 48, 60, or 99 nucleotides encoding all or a fragment of IgA and IgB. In some sub-embodiments of the above embodiments, the transmembrane domain is at least 75%, 80%, 90%, 95% or 100% identical to the transmembrane domain from IgD.

In some embodiments of any aspect and embodiment herein including polynucleotides (including nucleic acids encoding anti-idiotype polypeptides) comprising polynucleotide vectors or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the anti-idiotype polypeptides are 4 to 250, 4 and 200 or 4 to 100 amino acids in length. In some embodiments of any aspect herein, the handle domain comprises a polypeptide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids of one or more of the following amino acid sequences: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 2), FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3), CPPC (SEQ ID NO: 4), DKTHT (SEQ ID NO: 5); CPEPKSCDTPPPCPR (SEQ ID NO: 6), ELKTPLGDTTHT (SEQ ID NO: 7), KSCDKTHTCP (SEQ ID NO: 8), KCCVDCP (SEQ ID NO: 9), KYGPPCP (SEQ ID NO: 10), EPKSCDKTHTCPPCP (SEQ ID NO: 11), ERKCCVECPPCP (SEQ ID NO: 12), ELKTPLGDTTHTCPRCP (SEQ ID NO: 13), SPNMVPHAHHAQ (SEQ ID NO: 14), EPKSCDKTYTCPPCP (SEQ ID NO: 15), TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16).

In some embodiments of any aspect herein, the stem domain is derived from an antibody. In some embodiments, the handle domain comprises a heavy chain Fc domain. In some embodiments, the handle domain encodes a dimerization moiety, wherein the dimerization moiety is constitutively dimerized. In some embodiments, the constitutively dimerized dimerization domain comprises a leucine zipper.

In some embodiments of any aspect and embodiment herein including polynucleotides (including nucleic acids encoding anti-idiotype polypeptides) comprising polynucleotide vectors or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, polynucleotides herein including nucleic acids encoding and anti-id ERD also include nucleic acids encoding one or more inhibitory RNA molecules. In some embodiments, one or more inhibitory RNA molecules reduce cytokine release syndrome or enhance proliferation. In some embodiments, one of the one or more inhibitory RNA molecules targets ifnγ.

In some embodiments of any aspect and embodiment herein including polynucleotides (including nucleic acids encoding anti-idiotype polypeptides) comprising polynucleotide vectors or embodiments directed against polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the target antibody is structured such that binding of the anti-idiotype polypeptide to the target antibody induces antibody-dependent cellular cytotoxicity. In some embodiments of any aspect and embodiment herein including anti-id ERD or nucleic acid encoding the same, the target antibody is structured such that binding of the anti-idiotype polypeptide to the target antibody induces complement-dependent cytotoxicity. In some embodiments of any aspect and embodiment herein including anti-id ERD or nucleic acid encoding the same, the target antibody is structured such that binding of the anti-idiotype polypeptide to the target antibody induces opsonization. In some embodiments of any aspect and embodiment herein including anti-id ERD or nucleic acid encoding the same, the target antibody is structured such that binding of the anti-idiotype polypeptide to the target antibody does not induce antibody-dependent cytotoxicity.

In some embodiments of any aspect and embodiment herein including polynucleotides comprising polynucleotide vectors (including nucleic acids encoding anti-idiotype polypeptides comprising ICD) or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the ICD comprises an intracellular apoptosis domain capable of inducing or promoting or transmitting an apoptosis signal. In some sub-embodiments of these embodiments, the target antibody is incapable, structured, configured, or adapted to induce cytotoxicity. In some sub-embodiments of these embodiments, the target antibody is capable of, structured, configured or adapted to induce cytotoxicity.

In some embodiments of any aspect and embodiment herein including polynucleotides comprising a polynucleotide vector (including nucleic acids encoding anti-idiotype polypeptides comprising ICD) or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the ICD comprises an intracellular apoptotic domain capable of inducing an apoptotic signal upon binding of an anti-idiotype extracellular recognition domain to a target antibody or antibody mimetic comprising the idiotype. In some sub-embodiments of these embodiments, the anti-idiotype polypeptide further comprises a proteolytic cleavage site as part of and/or between the transmembrane domain and the intracellular apoptotic domain, wherein the proteolytic cleavage site has a cleavage site amino acid sequence such that when the anti-idiotype polypeptide is dimerized by binding of a target antibody or antibody mimetic to the anti-idiotype extracellular recognition domain, the anti-idiotype polypeptide is cleaved at the proteolytic cleavage site. In further sub-embodiments of these embodiments, the proteolytic cleavage site is effective for cleavage by a gamma secretase complex or a Notch receptor following dimerization of the anti-idiotype polypeptide. In further sub-embodiments of these embodiments, the proteolytic cleavage site is effective for cleavage by a gamma secretase complex or a Notch receptor following dimerization of the anti-idiotype polypeptide. In further sub-embodiments of these embodiments, the intracellular apoptosis domain comprises one or more of a caspase 2 polypeptide, a caspase 8 polypeptide, a caspase 9 polypeptide, and a caspase 10 polypeptide, wherein such an intracellular apoptosis domain is capable of activating an effector caspase upon cleavage from the anti-idiotype polypeptide. In further sub-embodiments of these embodiments, the intracellular apoptosis domain comprises one or more caspase polypeptides, wherein the caspase polypeptides are amino acids 327 to 452 of SEQ ID No. 680 (caspase 2), amino acids 384 to 496 of SEQ ID No. 681 (caspase 8), amino acids 294 to 416 of SEQ ID No. 682 (caspase 9), and amino acids 365 to 478 of SEQ ID No. 683 (caspase 10). In further sub-embodiments of these embodiments, the intracellular apoptosis domain comprises one or more caspase polypeptides, wherein each of the caspase polypeptides is a polypeptide having promoter caspase function when dimerized and is at least 90% identical to amino acids 327 to 452 of SEQ ID NO:680 (caspase 2), amino acids 384 to 496 of SEQ ID NO:681 (caspase 8), amino acids 294 to 416 of SEQ ID NO:682 (caspase 9), and amino acids 365 to 478 of SEQ ID NO: 683 (caspase 10).

In further sub-embodiments of these embodiments wherein the ICD comprises an intracellular apoptosis domain, including certain embodiments wherein the anti-idiotype polypeptide comprises a protease cleavage site and other embodiments wherein the anti-idiotype polypeptide does not comprise a protease cleavage site (e.g., activated by binding of the anti-id ERD to a target antibody), the ICD comprises one or more Caspase Activation and Recruitment Domains (CARD), death Domains (DD), death Effector Domains (DED), thermal protein domains (PYD), and/or caspase proteolytic domains that are activated to transmit an apoptosis-inducing signal upon dimerization. For example, these domains may be from a caspase, e.g., a promoter caspase, e.g., caspase 2, 8, 9, or 10. In some sub-embodiments of these embodiments, the one or more CARD, DD, DED, PYD and/or caspase proteolytic domains comprise one or more CARD from Apaf-1, DARK, CED-4, CED-3, dronc, CARMA1, bcl-10, nod1, nod2, RIP2, ICEBERG, RIG-I, MDA5, MAV5, ASC, NALP1, caspase 2, caspase 5, and/or caspase 9. In some sub-embodiments of these embodiments, the intracellular apoptosis domain may include one or more DDs and/or functional fragments thereof from TNF-R1, fas, p75, TRADD, FADD, RIP, myD88, IRAK, pelle, tube, PIDD, RAIDD, and/or MALT 1. In some sub-embodiments of these embodiments, the intracellular apoptosis domain may include one or more DEDs from FADD, caspase 8, caspase 10, c-FLIP, v-FLIP, MC159, PEA-15, DEDD, and/or DEDD2 and/or functional fragments thereof. In some sub-embodiments of these embodiments, the intracellular apoptosis domain may include one or more PYDs and/or functional fragments thereof from ASC, ASC2, NALP1, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, and/or NALP 12.

In some embodiments of any aspect and embodiment herein including polynucleotides comprising polynucleotide vectors (including nucleic acids encoding anti-idiotype polypeptides comprising ICD) or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, the ICD comprises a dimerizing moiety, wherein the dimerizing moiety is constitutively dimerizing, and/or wherein the target antibody is an IgM antibody. In some of these embodiments and other embodiments wherein the ICD comprises an apoptosis domain, the intracellular apoptosis domain comprises one or more Death Domains (DD) from FAS, TNF-R1, DR3, DR4, and/or DR 5.

In some embodiments of any aspect and embodiment herein including polynucleotides comprising polynucleotide vectors (including nucleic acids encoding anti-idiotype polypeptides comprising ICD) or embodiments directed to polypeptides encoded by such nucleic acids or cells comprising and optionally expressing polynucleotides, wherein the membrane associated domain is a transmembrane domain, the transmembrane domain is a transmembrane domain of a CAR and the ICD is an ICD of a CAR. In some sub-embodiments of these and other embodiments, the CAR is a bispecific CAR, wherein one astm of the CAR comprises an anti-idiotype extracellular recognition domain. In some sub-embodiments of these and other embodiments, another astm of the CAR is capable of binding a tumor-associated antigen or a tumor-specific antigen. In some sub-embodiments of these and other embodiments, one and the other of the astm s are scFV antibodies. In some sub-embodiments of these and other embodiments, one and the other of the astm s are scFV antibodies. One and the other ASTR of the CAR comprise scFv-FC, scFv-CH and scFv-zipper antibodies.

In one aspect, provided herein is a retroviral particle comprising:

a) A polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain, a membrane association domain, and a handle connecting the anti-idiotype extracellular recognition domain to the membrane association domain, wherein the anti-idiotype extracellular recognition domain comprises an anti-idiotype antibody or antibody mimetic idiotype binding variable region that recognizes the idiotype of a target antibody or target antibody mimetic; and

b) An activating element on the surface of a retroviral particle, wherein the activating element is fused to a heterologous membrane attachment sequence, and wherein the activating element is a polypeptide capable of binding CD3 on the surface of a resting T cell and activating the resting T cell and is not encoded by a polynucleotide in the retroviral particle.

In some embodiments of any aspect or embodiment herein comprising an anti-id ERD or a polynucleotide having a nucleic acid encoding the same, the idiotype is present on two target antibodies: a first target antibody that promotes cytotoxicity; and a second target antibody that promotes less cytotoxicity than the first target antibody. In some embodiments, wherein the idiotype is present on two target antibodies, the two target antibodies differ in their glycosylation pattern. In some embodiments, including embodiments in which an idiotype is present on two target antibodies, one or both of the target antibodies or the target antibodies comprise a glycosylated residue. In some embodiments of any aspect herein, the idiotype recognized by the anti-id ERD comprises a glycosylated residue. In some embodiments, including embodiments wherein the idiotype is present on two target antibodies, one or both of the target antibodies or the target antibodies comprise an a-Gal epitope. In some embodiments, including embodiments in which an idiotype is present on two target antibodies, the target antibodies comprise one or more glycoforms, or the target antibodies are different glycoforms. In some embodiments, the glycoform comprises a target antibody having a-1,3-Gal residues. In some embodiments, the glycoform comprises a target antibody having an N-glycolylneuraminic acid residue. In some embodiments, the glycoform comprises a target antibody having a mannooligosaccharide.

In some embodiments, the target antibody is produced by a cell line such that the target antibody is glycosylated. In some embodiments, including embodiments in which the idiotype is present on two target antibodies, the target antibodies are produced in SP2/0 cells, and/or the two target antibodies are produced in different cell lines. In some embodiments, including embodiments in which the idiotype is present on two target antibodies, the target antibodies are produced in NS0 cells, and the second target antibodies are produced in a different cell line. In some embodiments, including embodiments in which the idiotype is present on two target antibodies, the target antibodies are produced in Chinese Hamster Ovary (CHO) cells, and the second target antibody is produced in a different cell line.

In one aspect, provided herein is a method for delivering a modified T cell and/or NK cell to a subject, the method comprising administering the modified cell to the subject, wherein the modified cell is modified with a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide and a nucleic acid encoding a CAR.

In one aspect, provided herein is a use of a modified T cell and/or NK cell in the preparation of a kit, wherein the use of the kit comprises: administering a modified T cell and/or NK cell to a subject, wherein the modified T cell and/or NK cell comprises a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide and a nucleic acid encoding a CAR.

In one aspect, provided herein is a population of modified cells, in illustrative embodiments, modified T cells and/or NK cells, wherein at least some of the modified cells of the population of cells comprise a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide and a nucleic acid encoding a CAR.

In one illustrative embodiment, provided herein is a population of modified cells, in illustrative embodiments, a population of modified T cells and/or NK cells, wherein at least some of the modified cells of the modified cell population comprise a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide, and in illustrative embodiments, express an anti-idiotype polypeptide. In some embodiments of this illustrative embodiment, as well as other embodiments herein comprising a population of cells, at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75% of the modified cells in the population comprise the polynucleotide, and in the illustrative embodiment the anti-idiotype polypeptide is expressed. In some embodiments, from 1%, 2%, 5%, 10%, 20%, or 25% of the population at the low end of the range to 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% of the modified cells at the high end of the range comprise the polynucleotide, and in illustrative embodiments the anti-idiotype polypeptide is expressed. In some embodiments of this illustrative embodiment and any other embodiments herein, the modified cells of the population are primary cells, cells cultured ex vivo, cells that are not derived from a cell line, and/or cells that are not immortalized. In some illustrative embodiments of this illustrative embodiment, the anti-idiotype polypeptide comprises a membrane association domain, which in some illustrative embodiments is a transmembrane domain.

In some exemplary embodiments of the illustrative embodiments immediately above and any other embodiments or aspects herein, the anti-idiotype polypeptide lacks an intracellular domain (ICD). In some exemplary embodiments of the illustrative embodiments immediately above and any other embodiments or aspects herein, the anti-idiotype polypeptide comprises ICD. In some exemplary embodiments, the ICD has primarily or exclusively structural functions, and/or in related embodiments does not include a signaling domain, or does not include a proliferation, survival and/or apoptosis signaling domain. In some exemplary embodiments, the ICD comprises a signaling domain, which in illustrative embodiments is a proliferation, survival and/or apoptosis signaling domain. Anti-idiotype polypeptides and polypeptides comprising nucleic acids encoding the same themselves represent separate illustrative embodiments herein, whether they are intracellular or not.

In one aspect, provided herein is a polypeptide or polynucleotide having a nucleic acid encoding the same, comprising a lymphoproliferative element according to any embodiment herein, wherein the extracellular domain comprises an anti-id ERD. In one aspect, provided herein is a polypeptide or polynucleotide having a nucleic acid encoding the same, comprising a chimeric antigen receptor according to any embodiment herein, wherein the ASTR of the CAR comprises an anti-id ERD.

The inventors have observed that delivery of modified T cells and/or NK cells can subcutaneously form subcutaneous lymphoid structures. Thus, in another aspect, provided herein is a subcutaneous lymphoid structure, which may be considered a tertiary lymphoid structure, comprising at least some modified lymphocytes in a population of modified lymphocytes and in the illustrative embodiment genetically modified lymphocytes, including polynucleotides comprising nucleic acids encoding an anti-idiotype polypeptide and one or more of a general CAR or TCR and one or more of an LE or cytokine in the illustrative embodiment. In some embodiments, some of the genetically modified lymphocytes that express the CAR are located in the lymphatic vasculature. In some embodiments, the other leukocytes include B cells, macrophages, dendritic cells, T cells, and/or NK cells. In some embodiments, some of the modified lymphocytes in the population are in the lymphatic vasculature, in some embodiments, within 25, 50, 75, 100, 125, 150, 200, 250, 500, or 1,000 μm from the subcutaneous lymphoid structure. In some embodiments, the population of subcutaneous lymphoid structures or genetically modified lymphocytes further comprises actively dividing lymphocytes native to the subject and not expressing a CAR. In some embodiments, the genetically modified lymphocyte expresses a lymphoproliferative element. In some embodiments, the subcutaneous lymphoid structure is an artificial lymph node. In some embodiments, the population of genetically modified lymphocytes is in an artificial lymph node.

In some embodiments, the T cells comprise cd4+ and cd8+ cells, and wherein at least 50% of the genetically modified lymphocytes that are cd4+ and/or cd8+ are cd3-;

in some embodiments of any aspect or embodiment of the population of genetically modified lymphocytes herein,

i) The population of genetically modified lymphocytes comprises a durable population of genetically modified lymphocytes expressing a transgenic, engineered T cell receptor or Chimeric Antigen Receptor (CAR) that remains in the subject for at least 7, 14, 21 or 28 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or 1, 2, 3, 4 or 5 years after administration.

ii) the genetically modified lymphocytes produce a population of offspring lymphocytes, wherein the population of offspring lymphocytes comprises at least 1 x 10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ 、1×10 ¹⁰ Or 1X 10 ¹¹ Or 1X 10 ⁶ Up to 1X 10 ¹⁰ Or 1X 10 ⁸ Up to 1X 10 ¹² A cell;

iii) The population of genetically modified lymphocytes comprises at least 100 subcutaneously located genetically modified lymphocytes and the subcutaneous region does not comprise an artificial matrix component; and/or

iv) at least 10 genetically modified lymphocytes in the population remain localized subcutaneously for at least 7, 14, 21, or 28 days, and in an illustrative embodiment, the subcutaneous region does not comprise an artificial matrix component.

In some embodiments, at least 1×10 of the genetically modified lymphocytes ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ 、1×10 ¹⁰ Or 1X10 ¹¹ Or 1X10 ⁶ Up to 1X10 ¹⁰ Or 1X10 ⁸ Up to 1X10 ¹² The individual cells are located subcutaneously. In some embodiments, at least 1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ 、1×10 ¹⁰ Or 1X10 ¹¹ Or 1X10 ⁶ Up to 1X10 ¹⁰ Or 1X10 ⁸ Up to 1X10 ¹² Individual cells are not in the subcutaneous region and are illustratively embodiedIn examples circulating in the blood of the subject and/or at the site of the tumor.

In one aspect, provided herein is a subcutaneous lymphoid structure comprising:

a cell aggregate, wherein the cell aggregate comprises:

a) At least 10, 100, 1x10 ³ 、1x10 ⁴ 、1x10 ⁵ 、1x10 ⁶ 、1x10 ⁷ 、1x10 ⁸ 、1x10 ⁹ 、1x10 ¹⁰ Or 1x10 ¹¹ A genetically modified lymphocyte expressing an anti-idiotype polypeptide (in an illustrative embodiment an antigen, an engineered T cell receptor, or a Chimeric Antigen Receptor (CAR)), wherein the cell aggregate is subcutaneously localized in the subject, and wherein the genetically modified lymphocyte comprises a T cell and/or an NK cell; and

b) Other white blood cells that do not express the CAR, wherein at least 10% of the cells in the cell aggregate are other white blood cells.

In any aspect provided herein that includes intramuscular administration and in illustrative embodiments subcutaneous administration of modified lymphocytes (e.g., modified T cells and/or NK cells), in certain embodiments such methods can include a step in which the modified cells are expanded subcutaneously (e.g., the modified cells are expanded subcutaneously), e.g., at or near the site of subcutaneous administration (e.g., at 10, 5, 4, 3, 2, or 1 cm), for days (e.g., for up to 5, 7, 14, 17, 21, or 28 days), or months (e.g., for up to 1, 2, 3, 6, 12, or 24 months). In some embodiments herein comprising intraperitoneal administration, intramuscular administration, and in illustrative embodiments subcutaneous administration of modified T cells and/or NK cells or RIP to modify T cells and/or NK cells in vivo, the modified T cells and/or NK cells (e.g., genetically modified T cells and/or NK cells) migrate from the subcutaneous administration site to other sites of the body, such as a tumor. Thus, in some modified embodiments and in illustrative embodiments, such methods can include a step in which the genetically modified T cells and/or NK cells appear in circulation migrating from the subcutaneous administration site for days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 4, or 4 weeks), or months (e.g., 1, 2, 3, 6, 12, or 24 months) after the modified T cells are subcutaneously injected into the subject. In certain embodiments, at these time points, such methods may include a step in which a regional gradient or in the illustrative embodiment a concentration gradient of modified T cells and/or NK cells, and in the illustrative embodiment genetically modified T cells and/or NK cells, is formed from the site of intramuscular administration or in the illustrative embodiment subcutaneously.

In any aspect provided herein, including intraperitoneal administration, intramuscular administration, and in illustrative embodiments subcutaneous administration of modified lymphocytes (e.g., modified T cells and/or NK cells), certain embodiments may include the step of subcutaneously delivering, in the same or different formulations, at or near the delivery site of the modified T cells and/or NK cells, another component that may affect the modified T cells and/or NK cells, such as a molecule (ion), macromolecule (e.g., DNA, RNA, peptide, and polypeptide), and/or other cells, such as other modified cells (e.g., genetically modified cells). In certain illustrative embodiments, the other component comprises an antigen, a recombinant cell encoding a recombinant antigen, or an RNA encoding an antigen, or a cytokine that drives proliferation of T cells and/or NK cells. These other components disclosed in more detail herein may be delivered in the same formulation as the modified T cells and/or NK cells or in a different formulation. Further, these other components may be delivered with the modified T cells and/or NK cells, or may be delivered days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 4, or 4 weeks), or even months (e.g., 1, 2, 3, 6, 12, or 24 months) before or after delivery of the modified T cells and/or NK cells. In some embodiments, one or more of these other components is delivered at more than one point in time, e.g., on the same day as the modified T cells and/or NK cells, or simultaneously with the modified T cells and/or NK cells, and at one or more times as described above in this paragraph. Thus, in some embodiments, the second formulation is administered to the subject at a second time point from 1 day to 1 month, 2 months, 3 months, 6 months, or 12 months after administration of the cell formulation. In addition to modified lymphocytes or substantially purified or purified RIP, other components administered to a subject may include cytokines, such as IL-2, ii) antibodies or polypeptides capable of binding CD2, CD3, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, and/or CD82, and/or iii) a source of homologous antigen recognized by the CAR). In certain embodiments, subcutaneous administration of modified T cells and/or NK cells is performed near (e.g., within 1, 2, 3, 4, 5, 10, 20, or 30 cm) the site of a tumor (e.g., cancerous) cell, e.g., a tumor or tumor-containing organ, including, e.g., the spleen in the case of a hematologic cancer, or where multiple administrations of the same formulation or different formulations are performed near or remote from such site of prior administration. In some embodiments, the cell preparation comprises a source of a cognate antigen of the CAR, wherein the source of the cognate antigen is a cognate antigen, an mRNA encoding the cognate antigen, or a cell expressing the cognate antigen. In some embodiments, the cell preparation comprises a cytokine, and wherein the cytokine is IL-2, IL-7, IL-15, or IL-21, or a modified form of these cytokines that is capable of binding to and activating the natural receptor of the cytokine. The homologous antigen used in this example and any of the examples herein (including in this illustrative examples section) can be any of the tumor-associated antigens or tumor-specific antigens provided herein.

In one aspect, provided herein is a cell preparation comprising modified T cells and/or NK cells, wherein the modified T cells and/or NK cells are suspended in a delivery solution and are either or both,

i) Genetic modification with a polynucleotide comprising one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter active in T cells and/or NK cells, or

ii) associated with a RIP comprising said polynucleotide,

wherein the one or more transcriptional units encode an anti-idiotype polypeptide and in an illustrative embodiment a first polypeptide comprising a CAR, and wherein the cell preparation is contained within a syringe in an illustrative embodiment and has a volume of 0.5ml to 20ml, or 2ml to 10ml, or another subcutaneous or intramuscular cell preparation volume provided herein, and further comprises at least one, e.g., two or more, neutrophils, B cells, monocytes, basophils, and eosinophils. In an illustrative embodiment, the cell formulation is compatible with, effective for, and/or suitable for intramuscular delivery (and in a further illustrative embodiment subcutaneous delivery).

In some embodiments and any reaction mixture embodiments herein, or including any aspect of the cell preparation, particularly embodiments including subcutaneous, intramuscular, or intraperitoneal reaction mixtures, the cell preparation or reaction mixture further comprises i) a cytokine, ii) an antibody, antibody mimetic, or polypeptide capable of binding CD3, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, and/or CD82, and/or iii) a source of homologous antigen recognized by the CAR.

Additional cell preparation aspects and embodiments are provided below and in the detailed description herein beyond the illustrative embodiment section. Various volumes of cell preparations are provided herein for any cell preparation aspect. In some embodiments, the cell preparation has a volume of 3ml or greater, for example a volume of 3ml to 600ml, or between 50ml to 500ml, or between 100ml to 500 ml. In some embodiments, the cell preparation comprises hyaluronidase. In some embodiments, the cell preparation is 1ml to 10ml, 1ml to 5ml, 1ml to 3ml or 10ml, 5ml, 4ml, 3ml or 2ml or less, or less than 3ml, or any low volume element provided herein. In an illustrative embodiment, the cell preparation does not comprise hyaluronidase. Other volumes and formulations are provided herein. In some embodiments of any of the cell preparation aspects herein, the cell preparation is contained within a syringe. In some embodiments, for any of the cell preparations provided herein, the cell preparation is in an incubation bag or a blood processing bag. In an illustrative embodiment, the syringe is manufactured using Good Manufacturing Practice (GMP) and is GMP grade and quality.

In another aspect, provided herein is a method for modifying, genetically modifying, and/or transducing a lymphocyte (e.g., a T cell or NK cell) or population thereof, comprising contacting ex vivo a blood cell comprising a lymphocyte (e.g., a T cell or NK cell) or population thereof with a RIP comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in the lymphocyte (e.g., a T cell and/or NK cell), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes an anti-idiotype polypeptide and a CAR comprising an astm, transmembrane domain, and intracellular activation domain, one or more (e.g., two or more) inhibitory RNA molecules and polypeptide lymphoproliferative elements directed against one or more RNA targets, wherein the contacting promotes genetic modification and/or transduction of the lymphocyte (e.g., a T cell or NK cell) or at least some lymphocyte (e.g., a T cell and/or NK cell) by a promoter active in the lymphocyte, thereby producing a modified, genetically modified and/or transduced, e.g., a T cell and/or a transduced cell (e.g., a T cell and/or NK cell). In such methods, contacting is typically performed in a reaction mixture (sometimes referred to herein as a transduction reaction mixture) that comprises a population of lymphocytes (e.g., T cells and/or NK cells) and is contacted with a population of RIP. Various contact times are provided herein (including but not limited to in this illustrative example section) that may be used in this regard to facilitate membrane association and final membrane fusion of lymphocytes (e.g., T cells and/or NK cells) with RIP. In an illustrative embodiment, the contacting is performed for less than 15 minutes. In one aspect, provided herein is the use of RIP in the manufacture of a kit for modifying lymphocytes (e.g., T cells or NK cells) of a subject, wherein the use of the kit includes the aspects disclosed above in this paragraph and any of the related embodiments herein.

In some embodiments, the method may further comprise introducing the modified T cells and/or NK cells into the subject. In an illustrative embodiment, the blood cells comprising lymphocytes (e.g., T cells and/or NK cells) are from a subject, and thus the introduction is reintroduction. In this aspect, in some embodiments, a population of lymphocytes (e.g., T cells and/or NK cells) is contacted during the contacting step, modified, genetically modified, and/or transduced during the introducing step, and introduced into the subject.

In another aspect, provided herein is the use of RIP in the manufacture of a medicament for modifying lymphocytes, such as T cells and/or NK cells, of a subject, wherein the use of the medicament comprises the method aspects provided above in this paragraph.

In another aspect, provided herein is a kit for modifying NK cells and/or T cells comprising:

one or more first containers containing polynucleotides, typically substantially pure polynucleotides (e.g., found in recombinant retroviral particles according to any embodiment herein), comprising a first transcription unit operably linked to a promoter active in T cells and/or NK cells, wherein the first transcription unit encodes a first polypeptide comprising an anti-idiotype polypeptide and one or more of a CAR, a cytokine, and a lymphoproliferative element.

Provided in the following paragraphs are illustrative aspects and embodiments that may be used in or combined with any of the aspects or embodiments provided herein unless incompatible or otherwise indicated, as will be appreciated by one of ordinary skill in the art. In another aspect, provided herein are stably transfected or stably transcribed lymphocytes (e.g., T cells or NK cells) modified, in illustrative embodiments genetically modified, and in further illustrative embodiments prepared by modifying lymphocytes (e.g., T cells and/or NK cells) according to any of the methods herein.

In another aspect, provided herein is the use of RIP in a kit for modifying T cells and/or NK cells of a subject or in the preparation of a kit for modifying T cells and/or NK cells of a subject, wherein the use of the kit comprises any of the methods provided herein for delivering cells or modifying T cells and/or NK cells. In another aspect, provided herein is a use of RIP in a kit, or in the preparation of a kit for delivering modified lymphocytes to a subject, administering modified lymphocytes to a subject, injecting modified lymphocytes into a subject, and/or implanting modified lymphocytes in a subject, wherein the use of the kit comprises any of the methods provided herein for delivering to a subject, administering to a subject, injecting into a subject, and/or implanting in a subject. In another aspect, provided herein is the use of RIP in a kit or in the preparation of a kit for preparing a cell preparation, wherein the use of the kit comprises any of the methods provided herein for preparing a cell preparation comprising modifying T cells and/or NK cells. In another aspect, provided herein is RIP for subcutaneous delivery to a subject, wherein use of RIP includes any of the methods provided herein for subcutaneous delivery including RIP.

Exemplary embodiments, such as exemplary ranges and lists, are provided in the following paragraphs that may be used for any aspect provided above or otherwise provided herein unless incompatible with or otherwise indicated as would be recognized by one of skill in the art. In this specification, further aspects and embodiments are provided outside of this illustrative embodiment section.

In any aspect herein, the cell or lymphocyte is an NK cell, or in an illustrative embodiment, a T cell. It is to be understood that in aspects including collecting blood, such methods may include collecting a blood-derived product or a peripheral blood-derived product, which may be a blood sample, such as an unfractionated blood sample, or may include blood cells (e.g., white blood cells or lymphocytes) collected by apheresis.

In any aspect herein comprising a polynucleotide comprising one or more transcriptional units, the one or more transcriptional units may encode a polypeptide comprising LE. In some embodiments, the extracellular domain of LE is an anti-idiotype polypeptide. In some embodiments, the extracellular domain of LE further comprises any of the dimerization moieties disclosed herein, which in some embodiments may be considered a handle domain. In some embodiments, the dimerization motif may be a leucine-containing zipper motif polypeptide, CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271, or CD324, as well as mutants and/or active fragments thereof that retain the ability to dimerize.

In some embodiments, the lymphoproliferative element comprises an intracellular signaling domain from a cytokine receptor, which in illustrative embodiments activates the Janus kinase/signal transducer and transcription activator (JAK/STAT) pathway and/or the tumor necrosis factor receptor (TNF-R) related factor (TRAF) pathway. In illustrative embodiments, the lymphoproliferative element is generally constitutively active in that it constitutively activates one or more signaling pathways. In an illustrative embodiment, the lymphoproliferative element comprises a Box1 and optionally a Box2 JAK binding motif, and a STAT binding motif comprising a tyrosine residue. In some illustrative embodiments, the lymphoproliferative element does not comprise an extracellular ligand binding domain or a small molecule binding domain. In some embodiments, the constitutive active signaling pathway comprises activation of the PI3K pathway. In some embodiments, the constitutive active signaling pathway comprises activation of a PLC pathway. Thus, in certain embodiments, the lymphoproliferative elements used in any of the kits, methods, uses, or compositions herein are constitutively active and include intracellular signaling domains that activate the Jak/Stat pathway, TRAF pathway, PI3K pathway, and/or PLC pathway. Any of the polypeptide lymphoproliferative elements disclosed herein, such as, but not limited to, those disclosed herein in the section "lymphoproliferative element," or functional mutants and/or fragments thereof, may be encoded. In some embodiments, LE comprises a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids or to an intracellular domain from: IL7RA, IL 9D, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL13RA2, IL17RC, IL17RD, IL17RE IL20RA, IL20RB, IL 21D, CD RA1, IL 23D, CD RA, IL31RA, ITGA4, ITGA6, D, CD1, ITGB2, ITGB7, D, CD-1 (CD 11a/CD 18), D, CD1, D, CD 88, NKG 2D, CD80 (KLRF 1), A IL7RA, IL 9D, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21D, CD RA1, IL 23D, CD RA, IL31RA, ITGA4, ITGA6, D, CD1, ITGB2, ITGB7, D, CD-1 (CD 11a/CD 18), D, CD1, D, CD 88, NKG 2D, CD80 (KLRF 1) OSMR, OX40, PD-1, PRLR, PSGL1, PAG/Cbp, SLAM (SLAMF 1, CD150, IPO-3), SLAMF4 (C244, 2B 4), SLAMF6 (NTB-D, CD), SLAMF7, SLAMF8 (BLAME), SLP-76, TILR2, TILR4, TILR7, TILR9, TNFR2, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, TNFRSF18, TRANCE/RANKL, VLA1 or VLA-6, or functional mutants and/or fragments thereof, or a functional mutant and/or fragment thereof. In any of the embodiments disclosed herein, the lymphoproliferative element can comprise an extracellular ligand binding domain or a small molecule binding domain. In some embodiments, the lymphoproliferative element may include a transmembrane domain. In some embodiments, the transmembrane domain may include a transmembrane domain from: BAFFR, C3Z, CEACAM1, CD2, CD3A, CD3B, CD3D, CD3E, CD3G, CD3Z, CD, CD5, CD7, CD8A, CD8B, CD, CD11A, CD11A, CD11A, CD27, CD16, CD18, CD19, CD22, CD28, CD29, CD33, CD37, CD40, CD45, CD49A, CD49A, CD49A, CD64, CD79A, CD79A, CD80, CD84, CD86, CD96 (tactile), CD100 (SEMA 4D), CD103, C134 CD137, CD154, CD160 (BY 55), CD162 (SELPLG), CD226 (DNAM 1), CD229 (Ly 9), CD247, CRLF2, CRTAM, CSF2RA, CSF2RB, CSF 3A, CD 1A, CD 2A, CD2, GHR, HVEM (LIGHTR), IA4, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA IL 4A, CD RA, IL 6A, CD ST, IL7RA Ins PPCL, IL 9A, CD RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL 21A, CD RA1, IL 23A, CD RA, IL31RA, ITGA1, ITGA4, ITGA6, A, CD1, ITGB2, ITGB7, KIRDS2, LEPR, LFA-1 (CD 11a, CD 18), A, CD (KLRF 1), OSMR, PAG/A, CD1, SLAM (SLAMF 1, CD150, IPO-3), AMF4 (CD 244, 2B 4), SLF 6 (NTB-A, CD 108), SLAMF7, SLAMF8 (SLSF 8, TNSF 2, TNSF 6, or a mutant thereof.

In some embodiments herein including any aspect of the RIP, the RIP may comprise a binding polypeptide and a fusogenic element, and may include a polynucleotide encoding any of the anti-idiotype polypeptides disclosed herein, which in illustrative embodiments is the genome of the RIP. In some embodiments, the polynucleotide in the RIP encodes an anti-idiotype polypeptide provided herein, which generally further encodes a CAR, a lymphoproliferative element, one or more inhibitory RNA molecules, and/or a cytokine. In some embodiments, the polynucleotide in the RIP encodes an anti-idiotype polypeptide and CAR provided herein, and optionally further encodes a lymphoproliferative element, one or more inhibitory RNA molecules, and/or a cytokine. In some embodiments, the one or more viral envelope proteins include a binding polypeptide and a fusogenic element. In some embodiments, the viral envelope protein is a mutant viral envelope protein, wherein the binding polypeptide of the viral envelope protein has been mutated to reduce/eliminate binding to a target cell (e.g., a T cell), but wherein such binding is provided by another (e.g., heterologous) binding polypeptide, which in further illustrative embodiments is also an activating element (e.g., a CD3 binding polypeptide) as provided herein. In some embodiments, the viral envelope protein comprises a feline endogenous virus (RD 114) envelope protein, a tumor retrovirus amphotropic envelope protein, a tumor retrovirus mono-amphotropic envelope protein, a vesicular stomatitis virus envelope protein (VSV-G), baboon retrovirus envelope glycoprotein (BaEV), murine leukemia envelope protein (MuLV), influenza glycoprotein HA surface glycoprotein (HA), influenza glycoprotein Neuraminidase (NA), paramyxovirus measles envelope protein H, paramyxovirus measles envelope protein F, tree shrew paramyxovirus (TPMV) envelope protein H, TPMV envelope protein F, nipah virus (NiV) envelope protein H, niV envelope protein G, SINV protein E1, SINV protein E2, and/or a functional variant or fragment of any of these envelope proteins. In some embodiments, the viral envelope protein is NiV envelope protein G, wherein the NiV envelope protein G comprises one or more mutations in residues Y389, E501, W504, E505, V507, Q530, E533, or I588 of SEQ ID NO:375. In some embodiments, the henipav virus-G protein is SEQ ID No. 375 having mutations E533A and/or Q530A. In some embodiments, one or more N-glycosylation sites or O-glycosylation sites are mutated to improve pseudotyping and fusion. In some embodiments, one or more N-glycosylation sites are mutated to another amino acid, such as glutamine, for example, but not limited to, at one or more of N72, N159, N306, N378, N417, N481, or N529 of SEQ ID NO:375, or at the corresponding glutamine of other Huntington's virus-G protein. In some embodiments, one or more O-glycosylation sites are mutated from serine or threonine to another amino acid, such as alanine. In some embodiments, one or more serine or threonine residues in the highly O-glycosylated stem domain of amino acids 103 to 137 from SEQ ID NO. 375 are mutated to, for example, alanine. In other embodiments, the C-terminus of the henipav-G protein may be modified and fused to a binding polypeptide (and in the illustrative embodiment an activating element), such as an antibody or antibody mimetic, which in the illustrative embodiment may be an anti-CD 3 antibody or antibody mimetic.

In any of the aspects and embodiments provided herein that include a RIP, the RIP includes on its surface a pseudotyped element that is capable of binding to T cells and/or NK cells and promoting membrane fusion of the RIP therewith. In some embodiments, the pseudotyped element is a viral envelope protein. In some embodiments, the viral envelope protein is one or more of the following: feline endogenous virus (RD 114) envelope protein, tumor retrovirus amphotropic envelope protein, vesicular stomatitis virus envelope protein (VSV-G), baboon retrovirus envelope glycoprotein (BaEV), murine leukemia envelope protein (MuLV) and/or paramyxovirus measles envelope proteins H and F, tree shrew paramyxovirus (TPMV) envelope protein H, TPMV envelope protein F, nipah virus (NiV) envelope protein F, niV envelope protein G, sindbis virus (SINV) protein E1, SINV protein E2, or any fragment thereof that retains the ability to bind to resting T cells and/or resting NK cells. In an illustrative embodiment, the pseudotyped element is VSV-G. As discussed elsewhere herein, a pseudotyped element can include a fusion with a T cell activating element, which in an illustrative embodiment can be a fusion with any envelope protein pseudotyped element (e.g., muLV or VSV-G) and an anti-CD 3 antibody. In other illustrative embodiments, pseudotyped elements include VSV-G and fusions of anti-CD 3scFv with MuLV.

In any aspect herein that includes a RIP, the RIP may include an activation element on its surface. In some embodiments, the activating element on the surface is a membrane-bound T cell activating element. In some embodiments, the activating element is a polypeptide capable of binding to a polypeptide on the surface of a lymphocyte and in illustrative embodiments a T cell and/or NK cell. In some sub-embodiments of these and embodiments of any aspect provided herein,

in some embodiments, the T cell activating element comprises one or more of an antibody or antibody mimetic or mitogenic tetraspanin capable of binding to CD28, CD3, tcra/β, CD28, or wherein the T cell activating element is mitogenic tetraspanin. In some embodiments, the T cell activating element comprises an antibody or antibody mimetic capable of binding to CD3, and wherein the T cell activating element is bound to a membrane of the RIP. In some embodiments, the membrane-bound anti-CD 3 antibody or anti-CD 3 antibody mimetic is an anti-CD 3 scFv, an anti-CD 3 scffc, or an anti-CD 3 DARPin. In some embodiments, the anti-CD 3 antibody or anti-CD 3 antibody mimetic is bound to the membrane by a GPI anchor, such as a heterologous GPI anchor linker, wherein the anti-CD 3 antibody or anti-CD 3 antibody mimetic is a recombinant fusion protein with a MuLV viral envelope protein, with or without a mutation at the furin cleavage site, or wherein the anti-CD 3 antibody or anti-CD 3 antibody mimetic is a recombinant fusion protein with a VSV viral envelope protein, or wherein the anti-CD 3 antibody or anti-CD 3 antibody mimetic is a recombinant fusion protein with a henipa virus-G envelope protein, or wherein the anti-CD 3 antibody is a recombinant fusion protein with a NiV viral envelope protein. In some embodiments, the polypeptide capable of binding to CD28 is CD80 or an extracellular domain thereof that binds to a CD16B GPI anchor linkage sequence.

In an illustrative embodiment, the activating element is a T cell activating element capable of binding to a TCR complex polypeptide. In some embodiments, the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCR a or tcrp. In some embodiments, the activating element capable of binding to a TCR complex polypeptide is a polypeptide capable of binding to one or more of CD3D, CD3E, CD3G, CD3Z, TCR a or TCR β. In an illustrative embodiment, the activation element activates ZAP-70. In some embodiments, the activating element comprises a polypeptide capable of binding to CD16A, NKG2C, NKG2E, NKG2F or NKG 2H. In further embodiments, polypeptides capable of binding to CD16A include polypeptides capable of binding to one or more of NKp46, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG2F, or NKG 2H. In some embodiments, the activating element is a polypeptide capable of binding to one or more of the following combinations: NKp46 and 2B4, NKp46 and CD2, NKp46 and DNAM, NKp46 and NKG2D, 2B4 and DNAM, or 2B4 and NKG2D. In some embodiments, the activating element may be two or more polypeptides capable of binding to polypeptides on the surface of lymphocytes. In some embodiments, the activating element may be two or more polypeptides capable of binding to at least one of the following combinations: NKp46 and 2B4, NKp46 and CD2, NKp46 and DNAM, NKp46 and NKG2D, 2B4 and DNAM, or 2B4 and NKG2D.

In some embodiments, provided herein as a separate aspect, or as a component of, or as a result of, or used in, the methods, uses, and compositions provided herein are modified cells (in the illustrative embodiments T cells) having a darkened surface polypeptide, or a population of any of the foregoing modified cells having a darkened surface polypeptide. Such modified cd4+ cells or cd8+ cells or populations thereof may be CD 3-darkened and may have the following characteristics (referred to herein as "darkened T cell characteristics") and, in illustrative embodiments, the following characteristics when forming and/or administering a cell preparation:

i) At least 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 10% and 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 50% and 60%, 70%, 80%, 90%, 95% or 99%, or between 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, as the low end, and 99% as the high end of the cd4+ cells in the cell preparation are surface CD3-;

ii) at least 18%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 20% and 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 50% and 60%, 70%, 80%, 90%, 95% or 99%, or between 18%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as the low end to 99% of the cd8+ cells as the high end are surface CD3-;

iii) At least 10.5%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 10% and 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 25% and 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 50% and 60%, 70%, 80%, 90%, 95% or 99%, or between 10.5%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as the lower end and 99% as the upper end of the population of cells that are cd4+ or cd8+ in the cell preparation;

iv) at least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 11%, or between 1.5% and 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 11%, or between 5% and 6%, 7%, 8%, 9%, 10% or 11% of the cell preparation; or between 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% to 11% of the cells (excluding RBCs) are surface CD3-cd4+;

v) at least 0.65%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% in the cell preparation; between 0.65% and 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%; between 1% and 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%; or between 0.65%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5% of the cells (excluding RBCs) as the low end to 5% as the high end are surface CD3-cd8+;

vi) less than 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the cells (excluding RBCs) in the cell preparation are surface cd3+ and cd4+ or cd8+ ("total cd3+ cell percentage");

vii) less than 89%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%, or between 89% and 50%, 40%, 30%, 20%, 10%, 5% or 1%, or between 75% and 50%, 40%, 30%, 20%, 10%, 5% or 1%, or between 50% and 40%, 30%, 20%, 10%, 5% or 1%, or between 1% and 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or 89% of the population of cells in the cell preparation are surface cd3+;

viii) the surface expression of CD3 of cd4+ and/or cd8+ cells in a cell preparation is lower than the surface expression of CD3 on cd4+ and/or cd8+ cells in blood collected from a healthy subject or population of healthy subjects, wherein the surface expression of CD3 in the cell preparation is reduced by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or 10% to 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or 50% to 60%, 70%, 80%, 90%, 95% or 99%, or 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as the low end to 99% as the high end; and/or

ix) the total cd3+ cell percentage is reduced by at least 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% after contacting the polynucleotide vector (and RIP in the illustrative embodiment) as compared to the total cd3+ cell percentage without contacting the polynucleotide vector (and RIP in the illustrative embodiment).

In some embodiments, provided herein as a separate aspect, or as a component of, or as a result of, or used in, the methods, uses, and compositions provided herein are modified cells (NK cells modified in the illustrative embodiments) with a darkened surface polypeptide, or a population of modified cells with a darkened surface polypeptide. Such modified cells, e.g., modified NK cells or populations thereof, may have one or more of darkened CD16A, NKp46, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG F, or NKG2H, and may have the following characteristics (referred to herein as "darkened NK cell characteristics"), and in illustrative embodiments the following characteristics when forming and/or administering a cell preparation:

i) At least 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or between 10% and 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or between 50% and 60%, 70%, 80%, 90%, 95%, or 99%, or between 9%, 10%15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as the low end and 99% as the high end of the cell preparation are cd56+ cells surface CD16A-, NKp46-, 2B4-, CD2-, DNAM-, NKG2C-, NKG2D-, NKG2E-, NKG2F-, and/or NKG2H-;

ii) surface CD16A-, NKp46-, 2B4-, CD2-, DNAM-, NKG2C-, NKG2D-, NKG2E-, NKG2F-, and/or NKG2H-, in at least 10.5%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or between 10% to 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or between 50% to 60%, 70%, 80%, 90%, 95%, or 99%, or between 10.5%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as the lower end to 99% as the upper end of the population of cells being cd56+ in the cell preparation;

iii) At least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 11%, or between 1.5% and 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 11%, or between 5% and 6%, 7%, 8%, 9%, 10% or 11% of the cell preparation; or between 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% to 11% of cells (excluding RBCs) are CD56+ and surface CD16A-, NKp46-, 2B4-, CD2-, DNAM-, NKG2C-, NKG2D-, NKG2E-, NKG 2F-and/or NKG 2H-and/or NKG 2-and/or the whole cell is a cell

iv) less than 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the cells (excluding RBCs) in the cell preparation are surface cd16a+, nkp46+, 2b4+, cd2+, dnam+, nkg2c+, nkg2d+, nkg2e+, nkg2f+ and/or nkg2h+ and cd56+ ("total percentage of CD16A, NKp, 2B4, CD2, DNAM, nkg2C, NKG2D, NKG2E, NKG F and/or NKG2H cells");

v) surface cd16a+, nkp46+, 2b4+, cd2+, dnam+, nkg2c+, nkg2d+, nkg2e+, nkg2f+ and/or nkg2h+ and cd56+ within less than 89%, 80%, 75%, 70%, 50%, 40%, 30%, 25%, 15%, 10%, 5%, or 1%, or between 89% to 50%, 40%, 30%, 20%, 10%, 5% or 1%, or between 50% to 40%, 30%, 20%, 10%, 5% or 1%, or between 1% to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or 89% of the population of cells in the cell preparation that are cd56+;

vi) CD56+ cells in the cell preparation have lower surface expression of CD16A, NKp46, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG2F and/or NKG2H, respectively, than surface expression of CD16A, NKp46, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG2F and/or NKG2H on CD56+ cells in blood collected from a healthy subject or population of healthy subjects, wherein the surface expression of CD16A, NKp46, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG2F and/or NKG2H in the cell preparation is reduced by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 10% to 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or between 50% to 60%, 70%, 80%, 90%, 95% or 99%, or between 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as low end to 99% as high end; and/or

vii) the percentage of total CD16A, NKp46, 2B4, CD2, NKG2C, NKG2D, NKG2E, NKG F and/or NKG2H cells is reduced by at least 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% after contacting the polynucleotide vector (and RIP in the illustrative embodiment) compared to the percentage of total CD16A, NKp, 2B4, CD2, DNAM, NKG2C, NKG2D, NKG2E, NKG F and/or NKG2H cells without contacting the polynucleotide vector (and RIP in the illustrative embodiment).

In any aspect herein, in an illustrative embodiment comprising a darkened T cell signature or a darkened NK cell signature, the modified cell may have been recently activated within the previous 7, 6, 5, 4, 3, 2, or 1 day.

In some embodiments of any cell preparation aspect or embodiment herein, or in any method or use aspect that includes a cell preparation, or in any population embodiment, some of the modified cd4+, modified cd8+, modified cd56+, modified T cells and/or modified NK cells are in a cell aggregate. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, or 25%, or between 1% and 10%, 15%, 20%, 25%, 50%, and 75% of the white blood cells, modified cd4+ cells, modified cd8+ cells, modified cd56+ cells, modified T cells, and/or modified NK cells in the cell aggregate. In some embodiments, the cell aggregates have a diameter greater than 15 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, or 60 μm, or a diameter between 25 μm and 50 μm, 60 μm, 75 μm, 100 μm, 125 μm, 150 μm, 200 μm, or 250 μm. In some embodiments, the modified cell is an aggregate comprising at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, or 10,000 white blood cells or 5 to 500, 5 to 250, 5 to 100, 10 to 500, 10 to 250, or 10 to 100 white blood cells. Furthermore, in some embodiments (including sub-embodiments of immediately preceding embodiments), at least 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, or 25%, or between 1% to 10%, 15%, 20%, 25%, 50%, and 75% of the white blood cells, modified T and/or NK cells in the cell preparation are aggregates comprising or consisting of at least 4, 5, 6, 8, or 5 to 500, 5 to 250, 5 to 100, 10 to 500, 10 to 250, or 10 to 100 white blood cells, modified T cells, and/or NK cells. Furthermore, in some embodiments, the cell preparation comprises an aggregate of modified T cells and/or NK cells, in some embodiments together with unmodified T cells and/or NK cells and/or other leukocytes, which can be retained by a coarse filter having a pore size of at least 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm or 60 μm. In certain illustrative embodiments, at least 5% of the leukocytes, T cells, NK cells, modified T cells, and/or modified NK cells are in the cell aggregate. In certain sub-embodiments, the cell aggregates have a diameter greater than 40 μm and/or can be retained by a coarse filter having a pore diameter of at least 40 μm. In some sub-embodiments, the cell aggregates comprise 5 to 500 white blood cells or modified T cells.

In some embodiments herein comprising administering any aspect or embodiment of a cell, population or cell preparation to a subject, and in illustrative embodiments genetically modified T cells and/or NK cells, or progeny cells derived from the modified cells or populations of genetically modified progeny cells, remain in the subject for at least 1, 2, 3, 4, 5, 6, 7, 14, 17, 21, or 28 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or 1, 2, 3, 4, or 5 years after administration. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the genetically modified cells (and in the illustrative embodiments CAR-T cells) express the first polypeptide comprising the transgene, and in the illustrative embodiments the engineered T cell receptor or CAR. In some embodiments, at least 50%, 60%, 70%, 80%, 90% or 95% of the genetically modified cells expressing the first polypeptide comprising the transgene circulate in the blood and/or at the site of a tumor (e.g., a solid tumor), and the remainder of the genetically modified cells are subcutaneous. In some embodiments, the persistent population is subcutaneous, circulates in the blood, and/or is located at a site of a tumor, such as a solid tumor. In some embodiments, the subcutaneous region does not contain an artificial matrix component.

In some embodiments, the persisting population is detectable by histology. In some embodiments, the persisting population remains subcutaneously for at least or up to 14, 21, 28, 50, 60, 90 days and is detectable by histology. In some embodiments, the persisting population can be detected by FAC, e.g., FACs for CAR or tag-removed (e.g., eTag), e.g., as 2 genetically modified cells/μl blood, or by qPCR, e.g., transgenic qPCR or sequencing or chimeric ligation through CAR, or for non-human subjects treated with human engineered cells, e.g., human CAR-T cells, human RNAse P (hrnase P). In some embodiments, a persistent population can be detected in the blood.

In some embodiments of any aspect provided herein, including but not limited to the methods and uses aspects provided herein above in this illustrative embodiment section, the modified cells, e.g., modified T cells and/or NK cells or populations thereof, have a darkened surface polypeptide, which in illustrative embodiments may be a TCR complex polypeptide, and in illustrative sub-embodiments CD3. In illustrative embodiments, such darkened cells (including populations thereof) exhibit any of the darkened T cell characteristics and/or darkened NK cell characteristics provided herein.

In some embodiments of any aspect provided herein, including but not limited to the methods and uses provided above in this illustrative embodiment section, as disclosed herein, some (e.g., at least 5%, 7.5%, or 10%) of the modified cells (e.g., modified T cells and/or NK cells or populations thereof) or populations thereof are in an aggregate.

In some embodiments of any aspect provided herein, including but not limited to the methods and uses aspects provided above in this illustrative embodiment section, the cell-forming population, which may be a persistent population as disclosed herein.

In or any aspect or embodiment herein including a population of a durable population or progeny cells, the modified cells (such as modified T cells and/or NK cells) and in the illustrative embodiments the number of genetically modified T cells and/or NK cells includes at least 100, 1 x 10 ³ 、1×10 ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ 、1×10 ¹⁰ 、1×10 ¹¹ Or 1X 10 ¹² Individual cells, or 1X 10 ³ Up to 1X 10 ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ Or 1X 10 ⁹ Individual cells. In some embodiments, the modified cells present in the cell preparation administered to the subject and the T cells and/or NK cells modified in the illustrative embodiments proliferate in the subject At least 5, 10, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, or 10,000 fold, e.g., to form a population of durable or progeny cells.

In some embodiments, the population of the durable population or progeny cells expresses an engineered T cell receptor or CAR, and the population of the durable population or progeny cells is indirectly detected by a durable clinical response. For example, such persistence may be detected by detecting a stable disease, a partial response, or a complete response, wherein the duration of the response is at least 3, 6, 9, 12, 18, or 24 months after initial observation of the clinical response, which in some embodiments is a stable disease for a patient experiencing a progressive disease prior to administration of the cells (and in illustrative embodiments, administration of the engineered T cells or CAR-T therapy).

In any aspect provided herein that includes the step of collecting blood, the volume of blood collected may be, for example, 5ml to 600ml. More volume and scope are provided elsewhere in this specification, and in some embodiments, include the low volume elements provided herein. In some embodiments, when the collected blood is treated with a filter (a leukopenia filter in the illustrative embodiment), the volume of the blood sample applied to the filter is 600, 500, 400, 300, 200, 150, 120, 100, 75, 50, 40, 30, 25, 20, 15, 10, or 5ml or less. In illustrative embodiments, the volume of blood sample applied to the filter is 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1ml or less.

In some embodiments of any aspect provided herein, the cell preparation that may be in a syringe in an illustrative embodiment has 0.5ml to 20ml, 15ml, 10ml, 5ml, or 1ml; or 1ml to 20ml, 15ml, 10ml, 5ml, 4ml, 3ml, 2ml or less; or 2ml to 20ml, 15ml, 10ml, 7ml or 5ml; or a volume of 5ml to 20ml, 15ml or 10ml, or 3ml to 12ml, or less than 3 ml. In some embodiments of any aspect or embodiment provided herein, wherein blood is collected from the subject, the collected blood has a concentration of between 2.5ml to 75ml, 60ml, 50ml, 40ml, 30ml, 25ml, 20ml, 15ml, 10ml, or 5mlOr between 5ml and 75ml, 60ml, 50ml, 40ml, 30ml, 25ml, 20ml, 15ml or 10ml, or between 10ml and 75ml, 60ml, 50ml, 40ml, 30ml, 25ml or 20ml, or between 15ml and 75ml, 60ml, 50ml, 40ml, 30ml, 25ml or 20ml, or between 20ml and 75ml, 60ml, 50ml, 40ml, 30ml or 25ml, or between 25ml and 75ml, 70ml, 60ml, 50ml, 40ml and 30ml, or between 5ml, 10ml or 15ml as the lower end and 20ml as the upper end. In some embodiments, when the collected blood is treated with a filter (a leukopenia filter in the illustrative embodiment), the volume of the blood sample or fraction thereof applied to the filter may be 2.5ml to 75, 50, 40, 30, 25, 20, 15, or 10. In an illustrative embodiment, the volume of the whole blood sample or fraction thereof applied to the filter is between 10ml and 50ml, 25ml, 20ml, 15 ml. In illustrative embodiments, the volume of the whole blood sample or fraction thereof applied to the filter is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1ml or less. In some embodiments of any aspect or embodiment provided herein, the volume of the reaction mixture is between 2.5ml and 75ml, 60ml, 50ml, 40ml, 30ml, 25ml, 20ml, 15ml, 10ml or 5ml, or between 5ml and 75ml, 70ml, 60ml, 50ml, 40ml, 30ml, 25ml, 20ml or 10ml, or between 10ml and 75ml, 70ml, 60ml, 50ml, 40ml, 30ml, 25ml, 20ml or 15ml, or between 15ml and 75ml, 70ml, 60ml, 50ml, 40ml, 30ml, 25ml or 20ml, or between 20ml and 75ml, 70ml, 60ml, 50ml, 40ml, 30ml or 25ml, or between 25ml and 75ml, 70ml, 60ml, 50ml, 40ml and 30 ml. The volumes of cell preparation, collected blood and reaction mixture in this paragraph are referred to herein as "small volume elements". In an illustrative sub-embodiment of the embodiment comprising a small volume element, the cell preparation is suitable for subcutaneous delivery, wherein the number of modified cells (such as modified T cells and/or NK cells in the modified cell preparation) is 1.5 x 10 ⁴ Up to 1.5X10 ⁹ 、1×10 ⁹ 、1×10 ⁸ Or 1X 10 ⁷ Or 1X 10 ⁵ Up to 1.5X10 ⁸ Or 1X 10 ⁵ Up to 1X 10 ⁷ Or 1X 10 ⁶ Up to 1X 10 ⁸ Or 2X 10 ⁶ Up to 1X 10 ⁸ Or in the illustrative embodiment 3 x 10 ⁴ Up to 3X 10 ⁷ ，1×10 ⁵ Up to 3X 10 ⁷ Or 1X 10 ⁶ Up to 3X 10 ⁷ The modified T cells, NK cells, cd4+ cells, cd8+ cells and/or cd56+ cells.

In some embodiments, the contacting step is performed in a blood processing bag or other incubation bag, for example, wherein whole blood or a fraction thereof is added to an incubation bag containing RIP to form a reaction mixture, or wherein RIP is added to an incubation bag containing whole blood to form a reaction mixture.

In illustrative embodiments of any of the aspects provided herein or any other aspects including polynucleotide vector particles (e.g., polynucleotide vector particles), and in illustrative embodiments, the polynucleotide vector particles are substantially free of protein transcripts encoded by nucleic acids of the polynucleotide vector particles, e.g., substantially free of engineered T cell receptors or CARs encoded by nucleic acids of the polynucleotide vector particles.

In some embodiments, a sample, such as a blood sample or reaction mixture, is applied to the leukoreduction filter, e.g., to remove RIP that is not associated with lymphocytes, either before, during, or after incubation. In some embodiments, at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the RIP not associated with lymphocytes is removed from the reaction mixture. In some embodiments, the reaction mixture is filtered on a leukoreduction filter at a flow rate of 0.25 to 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5ml/min, or 0.5ml/min to 2 ml/min. In some embodiments, the reaction mixture is filtered on a leukoreduction filter using a syringe. In some embodiments, the angle of the syringe relative to a channel (e.g., a conduit) in fluid communication with the leukoreduction filter is less than 80 °, 75 °, 70 °, 65 °, 60 °, 55 °, 50 °, or 45 ° when filtering the reaction mixture. In some embodiments, during RIP removal, blood cells and modifications Is not moved across the junction at an angle greater than 70 °, 75 °, or 80 °. In some embodiments, the leukopenia filter has 3cm ² To 5cm ² And in these or other embodiments, the pores in the filter have a diameter of between 2 μm and 6 μm. In some embodiments, the cells retained by the leukopenia filter after filtering the reaction mixture through the leukopenia filter are washed with a wash buffer having a volume that is 0.25 to 3 times the volume of the reaction mixture. In some embodiments, the removal of the RIP is performed within a filter assembly comprising a syringe, a leukoreduction filter in fluid communication with the syringe, and one or more bags in fluid communication with the leukoreduction filter. In some embodiments, the removal of the RIP is performed within a filter assembly comprising a second syringe and a second bag, wherein the second bag is in fluid communication with the leukoreduction filter.

In any aspect provided herein that includes a polynucleotide (e.g., an isolated polynucleotide encoding an anti-idiotype polypeptide and one or more of a CAR, a cytokine, one or more inhibitory RNAs, and/or LE), such a polynucleotide or isolated polynucleotide can be contained in one or more containers, and for example in a solution of 0.1ml to 10 ml. Such polynucleotides may comprise a substantially pure GMP-grade recombinant vector (e.g., replication-defective retroviral particles). In some embodiments, such polynucleotides comprise a recombinant naked DNA vector. In an illustrative embodiment, such polynucleotides are those having a size of 1X 10 ⁶ Up to 5X10 ⁹ 、1×10 ⁷ Up to 1X10 ⁹ 、5×10 ⁶ Up to 1X10 ⁸ 、1×10 ⁶ Up to 5X10 ⁷ 、1×10 ⁶ Up to 5X10 ⁶ Or 5x10 ⁷ Up to 1x10 ⁸ A retroviral Transduction Unit (TU) or TU/ml, or at least 100, 1,000, 2,000 or 2,500TU/ng p 24.

In any of the aspects provided herein, the contacting step, including in combination with optional incubation, can be (or can occur) for 14, 12, or 10 hours or less, or in the illustrative embodiments for 8, 6, 4, 3, 2, or 1 hour or less, or in certain other illustrative embodiments for less than 8 hours, less than 6 hours, less than 4 hours, 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, although in each case at least an initial contacting step is present in which the retroviral particles and cells are contacted in suspension in the transduction reaction mixture. In other embodiments, the reaction mixture may be incubated for 15 minutes to 12 hours, 15 minutes to 10 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1 hour, 15 minutes to 45 minutes, or 15 minutes to 30 minutes. In other embodiments, the reaction mixture may be incubated for 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, or 30 minutes to 45 minutes. In other embodiments, the reaction mixture may be incubated for 1 hour to 12 hours, 1 hour to 8 hours, 1 hour to 4 hours, or 1 hour to 2 hours. In another illustrative embodiment, the contacting is performed only between an initial contacting step (in the reaction mixture, including free retroviral particles in suspension and cells in suspension without any further incubation) and without any further incubation in the reaction mixture, or an incubation in the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour. In certain embodiments, the contacting may be performed (or may occur) for 30 seconds or 1, 2,5, 10, 15, 30, or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours as the low end of the range to 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours as the high end of the range. In illustrative embodiments, the contacting may occur (or may occur) only for 30 seconds or 1, 2,5, 10, 15, 30 or 45 minutes or 1 hour as the low end of the range to 2, 4, 6, and 8 hours as the high end of the range. In some embodiments, RIP may be washed off immediately after addition to the cells to be modified, genetically modified, and/or transduced, such that the contact time is implemented for the length of time it takes to wash off RIP. Thus, typically, contacting comprises at least an initial contacting step in which the retroviral particles are contacted with the cells in suspension in the transduction reaction mixture. Such methods can be performed without prior activation.

In illustrative embodiments of the methods provided herein, the contacting step with optional incubation is performed at a temperature of 32 ℃ to 42 ℃ (e.g., at 37 ℃ as provided in more detail herein). In other illustrative embodiments, the contacting step with optional incubation is performed at a temperature below 37 ℃, e.g., at 1 ℃ to 25 ℃, 2 ℃ to 20 ℃, 2 ℃ to 15 ℃, 2 ℃ to 6 ℃, or 3 ℃ to 6 ℃. The optional incubation associated with the contacting step at these temperatures may be performed for any of the lengths of time described herein. In illustrative embodiments, the optional incubations associated with these temperatures are performed for 1 hour or less, for example, 0 to 55 minutes (i.e., 55 minutes or less), 0 to 45 minutes (i.e., 45 minutes or less), 0 to 30 minutes (i.e., 30 minutes or less), 0 to 15 minutes (i.e., 15 minutes or less), 0 to 10 minutes (i.e., 10 minutes or less), 0 to 5 minutes (i.e., 5 minutes or less), 5 to 30 minutes, 5 to 15 minutes, or 10 to 30 minutes. In further illustrative embodiments, the cold contacting and incubating is performed at a temperature of 2 ℃ to 15 ℃ for 0 to 55 minutes, 0 to 45 minutes, 0 to 30 minutes, 0 to 15 minutes, 0 to 10 minutes, 0 to 5 minutes, 5 to 15 minutes, or 10 to 30 minutes. In other further illustrative embodiments, the cold contacting and incubating is performed at a temperature of 1 ℃ to 25 ℃, 2 ℃ to 20 ℃, 2 ℃ to 15 ℃, 2 ℃ to 6 ℃, or 3 ℃ to 6 ℃ for 5 to 30 minutes.

In certain embodiments, which include a contacting step at a colder temperature provided immediately above, the secondary incubation is typically performed by suspending the cells in a solution comprising the recombinant vector (in the illustrative embodiment, retroviral particles) after the optional washing step. In an illustrative embodiment, the secondary incubation is performed at a temperature between 32 ℃ and 42 ℃ (e.g., at 37 ℃). The optional secondary incubation may be performed for any length of time described herein. In illustrative embodiments, the optional secondary incubation is performed for 6 hours or less, for example, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, 2 to 4 hours, 30 minutes to 4 hours, 10 minutes to 4 hours, 5 minutes to 1 hour, 1 minute to 5 minutes, or less than 5 minutes. Thus, in some illustrative embodiments, optionally, the T cell and/or NK cell activating element is contacted on the surface of the RIP at 2 ℃ to 15 ℃, and optionally at 2 ℃ to 6 ℃ for less than 1 hour, optionally after which TNC is incubated at 32 ℃ to 42 ℃ for 5 minutes to 8 hours, or in illustrative embodiments, 5 minutes to 4 hours, and optionally after which modified T cells and/or NK cells are collected on a filter to form a cell preparation.

In some embodiments, no more than 16 hours, 14 hours, 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour, or 5, 10, 15, 30, 45, or 60 minutes as the low end of the range to 1.5, 2, 4, 6, 8, 10, 12, 14, and 16 hours as the high end of the range, e.g., 5 minutes to 16 hours, 5 minutes to 12 hours, 5 minutes to 8 hours, 5 minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 3 hours, 5 hours to 2 hours, or 5 minutes to 1 hour, between the time that blood, TNC, or PBMC are contacted with the recombinant nucleic acid vector (which in the illustrative embodiment is a replication-defective retroviral particle) and the time that the modified cells are suspended and thus formulated in the delivery solution to form a cell preparation. In some embodiments, the time between when the cells are contacted with the replication defective retroviral particle and when the modified cells are formulated in the delivery solution may be 1 to 16 hours, 1 to 14 hours, 1 to 12 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, or 1 to 2 hours. In some embodiments, no more than 16 hours, 14 hours, 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour elapse between the time blood is collected from the subject to the time the modified lymphocytes are reintroduced into the subject. In some embodiments, the time between when blood is collected from the subject and when the modified lymphocytes are reintroduced into the subject may be 1 to 16 hours, 1 to 14 hours, 1 to 12 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, or 1 to 2 hours.

In any aspect provided herein that includes an administration step, in illustrative embodiments, the administered cells are treated ex vivo for less than 24, 18, 12, 10, 8, 6, 4, 2, or 1 hour or 30 minutes or 15 minutes, or for 15 minutes to 24, 18, 12, 10, 8, 6, 4, 2, 1, or 0.5 hours, or for 1 hour to 24, 18, 12, 10, 8, 6, 4, or 2 hours, prior to administration, e.g., using any of the methods provided herein that include the contacting and formulating steps. Thus, in certain embodiments, such an ex vivo time may be the time between the collection of blood from the subject and intravenous, intramuscular, intratumoral, intraperitoneal administration to the subject, and in the illustrative embodiment subcutaneous administration of the modified lymphocytes (derived from the subject in the illustrative embodiment).

In some embodiments of any of the related aspects herein, some or all of the T cells and NK cells have not expressed the recombinant nucleic acid or have not integrated the recombinant nucleic acid into the genome of the cell prior to being used in or included in any of the methods or compositions provided herein, including but not limited to being introduced or reintroduced back into the subject, or prior to being used in or at the time of being used to prepare a cell preparation. In some embodiments, at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified T cells and NK cells do not express a CAR or a transglutaminase, and/or do not have a CAR associated with their cell membrane, when the modified lymphocytes are introduced or reintroduced back into the subject, and in illustrative embodiments are subcutaneously or intramuscularly introduced or reintroduced back into the subject, or when used to prepare a cell preparation. In other embodiments, provided herein are cell preparations wherein at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified T cells and/or NK cells in the cell preparations comprise recombinant viral reverse transcriptase and/or integrase. In illustrative embodiments, at least 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified T cells and NK cells do not express a CAR and/or do not have a CAR associated with their cell membrane when the modified lymphocytes are introduced or reintroduced back into the subject, and in illustrative embodiments when subcutaneously or intramuscularly introduced or reintroduced back into the subject, or when used to prepare a cell preparation. In illustrative embodiments, at least 25%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified T cells and NK cells do not express recombinant mRNA (e.g., encode a CAR) when lymphocytes are introduced or reintroduced into a subject, and in illustrative embodiments when introduced or reintroduced into a subject subcutaneously or intramuscularly, or when used to prepare a cell preparation. In some embodiments, greater than 50%, 60%, 70%, 75%, 80%, or 90% of the cells, NK cells, and/or T cells in the cell preparation are viable.

In some embodiments, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified T cells and NK cells do not have a recombinant nucleic acid stably integrated into their genomes when lymphocytes are introduced or reintroduced into a subject, and in illustrative embodiments are subcutaneously or intramuscularly introduced or reintroduced into a subject, or when used to prepare a cell preparation. In illustrative embodiments, at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified T cells and NK cells do not have a recombinant nucleic acid stably integrated into their genomes when lymphocytes are introduced or reintroduced into a subject, and in illustrative embodiments when introduced or reintroduced into a subject subcutaneously or intramuscularly, or when used to prepare cell preparations. In some embodiments herein, including any aspect of modified, genetically modified, transduced and/or stably transfected lymphocytes, any percentage of lymphocytes can be modified, genetically modified, transduced and/or stably transfected when the lymphocytes are introduced or reintroduced back into the subject, and in illustrative embodiments are subcutaneously or intramuscularly introduced or reintroduced back into the subject, or when preparing a cell preparation. In some embodiments, at least 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the lymphocytes are modified. In illustrative embodiments, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of lymphocytes that are the low end of the range are modified, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of lymphocytes that are the high end of the range are modified. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified lymphocytes are not genetically modified, transduced or stably transfected. In illustrative embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified lymphocytes are not genetically modified, transduced or stably transfected. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the modified lymphocytes at the low end of the range are not genetically modified, transduced, or stably transfected, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified lymphocytes (e.g., 10% to 95%) at the high end of the range are not genetically modified, transduced, or stably transfected. Genetically modified lymphocytes containing recombinant nucleic acids can place the recombinant nucleic acids extrachromosomally or integrated into the genome. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the genetically modified lymphocytes have extrachromosomal recombinant nucleic acid. In illustrative embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the genetically modified lymphocytes have extrachromosomal recombinant nucleic acids. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the modified or genetically modified lymphocytes at the low end of the range have extrachromosomal recombinant nucleic acid, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the modified or genetically modified lymphocytes (e.g., 10% to 95%) at the high end of the range have extrachromosomal recombinant nucleic acid. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified or genetically modified lymphocytes are not transduced or stably transfected. In illustrative embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or all of the modified or genetically modified lymphocytes are not transduced or stably transfected. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% and 70% of the modified or genetically modified lymphocytes at the low end of the range are transduced or stably transfected, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% or all of the modified or genetically modified lymphocytes at the high end of the range are not transduced or stably transfected.

In some embodiments of any aspect herein, wherein the formulation is administered to the subject, the second formulation is administered to the subject at a second, third, fourth, etc., time point between 1 day and 1 month, 2 months, 3 months, 6 months, or 12 months after administration of the first cell formulation, wherein the second formulation may be the same as the first formulation, or may include any of the formulations provided herein. i) A cytokine, ii) an antibody, antibody mimetic or polypeptide that is capable of binding to CD3, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81 and/or CD82, and/or iii) a source of homologous antigen recognized by the CAR, and optionally wherein the cytokine is IL-2, IL-7, IL-15 or IL-21, or a modified form of any of these cytokines that is capable of binding to and activating the natural receptor of the cytokine.

In some embodiments herein including any aspect of the cell mixture or cell preparation, any cells in the cell mixture may be enriched. For example, cells for adoptive cell therapy, such as a population of one or more T and/or NK cells, may be enriched prior to formulation for delivery. In some embodiments, the one or more cell populations may be enriched after the cell mixture is contacted with a recombinant nucleic acid vector, such as a replication defective retroviral particle. In some embodiments, enriching one or more cell populations may be performed concurrently with any of the methods of genetic modification disclosed herein, and in illustrative embodiments, genetic modification with replication defective retroviral particles.

In some embodiments herein including any aspect of the cell mixture or cell preparation, one or more undesirable cell populations may be depleted such that the desired cells in the cell mixture or cell preparation are enriched. In some embodiments, the one or more cell populations may be depleted by negative selection prior to contact with a recombinant nucleic acid vector, such as a replication defective retroviral particle. In some embodiments, one or more cell populations may be depleted by negative selection after the cell mixture is contacted with a recombinant nucleic acid vector, such as a replication-defective retroviral particle. In some embodiments, depleting one or more cell populations may be performed prior to or concurrent with any of the methods of genetic modification disclosed herein (and in illustrative embodiments, genetic modification with replication defective retroviral particles).

In some embodiments, the undesirable cells include cancer cells. Cancer cells from multiple types of cancer can enter the blood and can be unintentionally genetically modified with lymphocytes at low frequencies using the methods provided herein. In some embodiments, the cancer cells may be derived from any cancer, including, but not limited to: renal cell carcinoma, gastric cancer, sarcoma, breast cancer, lymphoma, B-cell lymphoma such as diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, non-hodgkin's B-cell lymphoma (B-NHL), neuroblastoma, glioma, glioblastoma, medulloblastoma, colorectal cancer, ovarian cancer, prostate cancer, mesothelioma, lung cancer (e.g., small cell lung cancer), melanoma, leukemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), or Chronic Myelogenous Leukemia (CML), or any of the cancers listed in this disclosure. In an illustrative embodiment, the CAR-cancer cells can be derived from lymphoma, and in an illustrative embodiment, from B cell lymphoma.

In certain embodiments herein including any aspect of blood cells, the blood cells in the reaction mixture comprise at least 10% neutrophils and at least 0.5% eosinophils, by weight of the percentage of leukocytes in the reaction mixture.

In certain embodiments herein including any aspect of the reaction mixture and/or cell preparation, the reaction mixture and/or cell preparation comprises at least 5%, 10%, 20%, 25%, 30% or 40% neutrophils by percentage of cells in the reaction mixture or cell preparation, or 20% to 80%, 25% to 75%, or 40% to 60% neutrophils by percentage of leukocytes in the reaction mixture or cell preparation.

In certain embodiments herein including any aspect of the reaction mixture and/or cell preparation, the reaction mixture and/or cell preparation comprises at least 0.1% eosinophils, or 0.25% to 8% or 0.5% to 4% eosinophils, by percentage of leukocytes in the reaction mixture or cell preparation.

In certain embodiments herein including any aspect of blood cells, the PBMC enrichment procedure is not performed on blood cells in the reaction mixture prior to contacting.

In certain embodiments herein including any aspect of the reaction mixture, the reaction mixture is formed by adding recombinant retroviral particles to whole blood.

In certain embodiments of any aspect herein including the reaction mixture, the reaction mixture is formed by adding recombinant retroviral particles to substantially whole blood comprising an effective amount of an anticoagulant.

In certain embodiments of any aspect included herein in the reaction mixture, the reaction mixture comprises an anticoagulant. For example, in certain embodiments, the anticoagulant is selected from the group consisting of glucose citrate, EDTA, or heparin. In certain embodiments, the anticoagulant is not citric acid gluconic acid. In certain embodiments, the anticoagulant comprises an effective amount of heparin.

In certain embodiments herein including any aspect of the reaction mixture, the reaction mixture is in a blood bag during the contacting.

In certain embodiments as or including any aspect of the methods herein, the method further comprises subcutaneously administering the modified T cells and/or NK cells to the subject. Optionally, in certain embodiments of this type, the modified T cells and/or NK cells are delivered in a cell preparation further comprising neutrophils. Further, optionally, in such certain embodiments, the neutrophils are present in the cell formulation at a concentration that is too high for safe intravenous delivery, and/or the cell formulation comprises 5%, 10%, 15%, 20% or 25% neutrophils. In some embodiments of any of the methods herein comprising the collecting, contacting, and administering steps, the modified lymphocytes are introduced back into the subject within 14 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes from the time the subject collects blood comprising lymphocytes. In illustrative embodiments, such methods include subcutaneous administration. In illustrative embodiments, such methods include collecting blood cells using apheresis, or filtering blood cells or modified lymphocytes through a filter, such as a leukopenia filter.

In certain embodiments of any aspect of the methods included herein, the method further comprises subcutaneously administering the modified T cells and/or NK cells to the subject in the presence of hyaluronidase. In further illustrative embodiments, the modified T cells and/or NK cells are obtained from a subject.

In further sub-embodiments of these embodiments (including subcutaneous administration of the T cells and/or NK cells modified and in the illustrative embodiments genetically modified to a subject in the presence of hyaluronidase), the modified T cells and/or NK cells are delivered subcutaneously to the subject in a volume of 1ml to 5 ml. In further sub-embodiments, the T cells and/or NK cells are in blood drawn from the subject and the modified T cells and/or NK cells are delivered back into the subject, and in further embodiments, within 1-14, 1-8, 1-6, 1-4, 1-2, or 1 hour from the time the subject draws blood.

In certain embodiments of any aspect herein that includes a reaction mixture, the reaction mixture is contacted with the leukoreduction filter assembly in the closed cell processing system prior to contacting, upon contacting the recombinant retroviral particles with the blood cells, during contacting including optional incubation in the reaction mixture, and/or after contacting including optional incubation in the reaction mixture.

In some embodiments of any aspect herein, at least 10%, 20%, 25%, 30%, 40%, 50%, most, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the T cells are resting T cells, or at least 10%, 20%, 25%, 30%, 40%, 50%, most, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the NK cells are resting NK cells when the T cells or NK cells are combined with the replication-defective retroviral particle to form a reaction mixture.

In any aspect herein including modified cells, one or more cells are not subjected to a centrifugal seeding procedure, e.g., at least 30 minutes without being subjected to at least 800g of centrifugal seeding.

In some embodiments of any aspect of the methods included herein, the method further comprises administering the modified T cells and/or NK cells to a subject, optionally wherein the subject is a source of blood cells. In some sub-embodiments of these, as well as embodiments of any of the methods and uses herein, including those in the present exemplary embodiment section, provided that they are not incompatible or stated, the modified, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or populations thereof undergo 4 or less ex vivo cell divisions prior to being introduced or reintroduced into a subject. In some embodiments, no more than 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour passes between the time blood is collected from the subject and the time the modified lymphocytes are reintroduced into the subject. In some embodiments, all steps after blood collection and before reintroduction of the blood are performed in a closed system, optionally wherein the closed system is monitored manually throughout the treatment. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the modified lymphocytes in solution may include a pseudotyped element or T cell activating antibody on their surface. In some embodiments, the pseudotyped element and/or the T cell activating antibody can bind to the surface of the modified lymphocyte through, for example, a T cell receptor, and/or the pseudotyped element and/or the T cell activating antibody can be present in the plasma membrane of the modified lymphocyte.

In any of the kits provided above, the first and/or second polynucleotide can comprise any of the self-driven CARs provided herein. Additional kit aspects and embodiments are provided below and in the detailed description herein beyond the section of this exemplary embodiment.

For any aspect provided herein that includes a syringe, in an illustrative embodiment, the syringe is compatible with, effective for, and/or suitable for intramuscular delivery (and in an illustrative embodiment, subcutaneous delivery), and/or effective for intramuscular injection, effective for subcutaneous injection, suitable for intramuscular injection, and/or suitable for subcutaneous injection. For example, the syringe may have a needle gauge between 20 and 22 and a length between 1 inch and 1.5 inches for intramuscular delivery, and a needle gauge between 26 and 30 and a length between 0.5 inch and 0.625 inch for subcutaneous delivery.

In certain embodiments of any aspect and other embodiments herein comprising a polynucleotide encoding a CAR and LE (e.g., a polynucleotide, RIP, cell preparation, population, genetically modified lymphocyte, reaction mixture, mammalian packaging cell line comprising a packagable RNA genome for replication-defective retroviral particles, kit, use of RIP in preparing a kit for genetically modifying and/or transducing a lymphocyte, method for genetically modifying and/or transducing a T cell or NK cell, method for administering a genetically modified lymphocyte to a subject), the polynucleotide can comprise or encode an anti-idiotype polypeptide and one or more of a CAR and LE, a cytokine, and one or more inhibitory RNA molecules.

In some embodiments, a self-driven CAR embodiment can be a polynucleotide comprising a first transcription unit operably linked to an inducible promoter inducible in at least one of a T cell or NK cell and a second transcription unit operably linked to a constitutive T cell or NK cell promoter, wherein the first transcription unit and the second transcription unit are divergently arranged, wherein the first transcription unit encodes LE, and wherein the second transcription unit encodes an anti-idiotype polypeptide and a CAR, wherein the CAR comprises an astm, a transmembrane domain, and an intracellular activation domain.

In any aspect herein that includes recombinant retroviral particles in a vessel and/or reaction mixture, the recombinant retroviral particles are present in the vessel and/or reaction mixture at an MOI of 0.1 to 50, 0.5 to 20, 0.5 to 10, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 2 to 15, 2 to 10, 2 to 7, 2 to 3, 3 to 10, 3 to 15, or 5 to 15, or at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15, or at an MOI of at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15. For the kit and isolated retroviral particle embodiments, such MOI can assume 1X 10 based on 1, 2.5, 5, 10, 20, 25, 50, 100, 250, 500 or 1,000ml ⁶ Target cells/ml, for example in the case of whole blood, 1X 10 is assumed ⁶ PBMC/ml blood.

In any aspect herein that includes contacting a cell with a retroviral particle, sufficient retroviral particle is present in the reaction to obtain an MOI of 0.1 to 50, 0.5 to 20, 0.5 to 10, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 2 to 15, 2 to 10, 2 to 7, 2 to 3, 3 to 10, 3 to 15, or 5 to 15, or at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15, or to obtain an MOI of at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15.

In any aspect herein including genetically modified T cells and/or NK cells, at least 5%, at least 10%, at least 15%, or at least 20% of the T cells and/or NK cells are genetically modified, or between 5% and 85%, or between 5% and 20%, 25%, 50%, 60%, 70%, 80%, or 85%, or between 5%, 10%, 15%, 20%, or 25% as the low end of the range and 20%, 25%, 50%, 60%, 70%, 80%, or 85% as the high end of the range.

In any aspect herein including RIP, the RIP is a lentiviral particle. In further illustrative embodiments, the modified cell is a modified T cell or a modified NKT cell.

In any aspect herein comprising a polynucleotide comprising one or more transcriptional units, the one or more transcriptional units can encode an anti-idiotype polypeptide and a polypeptide comprising a CAR. In some embodiments, the CAR is a microenvironmentally limited biological (MRB) -CAR. In other embodiments, the ASTR of the CAR binds to a tumor-associated antigen. In other embodiments, the astm of the CAR is a microenvironmentally limited living organism (MRB) -astm.

In certain embodiments, provided herein are any aspects and embodiments comprising a polynucleotide comprising a nucleic acid sequence operably linked to a promoter active in T cells and/or NK cells, the polynucleotide encoding at least one polypeptide lymphoproliferative element comprising an anti-idiotype extracellular recognition domain. In illustrative embodiments, the anti-idiotype extracellular recognition domain is capable of binding to an idiotype of an antibody that does not induce cytotoxicity. In an illustrative embodiment, the polypeptide lymphoproliferative element is any of the polypeptide lymphoproliferative elements disclosed herein. In some embodiments, any or all of the nucleic acid sequences provided herein can be operably linked to a riboswitch. In some embodiments, the riboswitch is capable of binding a nucleoside analog. In some embodiments, the nucleoside analog is an antiviral drug.

In certain illustrative embodiments herein including any aspect of the blood cells in the reaction mixture, the blood cells in the reaction mixture are blood cells produced by a PBMC enrichment procedure and comprising PBMCs, or the blood cells in the illustrative embodiments are PBMCs. In illustrative embodiments, such embodiments including PMBC enrichment are not combined with embodiments wherein the reaction mixture includes at least 10% whole blood. Thus, in certain illustrative embodiments herein, the blood cells in the reaction mixture are PBMC cell fractions from a PBMC enrichment procedure to which the retroviral particles are added to form the reaction mixture, and in other illustrative embodiments, the blood cells in the reaction mixture are from whole blood to which the retroviral particles are added to form the reaction mixture.

In any aspect and embodiment provided herein that includes or optionally includes a nucleic acid sequence encoding an inhibitory RNA molecule, the inhibitory RNA molecule targets any gene (e.g., encoding mRNA) identified, for example, in the inhibitory RNA molecule portion herein.

In the illustrative embodiments of any of the kits, delivery solutions, and/or cell preparations provided herein, particularly those that are effective for or suitable for intramuscular delivery (and in the illustrative embodiments subcutaneous delivery), the delivery solutions and/or cell preparations are depot preparations, or the cell preparations are emulsions of cells that promote cell aggregation. In some embodiments, the depot delivery solution comprises an effective amount of alginate, hydrogel, PLGA, cross-linked hyaluronic acid and/or polymeric hyaluronic acid, PEG, collagen, and/or dextran to form a depot formulation. In some embodiments, the delivery solution and/or cell formulation is designed for controlled release or delayed release. In some embodiments, the delivery solution and/or cell formulation includes components that form an artificial extracellular matrix, such as a hydrogel.

In any of the aspects and embodiments provided herein that include or optionally include a cell mixture, delivery solution, or cell formulation, the cell mixture, delivery solution, or cell formulation can have a pH and an ionic composition that provides an environment in which cells can survive, for example, for at least 1 hour, and typically can survive for at least 4 hours. In some embodiments, the pH may be between pH 6.5 to pH 8.0, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. In some embodiments, for example, when the RIP has a polynucleotide encoding a MRB-CAR, the pH may be between pH 6.0 and pH 7.0, such as pH 6.2 to pH 7.0, or pH 6.4 to pH 6.8. In some embodiments, the cell mixture, delivery solution, or cell preparation may be maintained by a buffer, such as a phosphate buffer or bicarbonate, present at a concentration effective to maintain a pH within a target range. In some embodiments, the cell mixture, delivery solution, or cell formulation may include a saline composition having, for example, 0.8 to 1.0 or about 0.9 or 0.9% salt, such as a salt of sodium chloride. In some embodiments, the delivery solution is or includes PBS. In some embodiments of the delivery solutions and resulting cell preparations herein, na ⁺ Is between 110mM and 204mM, cl ^- Between 98mM and 122mM, and/or K ⁺ Is between 3mM and 6 mM.

In some embodiments herein including modified or genetically modified T cells or NK cells or any aspect of the methods, compositions, and kits for genetically modifying T cells and/or NK cells, proliferation and survival of genetically modified T cells and/or NK cells expressing a CAR can be induced by crosslinking the CAR molecule within the genetically modified T cells or NK cells in the absence of the CAR molecule bound to their cognate antigen. Thus, in some embodiments, a T cell or NK cell can comprise an epitope tag that is bound by an antibody and crosslinked to an epitope tag of a second CAR on the same T cell or NK cell. In some embodiments, the extracellular domain of the CAR can include an epitope tag. In an illustrative embodiment, the epitope tag can be in the stem domain. In some embodiments, the epitope tag may be His5 (HHHHH; SEQ ID NO: 76), hisX6 (HHHHH; SEQ ID NO: 77), c-myc (EQKLISEEDL; SEQ ID NO: 75), flag (DYKDDDDK; SEQ ID NO: 74), strep tag (WSHPQFEK; SEQ ID NO: 78), HA tag (YPYVPDYA; SEQ ID NO: 73), RYIRS (SEQ ID NO: 79), phe-His-His-Thr (SEQ ID NO: 80) or WEAAAREACCRECCARA (SEQ ID NO: 81). In an illustrative embodiment, the epitope tag may be a HisX6 tag (SEQ ID NO: 77). In some embodiments, the CAR may be crosslinked and activated by adding a soluble antibody or antibody mimetic that binds to the epitope tag, or in illustrative embodiments by adding cells (also referred to herein as universal feeder cells) that express the antibody or antibody mimetic on their surface that binds to the epitope tag. In some embodiments, the same universal feeder cells (e.g., universal feeder cells expressing anti-HisX 6 antibodies) can be used with cells expressing CARs that bind to different antigens but include the same epitope tag (e.g., hisX 6). In some embodiments, the CAR may be crosslinked and activated by adding mRNA encoding one or more antibodies or antibody mimics that bind to the epitope tag. In some embodiments, the mRNA may encode an antibody or antibody mimetic that is soluble, membrane-bound, or both soluble and membrane-bound.

In one aspect, provided herein is a cell formulation (i.e., delivery composition) comprising a delivery solution formulated with Tumor Infiltrating Lymphocytes (TILs) and/or modified or unmodified lymphocytes (T cells and/or NK cells in the illustrative examples), wherein the cell formulation is compatible with, effective for, and/or suitable for subcutaneous or intramuscular delivery. In some embodiments provided herein for any one of the cell preparations, the cell preparation is positioned subcutaneously in the subject, or a majority of the cell preparation is positioned subcutaneously. In some embodiments, the cell preparation is positioned subcutaneously or intramuscularly in the subject, or a majority of the cell preparation is positioned subcutaneously or intramuscularly in the subject. In some embodiments, wherein the cell preparation comprises TIL, the cell preparation may further comprise modified lymphocytes modified by either or both of: associated with a recombinant nucleic acid vector (RIP in the illustrative embodiment) comprising a polynucleotide comprising one or more transcriptional units operably linked to a promoter active in T cells and/or NK cells; or by genetic modification with the polynucleotide, wherein the one or more transcriptional units encode a first polypeptide comprising a first CAR. In some embodiments, wherein the cell preparation comprises TIL, the cell preparation further comprises a source of tumor antigen recognized by the TIL. In some embodiments, the TIL is contacted with a nucleic acid vector.

In addition to any of the method aspects and embodiments provided herein, further provided herein are use aspects and embodiments, including the use of a kit for performing the method, or the use of a nucleic acid vector (RIP in the illustrative embodiment) in the preparation of a kit for performing the method, wherein the use of a kit is to perform the steps of the method aspects or embodiments. Similarly, for any of the use aspects and embodiments provided herein, further provided herein are method aspects and embodiments, including methods as recited in the use aspects or embodiments.

The following non-limiting examples are provided by way of illustration of exemplary embodiments only and in no way limit the scope and spirit of the disclosure. Furthermore, it is to be understood that any invention disclosed or claimed herein encompasses all variations, combinations, and permutations of any one or more of the features described herein. Any one or more features may be specifically excluded from the claims even if the specific exclusion is not explicitly set forth herein. It should also be understood that the disclosure of reagents for use in a method is intended to be synonymous with (and provide support for) a method involving the use of a reagent, according to the particular methods disclosed herein or other methods known in the art, unless one of ordinary skill in the art will understand. In addition, unless one of ordinary skill in the art will appreciate that the specification and/or claims disclose a method for which any one or more of the reagents disclosed herein may be used.

Examples

Example 1 preparation of phage display library of scFv from human CD19+ PBMC

Isolation of human immunoglobulin heavy chains and kappa antibody light chains

Frozen human PBMC (San Diego Blood Bank) previously isolated from whole blood of 13 well-approved anonymous donors was thawed and pooled (1.5x10 ⁸ Individual PBMCs). Using EasySep ^TM Human CD19 positive selection kit II (Stemcell Technologies, # 17854) for CD19 isolation ⁺ PBMC(1.2x10 ⁷ ). CD19 from CD19 using the RNeasy Plus Mini Prep kit (Qiagen) ⁺ PBMC isolate RNA (7. Mu.g). First strand cDNA was synthesized from mRNA transcripts using anchor oligo d (T) 20 using SuperScript IV (Invitrogen). The antibody variable chains were then amplified by PCR using degenerate primer pools specific for human heavy and kappa light chains. The degenerate primer pool includes 6 forward primer sequences and 3 reverse primer sequences directed to the heavy chain, and 6 forward primer sequences and 5 reverse primer sequences directed to the kappa light chain. 2 primers with different adaptors were used per sequence to allow directed cloning.

Assembly of Xphage2 scFv library

Each heavy chain PCR product and each kappa chain PCR product was gel purified and cloned into 2 targeting-specific cloning vectors. The heavy and kappa light chains were then assembled seamlessly with the linker into a complete scFv within the phagemid backbone to generate an xphage.2 library. Ligation of heavy using six unique sequences Antibody chains and light antibody chains to generate libraries: GGSSRSS ("L1", SEQ ID NO: 673); (G) ₄ S) ₂ (“L2”，SEQ ID NO:674)；(G ₄ S) ₃ (“L3”，SEQ ID NO:673)；(G ₄ S) ₄ (“L4”，SEQ ID NO:372)；(G ₄ S) ₅ ("L5", SEQ ID NO: 675); and (G) ₄ S) ₆ (“L6”，SEQ ID:64)。

Library diversity estimation

The diversity of single heavy and kappa light chain libraries is determined by a combination of NGS and bioinformatics. Briefly, heavy and light chain libraries were sequenced using paired-end sequencing (Illumina). Paired end reads were pre-processed by mass filtration using trimmatic v0.36, followed by read clipping and pairing. Clustering is then performed using custom scripts. By Recon (Kaplinsky)&Arnaout 2016) reconstruct the diversity of the starting population using read distribution. As shown in Table 6, the estimated total diversity of the heavy chain library and the light chain library was 3.7X10, respectively ⁶ And 1.5x10 ⁶ . The estimated total diversity of the Xphage2 scFv library was 6.7x10 ¹³ ((κ,1.5x10 ⁶ ) x (weight, 3.7x10) ⁶ ) x (linker, 6) x (orientation, 2)).

TABLE 6 diversity of estimated heavy and light (kappa) chain libraries

EXAMPLE 2 isolation of recombinant anti-idiotype antibodies from Xphage2 scFv library

Monoclonal antibodies were used as decoys (Ab 1) to panning the Xphage2 scFv library and identify antibodies (Ab 2) specific for the idiotype of the decoys (Ab 1). Two separate panning experiments were performed using different baits, ab1.1 in panning experiment 1 (PE 1) and ab1.2 in panning experiment 2 (PE 2). Both baits were anti-EGFR (cetuximab). One bait was made in CHO chinese hamster cells (Selleck Chemicals, a 2000) and the other in SP2/0 mouse myeloma cells (Merck, 226667). Each antibody used as a bait has an IgG1 isotype.

Liquid phase biopanning of the xpage 2 scFv library was performed using Ab1 antibodies coupled to magnetic Dynabead. In panning experiment 1 Dynabead was used in parallel ^TM Protein A (Thermo Fisher, # 10001D) and Dynabead ^TM Five rounds of panning were performed with M-270 epoxy (Thermo Fisher, # 14301). Briefly, in the first round, panning polyclonal human IgG antibodies conjugated to protein a or M-270 epoxy, which had been blocked with pbs+2% milk and washed by PBS, were mixed with phage particles at 1:1 volume and incubated in pbs+2% milk for 1 hour at room temperature, then the supernatant was collected in a new tube. This pre-clean step was performed to remove non-specific phage that bound to the beads and human IgG monoclonal antibodies (CrownBio, cat.C0001). Dynabeads coupled to Ab1.1 and blocked with PBS+2% milk were then added to pre-cleared phages and incubated for 1.5 hours at room temperature, then washed 10 times with PBST (PBS containing 0.05% Tween-20) and 2 times with PBS. Phage bound to Ab1.1 conjugate beads were eluted twice in a total of 1.2ml of 0.1M glycine-HCl, pH 2.2 ("elution buffer") and 0.6ml of 1M Tris-HCl, pH 8.0 ("neutralization buffer"). The eluted phage were then used to infect TG1 bacteria (Lucigen, 60502-2) grown to an OD600 of 0.5 in 40ml of 2XYT medium. After incubation for 1 hour at 37 ℃, 1ml of bacteria was removed for analysis (10 μl for phage titer determination and 50ul for picking individual colonies by serial dilution and the rest frozen for storage). The remaining TG1 was precipitated, resuspended in 1.5ml of 2XYT+Amp, and spread on 2YT+AMP+1% w/v glucose plates and incubated overnight at 37 ℃. The bacteria were then collected and used to inoculate 80ml 2YT+AMP+1% w/v glucose and incubated at 37℃until OD600 was 0.5. Super phage M13K 07 ΔpIII (Progen, PRHYPE) was added at MOI 20 and the mixture was incubated at 37℃for 1 hour. The bacteria were then collected, resuspended in 80ml of 2YT+Amp+Kan medium with 0.1mM IPTG, and incubated overnight at 30℃at 230 rpm. The supernatant was then collected and phage particles were precipitated in PEG8000/NaCl solution and resuspended in 1.8ml PBS ("phage out") and titrated.

Subsequent panning rounds were similarly performed using phage output from previous panning rounds, with some modifications. In the fourth and fifth rounds of panning, after pre-clearing the phage supernatant with human IgG polyclonal antibodies (thermosusher, cat.02-7102), a second pre-clearing step was performed using anti-human CD20 IgG antibodies (rituximab) conjugated to Dynabead (Selleck Chemicals, a 2009). In the fourth round of panning, pbs+2% HSA was used instead of pbs+2% milk to block the beads. For panning round 5b, 1ml PBS containing 20ug/ml EGFR-His was used to specifically compete and elute phage displaying Ab2 that recognizes the antigen (EGFR) binding site of Ab 1.1.

For each round of panning in PE1, FIGS. 6A and 6B show the use of Dynabead, respectively ^TM Protein A and Dynabead ^TM M-270 epoxy resin was subjected to phage input (pfu), bead volume, number of washes, phage output (pfu), recovery and enrichment from the previous round. Pool ELISA results showed that after 3 rounds of panning, panning with Dynabead M-270 epoxy showed better enrichment than with Dynabead protein A.

The phage output of each round of panning was analyzed for ab1.1 binding by ELISA. Wells of the 96-well plates were coated with ab1.1 and serial dilutions of phage output from each round of panning were added. HRP conjugated anti-M13 antibody (Hangzhous Hua' an Biotech, # EM 1902-18) and step 1 were used ^TM The Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, # 34028) detects bound phage. Absorbance at 450nm was measured and data was processed using GraphPad Prism 8. By coupling to Dynabeads ^TM The results of panning performed by Ab1.1 of M-270 epoxy resin are shown in FIG. 7. As shown in the figure, phage displaying antibody binding to ab1.1 were successfully isolated in each round of panning. Furthermore, there was a large enrichment of Ab1.1-specific phages between round 1 and round 2 and between round 2 and round 3. Minimal enrichment of Ab 1.1-specific phage was observed in the subsequent rounds. Also utilizing coupling to Dynabeads ^TM Phage displaying antibodies that bind to ab1.1 were isolated in each round of panning performed on ab1.1 of protein a.

Panning experiment 2 (PE 2) was performed using a method substantially similar to that used in PE1. Using coupling to Dynabead ^TM Ab1.2 of M-270 epoxy served as a bait for a total of 3 rounds of panning. PBS +2% milk was used for all blocking steps. Phage libraries were pre-cleared in sequence prior to the first round of panning with human IgG polyclonal antibodies, anti-human CD20 IgG antibodies (rituximab) (Selleck Chemicals, a 2009), and anti-human biotin-CD 3 (OKT 3) (bioleged, # 317320). In the next few rounds, pre-clearing was performed using human IgG polyclonal antibodies without any subsequent pre-clearing steps. 0.1M glycine-HCL, pH 2.2 was used to elute Ab 1.2-bound phage in each round. Similar to the results for PE1, phages displaying antibodies binding to ab1.2 were successfully isolated in each round of screening.

Example 3 identification of individual recombinant anti-idiotype antibodies specific for anti-idiotype-producing antibody variable Domain

Individual anti-idiotype scFv were cloned from phage pools and tested for specific binding by ELISA to the idiotype of antibodies used as panning baits. The sequence of the scFv was determined by Sanger sequencing.

281 clones from panning experiment 1 (PE 1) (from 4 ^th Round panning 89 clones and from 5 ^th Round panning 192 clones) and 95 clones from panning experiment 2 (PE 2) (from 2) ^nd Round panning 30 clones and from 3 ^rd 65 clones from round panning) were amplified in TG1 and packaged with super phage in 96 deep well plates. A monoclonal phage ELISA was performed, wherein the microtiter plates were coated with either ab1.1 (for PE 1) or ab1.2 (for PE 2) or control antibodies, and individual phage clones were added to each well. Binding was detected with anti-M13-HRP and absorbance was measured at OD450 nm.

The results of the monoclonal phage ELISA are shown in tables 2, 3, 4 and 5. The columns indicate whether binding to the bait (ab1.1 or ab1.2) or control antibody (rituximab). The binding was shown to be OD450 nm. "ratio" is the binding ratio of bait to control antibody. Since the bait and control antibodies were each chimeric monoclonal antibody of the human IgG1 isotype, effective binding of phage to the bait rather than the control (determined as a ratio > 3.0) indicated that phage expressed anti-idiotype antibodies specific for the idiotype of the bait (ab1.1 or ab1.2). 24 clones from round 4 of PE1 and 40 clones from round 5 of panning were identified as ab1.1 anti-idiotype antibodies. Similarly, 23 clones from round 2 panning of PE2 and 54 clones from round 3 panning were identified as ab1.2 anti-idiotype antibodies.

The sequence data for each unique scFv identified in PE1 is shown in fig. 8, and the sequence data for each unique scFv identified in PE2 is shown in fig. 9A, 9B, and 9C. Individual clones are shown in each row. Shown in the columns are the SEQ ID NO of the DNA and the amino acids of the intact scFv, the orientation of the heavy and light chains, and the SEQ ID NO of the linker. Panning the Xphage2 scFv library using ab1.1 as a decoy in PE1 resulted in 19 unique scFv clones. Similarly, panning the xpage 2 scFv library using ab1.2 as a decoy in PE2 resulted in 42 unique scFv clones. The amino acid sequence of each CDR and its SEQ ID NO are also shown. As shown in the table, some clones shared HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3. Furthermore, some clones share the same sequence for all of their CDRs. The CDR was determined on www.vbase2.org using VBASE2.

Isolation of many unique scFv clones in PE1 and PE2 demonstrated that the xpage 2 library and panning methods described herein can be effectively used to identify scFvs and antibody heavy and light chains capable of binding to the idiotypes of other antibodies.

Example 4. Identification of a single recombinant anti-idiotype antibody that recognizes a specific region of the idiotype of an antibody that produces an anti-idiotype antibody.

The anti-idiotype antibodies identified in example 3 were further screened by competition ELISA to identify anti-idiotype antibodies specific for the Antigen Binding Site (ABS) of its cognate Ab1 antibody, as well as other anti-idiotype antibodies specific for idiotype epitopes outside of ABS.

Cetuximab anti-EGFR antibodies were used as baits in panning experiment 2 (ab1.2). To identify anti-idiotype antibodies to ABS that bind to cetuximab and other antibodies that bind to outside of ABS, soluble EGFR (Acrobiosystems, cat#egr-H5222) was added to compete with monoclonal phage for binding to cetuximab in ELISA. Briefly, microtiter plates were coated with ab1.2 and 50 individual phage clones displaying anti-idiotype antibodies identified in PE2 were added to wells in duplicate. For each sample plated in duplicate, soluble EGFR was added to one well at a final concentration of 1 μg/ml. Binding of monoclonal phage was detected with anti-M13-HRP and absorbance was measured at OD450 nm.

The results of the competition ELISA for 50 monoclonal phages are shown in fig. 10. Binding inhibition (% inhibition) was calculated as (OD in the absence of EGFR-OD in the presence of EGFR)/(OD in the OD-plate background in the absence of EGFR) x 100. Soluble EGFR will compete with antibodies that recognize ABS that binds to cetuximab. Thus, if the anti-idiotype antibody recognizes ABS, the% inhibition will be high, and if the anti-idiotype antibody recognizes the cetuximab region outside of ABS, the% inhibition will be low. Selecting 75% inhibition greater than inhibition ^th The percentile monoclonal phage (92%) was an anti-idiotype antibody that recognized ABS. Of the 50 phages shown in this screen, 13 clones were determined to be anti-idiotype antibodies that recognized the ABS of cetuximab. In contrast, the% inhibition is less than 25% inhibition ^th The percentile monoclonal phage (67%) was considered to be an anti-idiotype antibody that recognized the variable domain region outside of ABS. Of the 50 phages shown in this screen, 6 clones were identified as anti-idiotype antibodies that recognized the variable domain region of cetuximab outside of ABS. The results in this example demonstrate that both an anti-idiotype antibody that recognizes ABS of a target antibody and an anti-idiotype antibody that recognizes an idiotype epitope of a target antibody that is external to ABS can be identified using the methods described herein. Similar methods can also be used to identify antibody mimics of ABS that bind to a target antibody or antibody mimetic.

Example 5 surface expression of anti-idiotype Polypeptides on mammalian cells

The selected anti-idiotype antibodies identified in example 3 are cloned into a vector such that the transcriptional units encode a first polypeptide comprising a CAR and a second polypeptide comprising an anti-idiotype polypeptide. These vectors are transiently transfected and expressed on mammalian cells.

As shown in figure 11, the vector into which the scFv was cloned encodes a CD19 CAR consisting of an anti-CD 19scFv, a CD8 handle and a transmembrane region, and an intracellular domain from CD3z, followed by T2A and an anti-idiotype scFv fused to a "stmid" consisting of a histidine tag (optional), a handle, a transmembrane domain (TM) and an intracellular domain (optional) ("ICD"). Many different stmids were tested. STMICD1 contains 8 amino acids of the hinge, TM and ICD PDGFR beta (SEQ ID NO: 676). STMICD2 contains the hinge, TM and 9 amino acids of ICD CD28 (SEQ ID NO: 677). STMICD3 contains all ICDs of hinge, TM and CD28 (SEQ ID NO: 678). STMICD4 contains a hinge, a TM, and all ICDs of CD28 fused to all ICDs of CD80 (SEQ ID NO: 679).

Transient transfection of the expression vector into mammalian cells using Lipofectamine 2000 (Thermo Fisher Scientific) at 37℃and 5% CO ₂ After 48 hours of incubation, harvested and stained for FAC analysis with anti-His (BioLegend, 362607) and cetuximab (Selleck Chemicals, a 2000), ab1 used as a bait for identification of anti-idiotype scFv.

Figure 12 shows the expression of an anti-idiotype polypeptide on the surface of mammalian cells as detected by cetuximab. The vectors of clone a and clone B encode unique scFv, while the control vector lacks an insert encoding an anti-idiotype polypeptide. In this experiment, clone a and clone B both contained smicd 4. As shown in FIG. 12, expression of the anti-idiotype polypeptide was detected by Ab1 on transiently transfected 293T cells at 48.4% (clone A) and 54.5% (clone B). Similarly, the anti-idiotype polypeptide was expressed and detected on transiently transfected CHO-S cells at 8.3% (clone A) and 10.6% (clone B). In separate experiments, when replacing smicd 4 with each of smicd 1, smicd 2 and smicd 3, surface expression of the anti-idiotype polypeptides was also detected on the cells, demonstrating that surface expression can be achieved using various handles, TMS and ICDs.

This example demonstrates that the methods and compositions disclosed herein can be used to generate anti-idiotype polypeptides that can be expressed on the surface of mammalian cells and are capable of binding to the variable domains of their target antibodies.

The disclosed embodiments, examples, and experiments are not intended to limit the scope of the invention or represent all or the only experiments performed by the following experiments. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. It should be understood that variations to the methods as described may be made without altering the basic aspects of the experimental intent specification.

Many modifications and other embodiments can be devised by those skilled in the art that will fall within the scope and spirit of the disclosure. Indeed, variations in the described materials, methods, figures, experiments, examples, and embodiments may be made by those of skill in the art without altering the basic aspects of the disclosure. Any of the disclosed embodiments may be used in combination with other disclosed embodiments.

In some cases, some concepts are described with reference to specific embodiments. However, it will be apparent to those skilled in the art that various modifications and changes may be made without departing from the scope of the inventive concepts as set forth in the following claims. The specification and figures are, accordingly, to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of present invention.

TABLE 1 parts, names and amino acid sequences of the domains of lymphoproliferative moieties P1-P2, P1, P2, P3 and P4.

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

TABLE 2 from 4 ^th Round panning Ab1.1 clone

/>

TABLE 3 from 5 ^th Round panning Ab1.1 clone

/>

/>

TABLE 4 from 2 ^nd Round panning Ab1.2 clone

TABLE 5 from 3 ^rd Round panning Ab1.2 clone

Ab1.2 clone	Ab1.2	Control	Ratio of	Ab1.2 clone	Ab1.2	Control	Ratio of
								31	0.73	0.15	5.0	64	1.11	0.10	11.3
32	0.91	0.11	8.6	65	0.59	0.09	6.8
								33	1.05	0.17	6.3	66	0.64	0.05	12.3
34	0.84	0.32	2.7	67	0.71	0.12	5.8
								35	1.01	0.18	5.8	68	1.15	0.06	17.6
36	0.40	0.11	3.8	69	1.01	0.09	11.5
								37	1.07	0.12	8.6	70	1.01	0.06	15.9
38	1.07	0.08	12.7	71	1.08	0.06	18.0
								39	0.75	0.13	5.9	72	0.54	0.08	6.5
40	1.08	0.08	13.1	73	0.87	0.09	9.5
								41	1.01	0.07	13.7	74	1.11	0.14	8.1
42	0.71	0.11	6.7	75	1.16	0.32	3.6
								43	1.09	0.09	12.0	76	0.40	0.26	1.5
44	0.79	0.11	6.9	77	1.06	0.10	10.6
								45	1.10	0.46	2.4	78	0.78	0.06	12.6
46	1.00	0.28	3.5	79	1.05	0.89	1.2
								47	0.71	0.21	3.4	80	0.62	0.05	13.0
48	1.08	0.32	3.3	81	0.87	0.16	5.5
								49	1.18	0.31	3.8	82	0.53	0.42	1.3
50	0.69	0.41	1.7	83	0.77	0.10	7.8
								51	0.75	0.71	1.1	84	1.10	0.07	16.7
52	0.84	0.41	2.1	85	0.90	0.07	12.3
								53	1.06	0.09	11.6	86	1.07	0.06	18.2
54	1.11	0.24	4.5	87	1.11	0.10	11.7
								55	0.72	0.20	3.6	88	1.08	0.08	14.4
56	0.80	0.26	3.1	89	1.18	0.21	5.6
								57	1.10	0.19	5.7	90	1.11	0.07	16.6
58	0.36	0.36	1.0	91	1.11	0.09	12.5
								59	1.13	0.63	1.8	92	1.13	0.51	2.2
60	0.18	0.05	3.7	93	1.03	0.20	5.2
								61	0.55	0.06	8.9	94	0.98	0.08	12.1
62	0.43	0.12	3.5	95	0.58	0.07	8.7
								63	1.05	0.09	12.1

Sequence listing

<110> EXUMA BIOTECH CORP.

FROST, Gregory Ian

SCHREIBER, Gregory Harold

KUNDU, Anirban

<120> anti-idiotype compositions and methods of use thereof

<130> F1.007.WO.01

<150> PCT/US2021/020922

<151> 2021-03-04

<150> PCT/US2020/048843

<151> 2020-08-31

<150> US 63/200,329

<151> 2021-03-01

<150> US 63/136,177

<151> 2021-01-11

<160> 698

<170> patent in version 3.5

<210> 1

<211> 3

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(3)

<223> integrin binding peptide fragment

<400> 1

Arg Gly Asp

1

<210> 2

<211> 43

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(43)

<223> wild type CD8 handle

<400> 2

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

35 40

<210> 3

<211> 42

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(42)

<223> wild type CD28 handle

<400> 3

Phe Cys Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro

35 40

<210> 4

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 4

Cys Pro Pro Cys

1

<210> 5

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 5

Asp Lys Thr His Thr

1 5

<210> 6

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 6

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

1 5 10 15

<210> 7

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 7

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr

1 5 10

<210> 8

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 8

Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10

<210> 9

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 9

Lys Cys Cys Val Asp Cys Pro

1 5

<210> 10

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 10

Lys Tyr Gly Pro Pro Cys Pro

1 5

<210> 11

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 11

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 12

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 12

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210> 13

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 13

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro

<210> 14

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 14

Ser Pro Asn Met Val Pro His Ala His His Ala Gln

1 5 10

<210> 15

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 15

Glu Pro Lys Ser Cys Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 16

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hinge

<400> 16

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 17

<211> 24

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(24)

<223> CD. Alpha. Transmembrane domain

<400> 17

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 18

<211> 23

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(23)

<223> CD8 beta transmembrane domain

<400> 18

Leu Gly Leu Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly

1 5 10 15

Val Ala Ile His Leu Cys Cys

20

<210> 19

<211> 25

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(25)

<223> CD4 transmembrane Domain

<400> 19

Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly

1 5 10 15

Leu Gly Ile Phe Phe Cys Val Arg Cys

20 25

<210> 20

<211> 23

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(23)

<223> CD3 zeta transmembrane domain

<400> 20

Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu

1 5 10 15

Thr Ala Leu Phe Leu Arg Val

20

<210> 21

<211> 27

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(27)

<223> CD28 transmembrane Domain

<400> 21

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 22

<211> 26

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(26)

<223> OX40 transmembrane Domain

<400> 22

Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro

1 5 10 15

Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu

20 25

<210> 23

<211> 24

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(24)

<223> CD7 transmembrane Domain

<400> 23

Ala Leu Pro Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly

1 5 10 15

Leu Gly Val Ala Cys Val Leu Ala

20

<210> 24

<211> 69

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(69)

<223> CD8a handle and transmembrane Domain

<400> 24

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 25

<211> 66

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(66)

<223> CD28 handle and transmembrane Domain

<400> 25

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 26

<211> 163

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(163)

<223> CD3Z activation Domain isoform 1

<400> 26

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

100 105 110

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

115 120 125

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

130 135 140

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

145 150 155 160

Pro Pro Arg

<210> 27

<211> 164

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(164)

<223> CD3Z activation Domain isoform 2

<400> 27

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

100 105 110

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

115 120 125

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

130 135 140

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

145 150 155 160

Leu Pro Pro Arg

<210> 28

<211> 112

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(112)

<223> CD3Z activation Domain isoform 3

<400> 28

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 29

<211> 113

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(113)

<223> CD3Z activation Domain isoform

<400> 29

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 30

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD3Z activation Domain isoform 4

<400> 30

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

1 5 10 15

Val Leu Asp Lys Arg

20

<210> 31

<211> 22

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(22)

<223> CD3Z activation Domain isoform 5

<400> 31

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

1 5 10 15

Ser Glu Ile Gly Met Lys

20

<210> 32

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD3Z activation Domain isoform 6

<400> 32

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

1 5 10 15

Ala Leu His Met Gln

20

<210> 33

<211> 171

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(171)

<223> CD3D activation Domain isoform 1

<400> 33

Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu

1 5 10 15

Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg

20 25 30

Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val

35 40 45

Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile

50 55 60

Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys

65 70 75 80

Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys

85 90 95

Val Glu Leu Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val

100 105 110

Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His

115 120 125

Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg

130 135 140

Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr

145 150 155 160

Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

165 170

<210> 34

<211> 127

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(127)

<223> CD3D activation Domain isoform 2

<400> 34

Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu

1 5 10 15

Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg

20 25 30

Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val

35 40 45

Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile

50 55 60

Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys

65 70 75 80

Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Thr Ala Asp Thr Gln

85 90 95

Ala Leu Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp

100 105 110

Asp Ala Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

115 120 125

<210> 35

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD3D activation Domain isoform 3

<400> 35

Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr Ser

1 5 10 15

His Leu Gly Gly Asn

20

<210> 36

<211> 206

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(206)

<223> CD3E activation Domain isoform 1

<400> 36

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Met Ser

115 120 125

Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu

130 135 140

Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro

145 150 155 160

Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys

165 170 175

Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys

180 185 190

Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 37

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD3E activation Domain isoform 2

<400> 37

Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser

1 5 10 15

Gly Leu Asn Gln Arg

20

<210> 38

<211> 182

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(182)

<223> CD3G activation Domain isoform 1

<400> 38

Met Glu Gln Gly Lys Gly Leu Ala Val Leu Ile Leu Ala Ile Ile Leu

1 5 10 15

Leu Gln Gly Thr Leu Ala Gln Ser Ile Lys Gly Asn His Leu Val Lys

20 25 30

Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu Thr Cys Asp Ala

35 40 45

Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys Met Ile Gly Phe

50 55 60

Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp

65 70 75 80

Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro

85 90 95

Leu Gln Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala

100 105 110

Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val

115 120 125

Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val Arg Gln

130 135 140

Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr

145 150 155 160

Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly

165 170 175

Asn Gln Leu Arg Arg Asn

180

<210> 39

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD3G activation Domain isoform 2

<400> 39

Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser

1 5 10 15

His Leu Gln Gly Asn

20

<210> 40

<211> 226

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(226)

<223> CD79A activation Domain isoform 1

<400> 40

Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe

1 5 10 15

Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala

20 25 30

Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu

35 40 45

Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val

50 55 60

Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe

65 70 75 80

Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val

85 90 95

Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn

100 105 110

Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro

115 120 125

Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile

130 135 140

Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly

145 150 155 160

Thr Leu Leu Leu Phe Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu

165 170 175

Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn

180 185 190

Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly

195 200 205

Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu

210 215 220

Lys Pro

225

<210> 41

<211> 188

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(188)

<223> CD79A activation Domain isoform 2

<400> 41

Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe

1 5 10 15

Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala

20 25 30

Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu

35 40 45

Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val

50 55 60

Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe

65 70 75 80

Leu Gly Pro Gly Glu Asp Pro Asn Glu Pro Pro Pro Arg Pro Phe Leu

85 90 95

Asp Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala Glu Gly Ile

100 105 110

Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu Leu Phe Arg

115 120 125

Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu Tyr

130 135 140

Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met

145 150 155 160

Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val Gly

165 170 175

Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys Pro

180 185

<210> 42

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD79A activation Domain isoform 3

<400> 42

Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met Tyr Glu

1 5 10 15

Asp Ile Ser Arg Gly

20

<210> 43

<211> 113

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(113)

<223> DAP12 activation Domain isoform 1

<400> 43

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys

<210> 44

<211> 107

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(107)

<223> DAP12 activation Domain isoform 2

<400> 44

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Thr Arg Lys

65 70 75 80

Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln

85 90 95

Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln

100 105

<210> 45

<211> 102

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(102)

<223> DAP12 activation Domain isoform 3

<400> 45

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu

20 25 30

Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu

35 40 45

Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala

50 55 60

Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr

65 70 75 80

Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr

85 90 95

Gln Arg Pro Tyr Tyr Lys

100

<210> 46

<211> 101

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(101)

<223> DAP12 activation Domain isoform 4

<400> 46

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu

20 25 30

Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu

35 40 45

Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala

50 55 60

Glu Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln

65 70 75 80

Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln

85 90 95

Arg Pro Tyr Tyr Lys

100

<210> 47

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> DAP12 activation Domain isoform 5

<400> 47

Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser

1 5 10 15

Asp Leu Asn Thr Gln

20

<210> 48

<211> 86

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(86)

<223> FCERlG activation Domain isoform 1

<400> 48

Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala

1 5 10 15

Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu

20 25 30

Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile

35 40 45

Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val

50 55 60

Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys

65 70 75 80

His Glu Lys Pro Pro Gln

85

<210> 49

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> FCERlG activation Domain isoform 2

<400> 49

Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu

1 5 10 15

Thr Leu Lys His Glu

20

<210> 50

<211> 20

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(20)

<223> DAP10 activation Domain

<400> 50

Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr Ile Asn Met

1 5 10 15

Pro Gly Arg Gly

20

<210> 51

<211> 68

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(68)

<223> CD28 activation Domain

<400> 51

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

<210> 52

<211> 619

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(619)

<223> ZAP70 activation Domain

<400> 52

Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser

1 5 10 15

Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly

20 25 30

Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu

35 40 45

Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln

50 55 60

Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro

65 70 75 80

Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys

85 90 95

Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro

100 105 110

Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg

115 120 125

Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser

130 135 140

Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg

145 150 155 160

Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys

165 170 175

Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg

180 185 190

Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val

195 200 205

Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro

210 215 220

Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys

225 230 235 240

Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn

245 250 255

Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala

260 265 270

His Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn

275 280 285

Ser Asp Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys

290 295 300

Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser

305 310 315 320

Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn

325 330 335

Leu Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val

340 345 350

Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile

355 360 365

Lys Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met

370 375 380

Arg Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg

385 390 395 400

Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met

405 410 415

Ala Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu

420 425 430

Ile Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly

435 440 445

Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala

450 455 460

Arg Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe

465 470 475 480

Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg

485 490 495

Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn

500 505 510

Phe Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr

515 520 525

Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys

530 535 540

Gly Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys

545 550 555 560

Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp

565 570 575

Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg

580 585 590

Met Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro

595 600 605

Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala

610 615

<210> 53

<211> 42

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(42)

<223> CD137 costimulatory Domain

<400> 53

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 54

<211> 41

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(41)

<223> CD28 Co-stimulatory Domain

<400> 54

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 55

<211> 41

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(41)

<223> IC costimulatory Domain

<400> 55

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Ala Tyr Ala Ala

20 25 30

Ala Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 56

<211> 35

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(35)

<223> ICOS costimulatory Domain

<400> 56

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210> 57

<211> 37

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(37)

<223> OX40 Co-stimulatory Domain

<400> 57

Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly

1 5 10 15

Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser

20 25 30

Thr Leu Ala Lys Ile

35

<210> 58

<211> 49

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(49)

<223> CD27 Co-stimulatory Domain

<400> 58

His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu

1 5 10 15

Pro Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser

20 25 30

Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser

35 40 45

Pro

<210> 59

<211> 114

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(114)

<223> BLTA costimulatory Domain

<400> 59

Cys Cys Leu Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr

1 5 10 15

Ala Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln

20 25 30

Thr Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr

35 40 45

Gly Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly

50 55 60

Ser Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile

65 70 75 80

Val Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu

85 90 95

Ala Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val

100 105 110

Arg Ser

<210> 60

<211> 187

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(187)

<223> CD30 Co-stimulatory Domain

<400> 60

Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys Tyr

1 5 10 15

Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg Pro

20 25 30

Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu Pro

35 40 45

Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr Cys

50 55 60

His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp Ala

65 70 75 80

Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro Arg

85 90 95

Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile Met

100 105 110

Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro Glu

115 120 125

Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu Leu

130 135 140

Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro Pro

145 150 155 160

Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly Lys

165 170 175

Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys

180 185

<210> 61

<211> 54

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(54)

<223> GITR costimulatory domain

<400> 61

His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln

1 5 10 15

Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln

20 25 30

Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg

35 40 45

Leu Gly Asp Leu Trp Val

50

<210> 62

<211> 60

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(60)

<223> HVEM costimulatory Domain

<400> 62

Cys Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val

1 5 10 15

Ser Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile

20 25 30

Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu

35 40 45

Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His

50 55 60

<210> 63

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 63

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 64

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 64

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 65

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 65

Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 66

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 66

Gly Gly Ser Gly

1

<210> 67

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 67

Gly Gly Ser Gly Gly

1 5

<210> 68

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 68

Gly Ser Gly Ser Gly

1 5

<210> 69

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 69

Gly Ser Gly Gly Gly

1 5

<210> 70

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 70

Gly Gly Gly Ser Gly

1 5

<210> 71

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 71

Gly Ser Ser Ser Gly

1 5

<210> 72

<211> 21

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(21)

<223> CD8 Signal peptide

<400> 72

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 73

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: HA epitope

<400> 73

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

<210> 74

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FLAG epitope

<400> 74

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 75

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: c-myc epitope

<400> 75

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 76

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: his5 affinity

<400> 76

His His His His His

1 5

<210> 77

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: hisX6 affinity

<400> 77

His His His His His His

1 5

<210> 78

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: streptavidin tag affinity

<400> 78

Trp Ser His Pro Gln Phe Glu Lys

1 5

<210> 79

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: affinity tag

<400> 79

Arg Tyr Ile Arg Ser

1 5

<210> 80

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: affinity tag

<400> 80

Phe His His Thr

1

<210> 81

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: affinity tag

<400> 81

Trp Glu Ala Ala Ala Arg Glu Ala Cys Cys Arg Glu Cys Cys Ala Arg

1 5 10 15

Ala

<210> 82

<211> 357

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(357)

<223> EGFR truncation

<400> 82

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 83

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: cutting signal

<400> 83

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 84

<211> 368

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG IL7RA Ins PPCL (interleukin 7 receptor)

<400> 84

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Glu Ile Asn Asn Ser Ser

325 330 335

Gly Glu Met Asp Pro Ile Leu Leu Pro Pro Cys Leu Thr Ile Ser Ile

340 345 350

Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu

355 360 365

<210> 85

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG IL7RA Ins PPCL (interleukin 7 receptor)

<400> 85

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro Ile Leu Leu

195 200 205

Pro Pro Cys Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu

210 215 220

Leu Val Ile Leu Ala Cys Val Leu

225 230

<210> 86

<211> 194

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc TAG LMP1 NC_007505_1

<400> 86

Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu His Asp Leu Glu

1 5 10 15

Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro Arg Gly Pro Pro Leu Ser

20 25 30

Ser Ser Leu Gly Leu Ala Leu Leu Leu Leu Leu Leu Ala Leu Leu Phe

35 40 45

Trp Leu Tyr Ile Val Met Ser Asp Trp Thr Gly Gly Ala Leu Leu Val

50 55 60

Leu Tyr Ser Phe Ala Leu Met Leu Ile Ile Ile Ile Leu Ile Ile Phe

65 70 75 80

Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu Gly Ala Leu Cys Ile Leu

85 90 95

Leu Leu Met Ile Thr Leu Leu Leu Ile Ala Leu Trp Asn Leu His Gly

100 105 110

Gln Ala Leu Phe Leu Gly Ile Val Leu Phe Ile Phe Gly Cys Leu Leu

115 120 125

Val Leu Gly Ile Trp Ile Tyr Leu Leu Glu Met Leu Trp Arg Leu Gly

130 135 140

Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe Leu Ala Phe Phe Leu Asp

145 150 155 160

Leu Ile Leu Leu Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr

165 170 175

Leu Leu Val Asp Leu Leu Trp Leu Leu Leu Phe Leu Ala Ile Leu Ile

180 185 190

Trp Met

<210> 87

<211> 174

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc LMP1 NC_007505_1

<400> 87

Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Ser Ser Ser Leu Gly

1 5 10 15

Leu Ala Leu Leu Leu Leu Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile

20 25 30

Val Met Ser Asp Trp Thr Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe

35 40 45

Ala Leu Met Leu Ile Ile Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg

50 55 60

Asp Leu Leu Cys Pro Leu Gly Ala Leu Cys Ile Leu Leu Leu Met Ile

65 70 75 80

Thr Leu Leu Leu Ile Ala Leu Trp Asn Leu His Gly Gln Ala Leu Phe

85 90 95

Leu Gly Ile Val Leu Phe Ile Phe Gly Cys Leu Leu Val Leu Gly Ile

100 105 110

Trp Ile Tyr Leu Leu Glu Met Leu Trp Arg Leu Gly Ala Thr Ile Trp

115 120 125

Gln Leu Leu Ala Phe Phe Leu Ala Phe Phe Leu Asp Leu Ile Leu Leu

130 135 140

Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu Leu Val Asp

145 150 155 160

Leu Leu Trp Leu Leu Leu Phe Leu Ala Ile Leu Ile Trp Met

165 170

<210> 88

<211> 184

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: lmp1nc_007505_1

<400> 88

Met Glu His Asp Leu Glu Arg Gly Pro Pro Gly Pro Arg Arg Pro Pro

1 5 10 15

Arg Gly Pro Pro Leu Ser Ser Ser Leu Gly Leu Ala Leu Leu Leu Leu

20 25 30

Leu Leu Ala Leu Leu Phe Trp Leu Tyr Ile Val Met Ser Asp Trp Thr

35 40 45

Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu Met Leu Ile Ile

50 55 60

Ile Ile Leu Ile Ile Phe Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu

65 70 75 80

Gly Ala Leu Cys Ile Leu Leu Leu Met Ile Thr Leu Leu Leu Ile Ala

85 90 95

Leu Trp Asn Leu His Gly Gln Ala Leu Phe Leu Gly Ile Val Leu Phe

100 105 110

Ile Phe Gly Cys Leu Leu Val Leu Gly Ile Trp Ile Tyr Leu Leu Glu

115 120 125

Met Leu Trp Arg Leu Gly Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe

130 135 140

Leu Ala Phe Phe Leu Asp Leu Ile Leu Leu Ile Ile Ala Leu Tyr Leu

145 150 155 160

Gln Gln Asn Trp Trp Thr Leu Leu Val Asp Leu Leu Trp Leu Leu Leu

165 170 175

Phe Leu Ala Ile Leu Ile Trp Met

180

<210> 89

<211> 162

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: lmp1nc_007505_1

<400> 89

Met Ser Leu Gly Leu Ala Leu Leu Leu Leu Leu Leu Ala Leu Leu Phe

1 5 10 15

Trp Leu Tyr Ile Val Met Ser Asp Trp Thr Gly Gly Ala Leu Leu Val

20 25 30

Leu Tyr Ser Phe Ala Leu Met Leu Ile Ile Ile Ile Leu Ile Ile Phe

35 40 45

Ile Phe Arg Arg Asp Leu Leu Cys Pro Leu Gly Ala Leu Cys Ile Leu

50 55 60

Leu Leu Met Ile Thr Leu Leu Leu Ile Ala Leu Trp Asn Leu His Gly

65 70 75 80

Gln Ala Leu Phe Leu Gly Ile Val Leu Phe Ile Phe Gly Cys Leu Leu

85 90 95

Val Leu Gly Ile Trp Ile Tyr Leu Leu Glu Met Leu Trp Arg Leu Gly

100 105 110

Ala Thr Ile Trp Gln Leu Leu Ala Phe Phe Leu Ala Phe Phe Leu Asp

115 120 125

Leu Ile Leu Leu Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr

130 135 140

Leu Leu Val Asp Leu Leu Trp Leu Leu Leu Phe Leu Ala Ile Leu Ile

145 150 155 160

Trp Met

<210> 90

<211> 363

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG CRLF2 transcript variant 1 NM_022148_3

<400> 90

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Ala Glu Thr Pro Thr Pro Pro

325 330 335

Lys Pro Lys Leu Ser Lys Cys Ile Leu Ile Ser Ser Leu Ala Ile Leu

340 345 350

Leu Met Val Ser Leu Leu Leu Leu Ser Leu Trp

355 360

<210> 91

<211> 227

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG CRLF2 transcript variant 1 NM_022148_3

<400> 91

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Ala Glu Thr Pro Thr Pro Pro Lys Pro Lys Leu Ser Lys Cys Ile

195 200 205

Leu Ile Ser Ser Leu Ala Ile Leu Leu Met Val Ser Leu Leu Leu Leu

210 215 220

Ser Leu Trp

225

<210> 92

<211> 354

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG CSF2RB NM-000395_2

<400> 92

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Thr Glu Ser Val Leu Pro Met

325 330 335

Trp Val Leu Ala Leu Ile Glu Ile Phe Leu Thr Ile Ala Val Leu Leu

340 345 350

Ala Leu

<210> 93

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG CSF2RB NM-000395_2

<400> 93

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Thr Glu Ser Val Leu Pro Met Trp Val Leu Ala Leu Ile Glu Ile

195 200 205

Phe Leu Thr Ile Ala Val Leu Leu Ala Leu

210 215

<210> 94

<211> 360

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG CSF3R transcript variant 1 NM-000760_3

<400> 94

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Thr Pro Glu Gly Ser Glu Leu

325 330 335

His Ile Ile Leu Gly Leu Phe Gly Leu Leu Leu Leu Leu Asn Cys Leu

340 345 350

Cys Gly Thr Ala Trp Leu Cys Cys

355 360

<210> 95

<211> 224

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG CSF3R transcript variant 1 NM-000760_3

<400> 95

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Thr Pro Glu Gly Ser Glu Leu His Ile Ile Leu Gly Leu Phe Gly

195 200 205

Leu Leu Leu Leu Leu Asn Cys Leu Cys Gly Thr Ala Trp Leu Cys Cys

210 215 220

<210> 96

<211> 359

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG EPOR transcript variant 1 NM_000121_3

<400> 96

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Thr Pro Ser Asp Leu Asp Pro

325 330 335

Cys Cys Leu Thr Leu Ser Leu Ile Leu Val Val Ile Leu Val Leu Leu

340 345 350

Thr Val Leu Ala Leu Leu Ser

355

<210> 97

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG EPOR transcript variant 1 NM_000121_3

<400> 97

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Thr Pro Ser Asp Leu Asp Pro Cys Cys Leu Thr Leu Ser Leu Ile

195 200 205

Leu Val Val Ile Leu Val Leu Leu Thr Val Leu Ala Leu Leu Ser

210 215 220

<210> 98

<211> 368

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG GHR transcript variant 1 NM_000163_4

<400> 98

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Thr Leu Pro Gln Met Ser Gln

325 330 335

Phe Thr Cys Cys Glu Asp Phe Tyr Phe Pro Trp Leu Leu Cys Ile Ile

340 345 350

Phe Gly Ile Phe Gly Leu Thr Val Met Leu Phe Val Phe Leu Phe Ser

355 360 365

<210> 99

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTAG GHR transcript variant 1 NM_000163_4

<400> 99

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Thr Leu Pro Gln Met Ser Gln Phe Thr Cys Cys Glu Asp Phe Tyr

195 200 205

Phe Pro Trp Leu Leu Cys Ile Ile Phe Gly Ile Phe Gly Leu Thr Val

210 215 220

Met Leu Phe Val Phe Leu Phe Ser

225 230

<210> 100

<211> 360

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: truncated eTAG after Fn F523C IL27RA nm_004843_3

<400> 100

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly His Leu Pro Asp Asn Thr Leu

325 330 335

Arg Trp Lys Val Leu Pro Gly Ile Leu Cys Leu Trp Gly Leu Phe Leu

340 345 350

Leu Gly Cys Gly Leu Ser Leu Ala

355 360

<210> 101

<211> 224

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: truncated eTAG after Fn F523C IL27RA nm_004843_3

<400> 101

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln His Leu Pro Asp Asn Thr Leu Arg Trp Lys Val Leu Pro Gly Ile

195 200 205

Leu Cys Leu Trp Gly Leu Phe Leu Leu Gly Cys Gly Leu Ser Leu Ala

210 215 220

<210> 102

<211> 359

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: truncated eTAG after Fn s505_nmpl_005373_2

<400> 102

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Glu Thr Ala Thr Glu Thr Ala

325 330 335

Trp Ile Ser Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Asn Ala

340 345 350

Val Leu Gly Leu Leu Leu Leu

355

<210> 103

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: truncated eTAG after Fn s505_nmpl_005373_2

<400> 103

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser Leu Val Thr Ala Leu

195 200 205

His Leu Val Leu Gly Leu Asn Ala Val Leu Gly Leu Leu Leu Leu

210 215 220

<210> 104

<211> 368

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTag 0A JUN NM_002228_3

<400> 104

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Leu Glu Arg Ile Ala Arg Leu

325 330 335

Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser

340 345 350

Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val

355 360 365

<210> 105

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTag 1A JUN NM_002228_3

<400> 105

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Leu Glu Arg Ile Ala Arg Leu

325 330 335

Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser

340 345 350

Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val

355 360 365

Ala

<210> 106

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTag 2A JUN NM_002228_3

<400> 106

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Phe Gly Thr Ser Gly Gln Lys Thr

165 170 175

Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln

180 185 190

Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro

195 200 205

Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val

210 215 220

Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn

225 230 235 240

Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn

245 250 255

Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His

260 265 270

Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met

275 280 285

Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val

290 295 300

Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly

305 310 315 320

Leu Glu Gly Cys Pro Thr Asn Gly Leu Glu Arg Ile Ala Arg Leu Glu

325 330 335

Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr

340 345 350

Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val Ala

355 360 365

Ala

<210> 107

<211> 371

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTag 3A JUN NM_002228_3

<400> 107

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Leu Glu Arg Ile Ala Arg Leu

325 330 335

Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser

340 345 350

Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val

355 360 365

Ala Ala Ala

370

<210> 108

<211> 372

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: eTag 4A JUN NM_002228_3

<400> 108

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Leu Glu Arg Ile Ala Arg Leu

325 330 335

Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser

340 345 350

Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val

355 360 365

Ala Ala Ala Ala

370

<210> 109

<211> 69

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc TAG 0A JUN NM_002228_3

<400> 109

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Glu

20 25 30

Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn

35 40 45

Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln

50 55 60

Leu Lys Gln Lys Val

65

<210> 110

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc TAG 1A JUN NM_002228_3

<400> 110

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Glu

20 25 30

Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn

35 40 45

Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln

50 55 60

Leu Lys Gln Lys Val Ala

65 70

<210> 111

<211> 71

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc TAG 2A JUN NM_002228_3

<400> 111

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Glu

20 25 30

Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn

35 40 45

Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln

50 55 60

Leu Lys Gln Lys Val Ala Ala

65 70

<210> 112

<211> 72

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc TAG 3A JUN NM_002228_3

<400> 112

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Glu

20 25 30

Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn

35 40 45

Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln

50 55 60

Leu Lys Gln Lys Val Ala Ala Ala

65 70

<210> 113

<211> 73

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: myc TAG 4A JUN NM_002228_3

<400> 113

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Glu

20 25 30

Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn

35 40 45

Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln

50 55 60

Leu Lys Gln Lys Val Ala Ala Ala Ala

65 70

<210> 114

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD2 transcript variant 1 NM_001328609_1

<400> 114

Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met Val Phe Val

1 5 10 15

Ala Leu Leu Val Phe Tyr Ile

20

<210> 115

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3D transcript variant 1 NM-000732-4

<400> 115

Gly Ile Ile Val Thr Asp Val Ile Ala Thr Leu Leu Leu Ala Leu Gly

1 5 10 15

Val Phe Cys Phe Ala

20

<210> 116

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3E NM-000733-3

<400> 116

Val Met Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly

1 5 10 15

Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser

20 25

<210> 117

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3G NM_000073_2

<400> 117

Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val Leu Ala Val Gly

1 5 10 15

Val Tyr Phe Ile Ala

20

<210> 118

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3Z CD247 transcript variant 1 NM_198053_2

<400> 118

Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu

1 5 10 15

Thr Ala Leu Phe Leu

20

<210> 119

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD4 transcript variants 1 and 2NM_000616_4

<400> 119

Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile

1 5 10 15

Gly Leu Gly Ile Phe Phe

20

<210> 120

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD8A transcript variant 1 NM_001768_6

<400> 120

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr

20

<210> 121

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD8B transcript variant 2 NM_172213_3

<400> 121

Leu Gly Leu Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly

1 5 10 15

Val Ala Ile His Leu Cys Cys

20

<210> 122

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD27 NM_001242_4

<400> 122

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

1 5 10 15

Ala Leu Phe Leu His

20

<210> 123

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD28 transcript variant 1 NM_006139_3

<400> 123

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 124

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD40 transcript variants 1 and 6 NM_001250_5

<400> 124

Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu

1 5 10 15

Leu Val Leu Val Phe Ile

20

<210> 125

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD79A transcript variant 1 NM_001783_3

<400> 125

Ile Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro

1 5 10 15

Gly Thr Leu Leu Leu Phe

20

<210> 126

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD79B transcript variant 3NM_001039933_2

<400> 126

Gly Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val

1 5 10 15

Pro Ile Phe Leu Leu Leu

20

<210> 127

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CRLF2 transcript variant 1 NM_022148_3

<400> 127

Phe Ile Leu Ile Ser Ser Leu Ala Ile Leu Leu Met Val Ser Leu Leu

1 5 10 15

Leu Leu Ser Leu Trp

20

<210> 128

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CRLF2 transcript variant 1 NM_022148_3

<400> 128

Cys Ile Leu Ile Ser Ser Leu Ala Ile Leu Leu Met Val Ser Leu Leu

1 5 10 15

Leu Leu Ser Leu Trp

20

<210> 129

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF2RA transcript variants 7 and 8 nm_001161529_1

<400> 129

Asn Leu Gly Ser Val Tyr Ile Tyr Val Leu Leu Ile Val Gly Thr Leu

1 5 10 15

Val Cys Gly Ile Val Leu Gly Phe Leu Phe

20 25

<210> 130

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF2RB nm_000395_2

<400> 130

Met Trp Val Leu Ala Leu Ile Val Ile Phe Leu Thr Ile Ala Val Leu

1 5 10 15

Leu Ala Leu

<210> 131

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF2RB nm_000395_2

<400> 131

Met Trp Val Leu Ala Leu Ile Glu Ile Phe Leu Thr Ile Ala Val Leu

1 5 10 15

Leu Ala Leu

<210> 132

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF3R transcript variant 1 nm_000760_3

<400> 132

Ile Ile Leu Gly Leu Phe Gly Leu Leu Leu Leu Leu Thr Cys Leu Cys

1 5 10 15

Gly Thr Ala Trp Leu Cys Cys

20

<210> 133

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF3R transcript variant 1 nm_000760_3

<400> 133

Ile Ile Leu Gly Leu Phe Gly Leu Leu Leu Leu Leu Asn Cys Leu Cys

1 5 10 15

Gly Thr Ala Trp Leu Cys Cys

20

<210> 134

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: EPOR transcript variant 1 nm_000121_3

<400> 134

Leu Ile Leu Thr Leu Ser Leu Ile Leu Val Val Ile Leu Val Leu Leu

1 5 10 15

Thr Val Leu Ala Leu Leu Ser

20

<210> 135

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: EPOR transcript variant 1 nm_000121_3

<400> 135

Cys Cys Leu Thr Leu Ser Leu Ile Leu Val Val Ile Leu Val Leu Leu

1 5 10 15

Thr Val Leu Ala Leu Leu Ser

20

<210> 136

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FCER1G NM-004106_1

<400> 136

Leu Cys Tyr Ile Leu Asp Ala Ile Leu Phe Leu Tyr Gly Ile Val Leu

1 5 10 15

Thr Leu Leu Tyr Cys

20

<210> 137

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FCGR2CNM_2015163_5

<400> 137

Ile Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala

1 5 10 15

Ala Val Val Ala Leu Ile Tyr

20

<210> 138

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FCGRA2 transcript variant 1 NM_001136219_1

<400> 138

Ile Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile Val Ala

1 5 10 15

Ala Val Val Ala Leu Ile Tyr

20

<210> 139

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: GHR transcript variant 1 NM_000163_4

<400> 139

Phe Pro Trp Leu Leu Ile Ile Ile Phe Gly Ile Phe Gly Leu Thr Val

1 5 10 15

Met Leu Phe Val Phe Leu Phe Ser

20

<210> 140

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: GHR transcript variant 1 NM_000163_4

<400> 140

Phe Pro Trp Leu Leu Cys Ile Ile Phe Gly Ile Phe Gly Leu Thr Val

1 5 10 15

Met Leu Phe Val Phe Leu Phe Ser

20

<210> 141

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ICOS nm_012092.3

<400> 141

Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu

1 5 10 15

Gly Cys Ile Leu Ile

20

<210> 142

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ifnar1nm_000629_2

<400> 142

Ile Trp Leu Ile Val Gly Ile Cys Ile Ala Leu Phe Ala Leu Pro Phe

1 5 10 15

Val Ile Tyr Ala Ala

20

<210> 143

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IFNAR2 transcript variant 1 NM 207585_2

<400> 143

Ile Gly Gly Ile Ile Thr Val Phe Leu Ile Ala Leu Val Leu Thr Ser

1 5 10 15

Thr Ile Val Thr Leu

20

<210> 144

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ifngr1nm_000416_2

<400> 144

Ser Leu Trp Ile Pro Val Val Ala Ala Leu Leu Leu Phe Leu Val Leu

1 5 10 15

Ser Leu Val Phe Ile

20

<210> 145

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IFNGR2 transcript variant 1 nm_001329128_1

<400> 145

Val Ile Leu Ile Ser Val Gly Thr Phe Ser Leu Leu Ser Val Leu Ala

1 5 10 15

Gly Ala Cys Phe Phe

20

<210> 146

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ifnlr1nm_170743_3

<400> 146

Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu Val Ile Ala Ala

1 5 10 15

Gly Gly Val Ile Trp

20

<210> 147

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1R1 transcript variant 2 NM_001288706_1

<400> 147

His Met Ile Gly Ile Cys Val Thr Leu Thr Val Ile Ile Val Cys Ser

1 5 10 15

Val Phe Ile Tyr Lys Ile Phe

20

<210> 148

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RAP transcript variant 1 NM_002182_3

<400> 148

Val Leu Leu Val Val Ile Leu Ile Val Val Tyr His Val Tyr Trp Leu

1 5 10 15

Glu Met Val Leu Phe

20

<210> 149

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RL1 transcript variant 1 NM_016232.4

<400> 149

Ile Tyr Cys Ile Ile Ala Val Cys Ser Val Phe Leu Met Leu Ile Asn

1 5 10 15

Val Leu Val Ile Ile

20

<210> 150

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RL2 NM-003854.2

<400> 150

Ala Tyr Leu Ile Gly Gly Leu Ile Ala Leu Val Ala Val Ala Val Ser

1 5 10 15

Val Val Tyr Ile Tyr

20

<210> 151

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL2RA transcript variant 1 NM-000417-2

<400> 151

Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu

1 5 10 15

Ser Gly Leu

<210> 152

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL2RB transcript variant 1 NM-000878-4

<400> 152

Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly

1 5 10 15

Phe Ile Ile Leu Val Tyr Leu Leu Ile

20 25

<210> 153

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL2RG nm—000206_2

<400> 153

Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys

1 5 10 15

Val Tyr Phe Trp Leu

20

<210> 154

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL3RA transcript variants 1 and 2NM_002183_3

<400> 154

Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Val Cys Val

1 5 10 15

Phe Val Ile Cys

20

<210> 155

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL4R transcript variant 1 NM-000418-3

<400> 155

Leu Leu Leu Gly Val Ser Val Ser Cys Ile Val Ile Leu Ala Val Cys

1 5 10 15

Leu Leu Cys Tyr Val Ser Ile Thr

20

<210> 156

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL5RA transcript variant 1 NM-000564-4

<400> 156

Phe Val Ile Val Ile Met Ala Thr Ile Cys Phe Ile Leu Leu Ile Leu

1 5 10 15

Ser Leu Ile Cys

20

<210> 157

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL6R transcript variant 1 NM-000565-3

<400> 157

Thr Phe Leu Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu Leu Cys

1 5 10 15

Ile Ala Ile Val Leu

20

<210> 158

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL6ST transcript variants 1 and 3NM_002184_3

<400> 158

Ala Ile Val Val Pro Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu

1 5 10 15

Gly Val Leu Phe Cys Phe

20

<210> 159

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL7RA nm_002185_3

<400> 159

Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu

1 5 10 15

Val Ile Leu Ala Cys Val Leu

20

<210> 160

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL7RA Ins PPCL (interleukin 7 receptor)

<400> 160

Ile Leu Leu Pro Pro Cys Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser

1 5 10 15

Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu

20 25

<210> 161

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL9R transcript variant 1 NM_002186_2

<400> 161

Gly Asn Thr Leu Val Ala Val Ser Ile Phe Leu Leu Leu Thr Gly Pro

1 5 10 15

Thr Tyr Leu Leu Phe

20

<210> 162

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL10RA transcript variant 1 NM_001558_3

<400> 162

Val Ile Ile Phe Phe Ala Phe Val Leu Leu Leu Ser Gly Ala Leu Ala

1 5 10 15

Tyr Cys Leu Ala Leu

20

<210> 163

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL10RB NM-000628_4

<400> 163

Trp Met Val Ala Val Ile Leu Met Ala Ser Val Phe Met Val Cys Leu

1 5 10 15

Ala Leu Leu Gly Cys Phe

20

<210> 164

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL11RA NM-001142784_2

<400> 164

Ser Leu Gly Ile Leu Ser Phe Leu Gly Leu Val Ala Gly Ala Leu Ala

1 5 10 15

Leu Gly Leu Trp Leu

20

<210> 165

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL12RB1 transcript variants 1 and 4NM_005535_2

<400> 165

Trp Leu Ile Phe Phe Ala Ser Leu Gly Ser Phe Leu Ser Ile Leu Leu

1 5 10 15

Val Gly Val Leu Gly Tyr Leu Gly Leu

20 25

<210> 166

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL12RB2 transcript variants 1 and 3NM_001559_2

<400> 166

Trp Met Ala Phe Val Ala Pro Ser Ile Cys Ile Ala Ile Ile Met Val

1 5 10 15

Gly Ile Phe Ser Thr

20

<210> 167

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL13RA1 NM-001560-2

<400> 167

Leu Tyr Ile Thr Met Leu Leu Ile Val Pro Val Ile Val Ala Gly Ala

1 5 10 15

Ile Ile Val Leu Leu Leu Tyr Leu

20

<210> 168

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL13RA2 NM-000640-2

<400> 168

Phe Trp Leu Pro Phe Gly Phe Ile Leu Ile Leu Val Ile Phe Val Thr

1 5 10 15

Gly Leu Leu Leu

20

<210> 169

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL15RA transcript variant 4 NM_001256765_1

<400> 169

Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val

1 5 10 15

Ser Leu Leu Ala Cys Tyr Leu

20

<210> 170

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RA nm_014339_6

<400> 170

Val Tyr Trp Phe Ile Thr Gly Ile Ser Ile Leu Leu Val Gly Ser Val

1 5 10 15

Ile Leu Leu Ile Val

20

<210> 171

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RB NM_018725_3

<400> 171

Leu Leu Leu Leu Ser Leu Leu Val Ala Thr Trp Val Leu Val Ala Gly

1 5 10 15

Ile Tyr Leu Met Trp

20

<210> 172

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RC transcript variant 1 NM_153460_3

<400> 172

Trp Ala Leu Val Trp Leu Ala Cys Leu Leu Phe Ala Ala Ala Leu Ser

1 5 10 15

Leu Ile Leu Leu Leu

20

<210> 173

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RD transcript variant 2 NM_017563_4

<400> 173

Ala Val Ala Ile Thr Val Pro Leu Val Val Ile Ser Ala Phe Ala Thr

1 5 10 15

Leu Phe Thr Val Met

20

<210> 174

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RE transcript variant 1 NM_153480_1

<400> 174

Leu Gly Leu Leu Ile Leu Ala Leu Leu Ala Leu Leu Thr Leu Leu Gly

1 5 10 15

Val Val Leu Ala Leu

20

<210> 175

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL18R1 transcript variant 1 NM-003855-3

<400> 175

Gly Met Ile Ile Ala Val Leu Ile Leu Val Ala Val Val Cys Leu Val

1 5 10 15

Thr Val Cys Val Ile

20

<210> 176

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL18RAP NM-003853_3

<400> 176

Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile Gly Thr Leu Val

1 5 10 15

Ala Val Leu Ala Ala

20

<210> 177

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL20RA transcript variant 1 NM_014432_3

<400> 177

Ile Ile Phe Trp Tyr Val Leu Pro Ile Ser Ile Thr Val Phe Leu Phe

1 5 10 15

Ser Val Met Gly Tyr

20

<210> 178

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL20RB NM-144717_3

<400> 178

Val Leu Ala Leu Phe Ala Phe Val Gly Phe Met Leu Ile Leu Val Val

1 5 10 15

Val Pro Leu Phe Val

20

<210> 179

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL21R transcript variant 2 NM_181078_2

<400> 179

Gly Trp Asn Pro His Leu Leu Leu Leu Leu Leu Leu Val Ile Val Phe

1 5 10 15

Ile Pro Ala Phe Trp

20

<210> 180

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL22RA1 NM-021258_3

<400> 180

Tyr Ser Phe Ser Gly Ala Phe Leu Phe Ser Met Gly Phe Leu Val Ala

1 5 10 15

Val Leu Cys Tyr Leu

20

<210> 181

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL23R nm_144701_2

<400> 181

Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser Ile Leu Ser

1 5 10 15

Leu Ile Gly Ile Phe

20

<210> 182

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL27RA NM-004843_3

<400> 182

Val Leu Pro Gly Ile Leu Phe Leu Trp Gly Leu Phe Leu Leu Gly Cys

1 5 10 15

Gly Leu Ser Leu Ala

20

<210> 183

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL27RA NM-004843_3

<400> 183

Val Leu Pro Gly Ile Leu Cys Leu Trp Gly Leu Phe Leu Leu Gly Cys

1 5 10 15

Gly Leu Ser Leu Ala

20

<210> 184

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL31RA transcript variant 1 NM_139017_5

<400> 184

Ile Ile Leu Ile Thr Ser Leu Ile Gly Gly Gly Leu Leu Ile Leu Ile

1 5 10 15

Ile Leu Thr Val Ala Tyr Gly Leu

20

<210> 185

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: LEPR transcript variant 1 nm_002303_5

<400> 185

Ala Gly Leu Tyr Val Ile Val Pro Val Ile Ile Ser Ser Ser Ile Leu

1 5 10 15

Leu Leu Gly Thr Leu Leu Ile

20

<210> 186

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: LIFR NM_001127671_1

<400> 186

Val Gly Leu Ile Ile Ala Ile Leu Ile Pro Val Ala Val Ala Val Ile

1 5 10 15

Val Gly Val Val Thr Ser Ile Leu Cys

20 25

<210> 187

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: mplnm_005373_2

<400> 187

Ile Ser Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val

1 5 10 15

Leu Gly Leu Leu Leu Leu

20

<210> 188

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: mplnm_005373_2

<400> 188

Ile Ser Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Asn Ala Val

1 5 10 15

Leu Gly Leu Leu Leu Leu

20

<210> 189

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: OSMR transcript variant 4 nm_001323505_1

<400> 189

Leu Ile His Ile Leu Leu Pro Met Val Phe Cys Val Leu Leu Ile Met

1 5 10 15

Val Met Cys Tyr Leu

20

<210> 190

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: PRLR transcript variant 1 nm_000949_6

<400> 190

Thr Thr Val Trp Ile Ser Val Ala Val Leu Ser Ala Val Ile Cys Leu

1 5 10 15

Ile Ile Val Trp Ala Val Ala Leu

20

<210> 191

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: tnfrsf4nm_003327_3

<400> 191

Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro

1 5 10 15

Leu Ala Ile Leu Leu

20

<210> 192

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF8 transcript variant 1 nm_001243_4

<400> 192

Pro Val Leu Asp Ala Gly Pro Val Leu Phe Trp Val Ile Leu Val Leu

1 5 10 15

Val Val Val Val Gly Ser Ser Ala Phe Leu Leu Cys

20 25

<210> 193

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: tnfrsf9nm_001561_5

<400> 193

Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu

1 5 10 15

Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val

20 25

<210> 194

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF14 transcript variant 1 nm_003820_3

<400> 194

Trp Trp Phe Leu Ser Gly Ser Leu Val Ile Val Ile Val Cys Ser Thr

1 5 10 15

Val Gly Leu Ile Ile

20

<210> 195

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF18 transcript variant 1 nm_004195_2

<400> 195

Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys Val Leu

1 5 10 15

Leu Leu Thr Ser Ala

20

<210> 196

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD2 transcript variant 1 NM_001328609_1

<400> 196

Thr Lys Arg Lys Lys Gln Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu

1 5 10 15

Thr Arg Ala His Arg Val Ala Thr Glu Glu Arg Gly Arg Lys Pro His

20 25 30

Gln Ile Pro Ala Ser Thr Pro Gln Asn Pro Ala Thr Ser Gln His Pro

35 40 45

Pro Pro Pro Pro Gly His Arg Ser Gln Ala Pro Ser His Arg Pro Pro

50 55 60

Pro Pro Gly His Arg Val Gln His Gln Pro Gln Lys Arg Pro Pro Ala

65 70 75 80

Pro Ser Gly Thr Gln Val His Gln Gln Lys Gly Pro Pro Leu Pro Arg

85 90 95

Pro Arg Val Gln Pro Lys Pro Pro His Gly Ala Ala Glu Asn Ser Leu

100 105 110

Ser Pro Ser Ser Asn

115

<210> 197

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3D transcript variant 1 NM-000732-4

<400> 197

Gly His Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu

1 5 10 15

Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala

20 25 30

Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

35 40 45

<210> 198

<211> 55

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3E NM-000733-3

<400> 198

Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala

1 5 10 15

Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro

20 25 30

Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser

35 40 45

Gly Leu Asn Gln Arg Arg Ile

50 55

<210> 199

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD3G NM_000073_2

<400> 199

Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys Gln Thr Leu

1 5 10 15

Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp

20 25 30

Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn

35 40 45

<210> 200

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD4 transcript variants 1 and 2NM_000616_4

<400> 200

Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln

1 5 10 15

Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His Arg

20 25 30

Phe Gln Lys Thr Cys Ser Pro Ile

35 40

<210> 201

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD8A transcript variant 1 NM_001768_6

<400> 201

Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg

1 5 10 15

Pro Val Val Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

20 25 30

<210> 202

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD8B transcript variant 2 NM_172213_3

<400> 202

Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Pro Gln Gly Glu

1 5 10 15

Gly Ile Ser Gly Thr Phe Val Pro Gln Cys Leu His Gly Tyr Tyr Ser

20 25 30

Asn Thr Thr Thr Ser Gln Lys Leu Leu Asn Pro Trp Ile Leu Lys Thr

35 40 45

<210> 203

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD8B transcript variant 3NM_172101_3

<400> 203

Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Leu Arg Leu His

1 5 10 15

Pro Leu Glu Lys Cys Ser Arg Met Asp Tyr

20 25

<210> 204

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD8B transcript variant 5 NM_004931_4

<400> 204

Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Phe Tyr Lys

1 5 10 15

<210> 205

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD27 NM_001242_4

<400> 205

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 206

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: mutant delta Lck CD28 transcript variant 1 NM_006139_3

<400> 206

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Ala Tyr Ala Ala

20 25 30

Ala Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 207

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD28 transcript variant 1 NM_006139_3

<400> 207

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 208

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD40 transcript variants 1 and 6 NM_001250_5

<400> 208

Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys Gln

1 5 10 15

Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr

20 25 30

Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln

35 40 45

Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

50 55 60

<210> 209

<211> 66

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis of CD40 transcript variant 5 NM_001322421_1

<400> 209

Ser Glu Ser Ser Glu Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro

1 5 10 15

His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro

20 25 30

Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln

35 40 45

Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu

50 55 60

Arg Gln

65

<210> 210

<211> 61

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD79A transcript variant 1 NM_001783_3

<400> 210

Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu

1 5 10 15

Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser

20 25 30

Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val

35 40 45

Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys Pro

50 55 60

<210> 211

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CD79B transcript variant 3NM_001039933_2

<400> 211

Leu Asp Lys Asp Asp Ser Lys Ala Gly Met Glu Glu Asp His Thr Tyr

1 5 10 15

Glu Gly Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu Asp Ile Val Thr

20 25 30

Leu Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu His Pro Gly Gln

35 40 45

Glu

<210> 212

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CRLF2 transcript variant 1 NM_022148_3

<400> 212

Lys Leu Trp Arg Val Lys Lys Phe Leu Ile Pro Ser Val Pro Asp Pro

1 5 10 15

Lys Ser Ile Phe Pro Gly Leu Phe Glu Ile His Gln Gly Asn Phe Gln

20 25 30

Glu Trp Ile Thr Asp Thr Gln Asn Val Ala His Leu His Lys Met Ala

35 40 45

Gly Ala Glu Gln Glu Ser Gly Pro Glu Glu Pro Leu Val Val Gln Leu

50 55 60

Ala Lys Thr Glu Ala Glu Ser Pro Arg Met Leu Asp Pro Gln Thr Glu

65 70 75 80

Glu Lys Glu Ala Ser Gly Gly Ser Leu Gln Leu Pro His Gln Pro Leu

85 90 95

Gln Gly Gly Asp Val Val Thr Ile Gly Gly Phe Thr Phe Val Met Asn

100 105 110

Asp Arg Ser Tyr Val Ala Leu

115

<210> 213

<211> 437

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF2RB nm_000395_2

<400> 213

Arg Phe Cys Gly Ile Tyr Gly Tyr Arg Leu Arg Arg Lys Trp Glu Glu

1 5 10 15

Lys Ile Pro Asn Pro Ser Lys Ser His Leu Phe Gln Asn Gly Ser Ala

20 25 30

Glu Leu Trp Pro Pro Gly Ser Met Ser Ala Phe Thr Ser Gly Ser Pro

35 40 45

Pro His Gln Gly Pro Trp Gly Ser Arg Phe Pro Glu Leu Glu Gly Val

50 55 60

Phe Pro Val Gly Phe Gly Asp Ser Glu Val Ser Pro Leu Thr Ile Glu

65 70 75 80

Asp Pro Lys His Val Cys Asp Pro Pro Ser Gly Pro Asp Thr Thr Pro

85 90 95

Ala Ala Ser Asp Leu Pro Thr Glu Gln Pro Pro Ser Pro Gln Pro Gly

100 105 110

Pro Pro Ala Ala Ser His Thr Pro Glu Lys Gln Ala Ser Ser Phe Asp

115 120 125

Phe Asn Gly Pro Tyr Leu Gly Pro Pro His Ser Arg Ser Leu Pro Asp

130 135 140

Ile Leu Gly Gln Pro Glu Pro Pro Gln Glu Gly Gly Ser Gln Lys Ser

145 150 155 160

Pro Pro Pro Gly Ser Leu Glu Tyr Leu Cys Leu Pro Ala Gly Gly Gln

165 170 175

Val Gln Leu Val Pro Leu Ala Gln Ala Met Gly Pro Gly Gln Ala Val

180 185 190

Glu Val Glu Arg Arg Pro Ser Gln Gly Ala Ala Gly Ser Pro Ser Leu

195 200 205

Glu Ser Gly Gly Gly Pro Ala Pro Pro Ala Leu Gly Pro Arg Val Gly

210 215 220

Gly Gln Asp Gln Lys Asp Ser Pro Val Ala Ile Pro Met Ser Ser Gly

225 230 235 240

Asp Thr Glu Asp Pro Gly Val Ala Ser Gly Tyr Val Ser Ser Ala Asp

245 250 255

Leu Val Phe Thr Pro Asn Ser Gly Ala Ser Ser Val Ser Leu Val Pro

260 265 270

Ser Leu Gly Leu Pro Ser Asp Gln Thr Pro Ser Leu Cys Pro Gly Leu

275 280 285

Ala Ser Gly Pro Pro Gly Ala Pro Gly Pro Val Lys Ser Gly Phe Glu

290 295 300

Gly Tyr Val Glu Leu Pro Pro Ile Glu Gly Arg Ser Pro Arg Ser Pro

305 310 315 320

Arg Asn Asn Pro Val Pro Pro Glu Ala Lys Ser Pro Val Leu Asn Pro

325 330 335

Gly Glu Arg Pro Ala Asp Val Ser Pro Thr Ser Pro Gln Pro Glu Gly

340 345 350

Leu Leu Val Leu Gln Gln Val Gly Asp Tyr Cys Phe Leu Pro Gly Leu

355 360 365

Gly Pro Gly Pro Leu Ser Leu Arg Ser Lys Pro Ser Ser Pro Gly Pro

370 375 380

Gly Pro Glu Ile Lys Asn Leu Asp Gln Ala Phe Gln Val Lys Lys Pro

385 390 395 400

Pro Gly Gln Ala Val Pro Gln Val Pro Val Ile Gln Leu Phe Lys Ala

405 410 415

Leu Lys Gln Gln Asp Tyr Leu Ser Leu Pro Pro Trp Glu Val Asn Lys

420 425 430

Pro Gly Glu Val Cys

435

<210> 214

<211> 54

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF2RA transcript variants 7 and 8 nm_001161529_1

<400> 214

Lys Arg Phe Leu Arg Ile Gln Arg Leu Phe Pro Pro Val Pro Gln Ile

1 5 10 15

Lys Asp Lys Leu Asn Asp Asn His Glu Val Glu Asp Glu Ile Ile Trp

20 25 30

Glu Glu Phe Thr Pro Glu Glu Gly Lys Gly Tyr Arg Glu Glu Val Leu

35 40 45

Thr Val Lys Glu Ile Thr

50

<210> 215

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF2RA transcript variant 9 nm_001161531_1

<400> 215

Lys Arg Phe Leu Arg Ile Gln Arg Leu Phe Pro Pro Val Pro Gln Ile

1 5 10 15

Lys Asp Lys Leu Asn Asp Asn His Glu Val Glu Asp Glu Met Gly Pro

20 25 30

Gln Arg His His Arg Cys Gly Trp Asn Leu Tyr Pro Thr Pro Gly Pro

35 40 45

Ser Pro Gly Ser Gly Ser Ser Pro Arg Leu Gly Ser Glu Ser Ser Leu

50 55 60

<210> 216

<211> 186

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF3R transcript variant 1 nm_000760_3

<400> 216

Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala

1 5 10 15

His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala

20 25 30

Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val

35 40 45

Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser

50 55 60

Ser Glu Thr Cys Gly Leu Pro Thr Leu Val Gln Thr Tyr Val Leu Gln

65 70 75 80

Gly Asp Pro Arg Ala Val Ser Thr Gln Pro Gln Ser Gln Ser Gly Thr

85 90 95

Ser Asp Gln Val Leu Tyr Gly Gln Leu Leu Gly Ser Pro Thr Ser Pro

100 105 110

Gly Pro Gly His Tyr Leu Arg Cys Asp Ser Thr Gln Pro Leu Leu Ala

115 120 125

Gly Leu Thr Pro Ser Pro Lys Ser Tyr Glu Asn Leu Trp Phe Gln Ala

130 135 140

Ser Pro Leu Gly Thr Leu Val Thr Pro Ala Pro Ser Gln Glu Asp Asp

145 150 155 160

Cys Val Phe Gly Pro Leu Leu Asn Phe Pro Leu Leu Gln Gly Ile Arg

165 170 175

Val His Gly Met Glu Ala Leu Gly Ser Phe

180 185

<210> 217

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF3R transcript variant 3nm_156039_3

<400> 217

Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala

1 5 10 15

His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Leu Pro

20 25 30

Gly Pro Arg Gln Gly Gln Trp Leu Gly Gln Thr Ser Glu Met Ser Arg

35 40 45

Ala Leu Thr Pro His Pro Cys Val Gln Asp Ala Phe Gln Leu Pro Gly

50 55 60

Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val Leu Glu Glu Asp Glu

65 70 75 80

Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser Ser Glu Thr Cys Gly

85 90 95

Leu Pro Thr Leu Val Gln Thr Tyr Val Leu Gln Gly Asp Pro Arg Ala

100 105 110

Val Ser Thr Gln Pro Gln Ser Gln Ser Gly Thr Ser Asp Gln Val Leu

115 120 125

Tyr Gly Gln Leu Leu Gly Ser Pro Thr Ser Pro Gly Pro Gly His Tyr

130 135 140

Leu Arg Cys Asp Ser Thr Gln Pro Leu Leu Ala Gly Leu Thr Pro Ser

145 150 155 160

Pro Lys Ser Tyr Glu Asn Leu Trp Phe Gln Ala Ser Pro Leu Gly Thr

165 170 175

Leu Val Thr Pro Ala Pro Ser Gln Glu Asp Asp Cys Val Phe Gly Pro

180 185 190

Leu Leu Asn Phe Pro Leu Leu Gln Gly Ile Arg Val His Gly Met Glu

195 200 205

Ala Leu Gly Ser Phe

210

<210> 218

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: CSF3R transcript variant 4 nm_172313_2

<400> 218

Ser Pro Asn Arg Lys Asn Pro Leu Trp Pro Ser Val Pro Asp Pro Ala

1 5 10 15

His Ser Ser Leu Gly Ser Trp Val Pro Thr Ile Met Glu Glu Asp Ala

20 25 30

Phe Gln Leu Pro Gly Leu Gly Thr Pro Pro Ile Thr Lys Leu Thr Val

35 40 45

Leu Glu Glu Asp Glu Lys Lys Pro Val Pro Trp Glu Ser His Asn Ser

50 55 60

Ser Glu Thr Cys Gly Leu Pro Thr Leu Val Gln Thr Tyr Val Leu Gln

65 70 75 80

Gly Asp Pro Arg Ala Val Ser Thr Gln Pro Gln Ser Gln Ser Gly Thr

85 90 95

Ser Asp Gln Ala Gly Pro Pro Arg Arg Ser Ala Tyr Phe Lys Asp Gln

100 105 110

Ile Met Leu His Pro Ala Pro Pro Asn Gly Leu Leu Cys Leu Phe Pro

115 120 125

Ile Thr Ser Val Leu

130

<210> 219

<211> 235

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: EPOR transcript variant 1 nm_000121_3

<400> 219

His Arg Arg Ala Leu Lys Gln Lys Ile Trp Pro Gly Ile Pro Ser Pro

1 5 10 15

Glu Ser Glu Phe Glu Gly Leu Phe Thr Thr His Lys Gly Asn Phe Gln

20 25 30

Leu Trp Leu Tyr Gln Asn Asp Gly Cys Leu Trp Trp Ser Pro Cys Thr

35 40 45

Pro Phe Thr Glu Asp Pro Pro Ala Ser Leu Glu Val Leu Ser Glu Arg

50 55 60

Cys Trp Gly Thr Met Gln Ala Val Glu Pro Gly Thr Asp Asp Glu Gly

65 70 75 80

Pro Leu Leu Glu Pro Val Gly Ser Glu His Ala Gln Asp Thr Tyr Leu

85 90 95

Val Leu Asp Lys Trp Leu Leu Pro Arg Asn Pro Pro Ser Glu Asp Leu

100 105 110

Pro Gly Pro Gly Gly Ser Val Asp Ile Val Ala Met Asp Glu Gly Ser

115 120 125

Glu Ala Ser Ser Cys Ser Ser Ala Leu Ala Ser Lys Pro Ser Pro Glu

130 135 140

Gly Ala Ser Ala Ala Ser Phe Glu Tyr Thr Ile Leu Asp Pro Ser Ser

145 150 155 160

Gln Leu Leu Arg Pro Trp Thr Leu Cys Pro Glu Leu Pro Pro Thr Pro

165 170 175

Pro His Leu Lys Tyr Leu Tyr Leu Val Val Ser Asp Ser Gly Ile Ser

180 185 190

Thr Asp Tyr Ser Ser Gly Asp Ser Gln Gly Ala Gln Gly Gly Leu Ser

195 200 205

Asp Gly Pro Tyr Ser Asn Pro Tyr Glu Asn Ser Leu Ile Pro Ala Ala

210 215 220

Glu Pro Leu Pro Pro Ser Tyr Val Ala Cys Ser

225 230 235

<210> 220

<211> 235

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: EPOR transcript variant 1 nm_000121_3

<400> 220

His Arg Arg Ala Leu Lys Gln Lys Ile Trp Pro Gly Ile Pro Ser Pro

1 5 10 15

Glu Ser Glu Phe Glu Gly Leu Phe Thr Thr His Lys Gly Asn Phe Gln

20 25 30

Leu Trp Leu Tyr Gln Asn Asp Gly Cys Leu Trp Trp Ser Pro Cys Thr

35 40 45

Pro Phe Thr Glu Asp Pro Pro Ala Ser Leu Glu Val Leu Ser Glu Arg

50 55 60

Cys Trp Gly Thr Met Gln Ala Val Glu Pro Gly Thr Asp Asp Glu Gly

65 70 75 80

Pro Leu Leu Glu Pro Val Gly Ser Glu His Ala Gln Asp Thr Tyr Leu

85 90 95

Val Leu Asp Lys Trp Leu Leu Pro Arg Asn Pro Pro Ser Glu Asp Leu

100 105 110

Pro Gly Pro Gly Gly Ser Val Asp Ile Val Ala Met Asp Glu Gly Ser

115 120 125

Glu Ala Ser Ser Cys Ser Ser Ala Leu Ala Ser Lys Pro Ser Pro Glu

130 135 140

Gly Ala Ser Ala Ala Ser Phe Glu Tyr Thr Ile Leu Asp Pro Ser Ser

145 150 155 160

Gln Leu Leu Arg Pro Trp Thr Leu Cys Pro Glu Leu Pro Pro Thr Pro

165 170 175

Pro His Leu Lys Phe Leu Phe Leu Val Val Ser Asp Ser Gly Ile Ser

180 185 190

Thr Asp Tyr Ser Ser Gly Asp Ser Gln Gly Ala Gln Gly Gly Leu Ser

195 200 205

Asp Gly Pro Tyr Ser Asn Pro Tyr Glu Asn Ser Leu Ile Pro Ala Ala

210 215 220

Glu Pro Leu Pro Pro Ser Tyr Val Ala Cys Ser

225 230 235

<210> 221

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FCER1G NM-004106_1

<400> 221

Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys

1 5 10 15

Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr

20 25 30

Glu Thr Leu Lys His Glu Lys Pro Pro Gln

35 40

<210> 222

<211> 77

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FCGR2CNM_2015163_5

<400> 222

Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala

1 5 10 15

Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg

20 25 30

Gln Pro Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr

35 40 45

Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr

50 55 60

Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

65 70 75

<210> 223

<211> 77

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: FCGRA2 transcript variant 1 NM_001136219_1

<400> 223

Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala

1 5 10 15

Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg

20 25 30

Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr

35 40 45

Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr

50 55 60

Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

65 70 75

<210> 224

<211> 350

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: GHR transcript variant 1 NM_000163_4

<400> 224

Lys Gln Gln Arg Ile Lys Met Leu Ile Leu Pro Pro Val Pro Val Pro

1 5 10 15

Lys Ile Lys Gly Ile Asp Pro Asp Leu Leu Lys Glu Gly Lys Leu Glu

20 25 30

Glu Val Asn Thr Ile Leu Ala Ile His Asp Ser Tyr Lys Pro Glu Phe

35 40 45

His Ser Asp Asp Ser Trp Val Glu Phe Ile Glu Leu Asp Ile Asp Glu

50 55 60

Pro Asp Glu Lys Thr Glu Glu Ser Asp Thr Asp Arg Leu Leu Ser Ser

65 70 75 80

Asp His Glu Lys Ser His Ser Asn Leu Gly Val Lys Asp Gly Asp Ser

85 90 95

Gly Arg Thr Ser Cys Cys Glu Pro Asp Ile Leu Glu Thr Asp Phe Asn

100 105 110

Ala Asn Asp Ile His Glu Gly Thr Ser Glu Val Ala Gln Pro Gln Arg

115 120 125

Leu Lys Gly Glu Ala Asp Leu Leu Cys Leu Asp Gln Lys Asn Gln Asn

130 135 140

Asn Ser Pro Tyr His Asp Ala Cys Pro Ala Thr Gln Gln Pro Ser Val

145 150 155 160

Ile Gln Ala Glu Lys Asn Lys Pro Gln Pro Leu Pro Thr Glu Gly Ala

165 170 175

Glu Ser Thr His Gln Ala Ala His Ile Gln Leu Ser Asn Pro Ser Ser

180 185 190

Leu Ser Asn Ile Asp Phe Tyr Ala Gln Val Ser Asp Ile Thr Pro Ala

195 200 205

Gly Ser Val Val Leu Ser Pro Gly Gln Lys Asn Lys Ala Gly Met Ser

210 215 220

Gln Cys Asp Met His Pro Glu Met Val Ser Leu Cys Gln Glu Asn Phe

225 230 235 240

Leu Met Asp Asn Ala Tyr Phe Cys Glu Ala Asp Ala Lys Lys Cys Ile

245 250 255

Pro Val Ala Pro His Ile Lys Val Glu Ser His Ile Gln Pro Ser Leu

260 265 270

Asn Gln Glu Asp Ile Tyr Ile Thr Thr Glu Ser Leu Thr Thr Ala Ala

275 280 285

Gly Arg Pro Gly Thr Gly Glu His Val Pro Gly Ser Glu Met Pro Val

290 295 300

Pro Asp Tyr Thr Ser Ile His Ile Val Gln Ser Pro Gln Gly Leu Ile

305 310 315 320

Leu Asn Ala Thr Ala Leu Pro Leu Pro Asp Lys Glu Phe Leu Ser Ser

325 330 335

Cys Gly Tyr Val Ser Thr Asp Gln Leu Asn Lys Ile Met Pro

340 345 350

<210> 225

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ICOS nm_012092.3

<400> 225

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 5 10 15

Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg

20 25 30

Leu Thr Asp Val Thr Leu

35

<210> 226

<211> 100

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ifnar1nm_000629_2

<400> 226

Lys Val Phe Leu Arg Cys Ile Asn Tyr Val Phe Phe Pro Ser Leu Lys

1 5 10 15

Pro Ser Ser Ser Ile Asp Glu Tyr Phe Ser Glu Gln Pro Leu Lys Asn

20 25 30

Leu Leu Leu Ser Thr Ser Glu Glu Gln Ile Glu Lys Cys Phe Ile Ile

35 40 45

Glu Asn Ile Ser Thr Ile Ala Thr Val Glu Glu Thr Asn Gln Thr Asp

50 55 60

Glu Asp His Lys Lys Tyr Ser Ser Gln Thr Ser Gln Asp Ser Gly Asn

65 70 75 80

Tyr Ser Asn Glu Asp Glu Ser Glu Ser Lys Thr Ser Glu Glu Leu Gln

85 90 95

Gln Asp Phe Val

100

<210> 227

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IFNAR2 transcript variant 1 NM 207585_2

<400> 227

Lys Trp Ile Gly Tyr Ile Cys Leu Arg Asn Ser Leu Pro Lys Val Leu

1 5 10 15

Asn Phe His Asn Phe Leu Ala Trp Pro Phe Pro Asn Leu Pro Pro Leu

20 25 30

Glu Ala Met Asp Met Val Glu Val Ile Tyr Ile Asn Arg Lys Lys Lys

35 40 45

Val Trp Asp Tyr Asn Tyr Asp Asp Glu Ser Asp Ser Asp Thr Glu Ala

50 55 60

Ala Pro Arg Thr Ser Gly Gly Gly Tyr Thr Met His Gly Leu Thr Val

65 70 75 80

Arg Pro Leu Gly Gln Ala Ser Ala Thr Ser Thr Glu Ser Gln Leu Ile

85 90 95

Asp Pro Glu Ser Glu Glu Glu Pro Asp Leu Pro Glu Val Asp Val Glu

100 105 110

Leu Pro Thr Met Pro Lys Asp Ser Pro Gln Gln Leu Glu Leu Leu Ser

115 120 125

Gly Pro Cys Glu Arg Arg Lys Ser Pro Leu Gln Asp Pro Phe Pro Glu

130 135 140

Glu Asp Tyr Ser Ser Thr Glu Gly Ser Gly Gly Arg Ile Thr Phe Asn

145 150 155 160

Val Asp Leu Asn Ser Val Phe Leu Arg Val Leu Asp Asp Glu Asp Ser

165 170 175

Asp Asp Leu Glu Ala Pro Leu Met Leu Ser Ser His Leu Glu Glu Met

180 185 190

Val Asp Pro Glu Asp Pro Asp Asn Val Gln Ser Asn His Leu Leu Ala

195 200 205

Ser Gly Glu Gly Thr Gln Pro Thr Phe Pro Ser Pro Ser Ser Glu Gly

210 215 220

Leu Trp Ser Glu Asp Ala Pro Ser Asp Gln Ser Asp Thr Ser Glu Ser

225 230 235 240

Asp Val Asp Leu Gly Asp Gly Tyr Ile Met Arg

245 250

<210> 228

<211> 67

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IFNAR2 transcript variant 2nm_000874_4

<400> 228

Lys Trp Ile Gly Tyr Ile Cys Leu Arg Asn Ser Leu Pro Lys Val Leu

1 5 10 15

Arg Gln Gly Leu Ala Lys Gly Trp Asn Ala Val Ala Ile His Arg Cys

20 25 30

Ser His Asn Ala Leu Gln Ser Glu Thr Pro Glu Leu Lys Gln Ser Ser

35 40 45

Cys Leu Ser Phe Pro Ser Ser Trp Asp Tyr Lys Arg Ala Ser Leu Cys

50 55 60

Pro Ser Asp

65

<210> 229

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ifngr1nm_000416_2

<400> 229

Cys Phe Tyr Ile Lys Lys Ile Asn Pro Leu Lys Glu Lys Ser Ile Ile

1 5 10 15

Leu Pro Lys Ser Leu Ile Ser Val Val Arg Ser Ala Thr Leu Glu Thr

20 25 30

Lys Pro Glu Ser Lys Tyr Val Ser Leu Ile Thr Ser Tyr Gln Pro Phe

35 40 45

Ser Leu Glu Lys Glu Val Val Cys Glu Glu Pro Leu Ser Pro Ala Thr

50 55 60

Val Pro Gly Met His Thr Glu Asp Asn Pro Gly Lys Val Glu His Thr

65 70 75 80

Glu Glu Leu Ser Ser Ile Thr Glu Val Val Thr Thr Glu Glu Asn Ile

85 90 95

Pro Asp Val Val Pro Gly Ser His Leu Thr Pro Ile Glu Arg Glu Ser

100 105 110

Ser Ser Pro Leu Ser Ser Asn Gln Ser Glu Pro Gly Ser Ile Ala Leu

115 120 125

Asn Ser Tyr His Ser Arg Asn Cys Ser Glu Ser Asp His Ser Arg Asn

130 135 140

Gly Phe Asp Thr Asp Ser Ser Cys Leu Glu Ser His Ser Ser Leu Ser

145 150 155 160

Asp Ser Glu Phe Pro Pro Asn Asn Lys Gly Glu Ile Lys Thr Glu Gly

165 170 175

Gln Glu Leu Ile Thr Val Ile Lys Ala Pro Thr Ser Phe Gly Tyr Asp

180 185 190

Lys Pro His Val Leu Val Asp Leu Leu Val Asp Asp Ser Gly Lys Glu

195 200 205

Ser Leu Ile Gly Tyr Arg Pro Thr Glu Asp Ser Lys Glu Phe Ser

210 215 220

<210> 230

<211> 69

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IFNGR2 transcript variant 1 nm_001329128_1

<400> 230

Leu Val Leu Lys Tyr Arg Gly Leu Ile Lys Tyr Trp Phe His Thr Pro

1 5 10 15

Pro Ser Ile Pro Leu Gln Ile Glu Glu Tyr Leu Lys Asp Pro Thr Gln

20 25 30

Pro Ile Leu Glu Ala Leu Asp Lys Asp Ser Ser Pro Lys Asp Asp Val

35 40 45

Trp Asp Ser Val Ser Ile Ile Ser Phe Pro Glu Lys Glu Gln Glu Asp

50 55 60

Val Leu Gln Thr Leu

65

<210> 231

<211> 271

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: ifnlr1nm_170743_3

<400> 231

Lys Thr Leu Met Gly Asn Pro Trp Phe Gln Arg Ala Lys Met Pro Arg

1 5 10 15

Ala Leu Asp Phe Ser Gly His Thr His Pro Val Ala Thr Phe Gln Pro

20 25 30

Ser Arg Pro Glu Ser Val Asn Asp Leu Phe Leu Cys Pro Gln Lys Glu

35 40 45

Leu Thr Arg Gly Val Arg Pro Thr Pro Arg Val Arg Ala Pro Ala Thr

50 55 60

Gln Gln Thr Arg Trp Lys Lys Asp Leu Ala Glu Asp Glu Glu Glu Glu

65 70 75 80

Asp Glu Glu Asp Thr Glu Asp Gly Val Ser Phe Gln Pro Tyr Ile Glu

85 90 95

Pro Pro Ser Phe Leu Gly Gln Glu His Gln Ala Pro Gly His Ser Glu

100 105 110

Ala Gly Gly Val Asp Ser Gly Arg Pro Arg Ala Pro Leu Val Pro Ser

115 120 125

Glu Gly Ser Ser Ala Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr

130 135 140

Val Asp Ser Ser Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu

145 150 155 160

Lys Gly Pro Gly Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu

165 170 175

Pro Pro Pro Glu Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu Pro

180 185 190

Glu Asp Asn Leu Ser Ser Trp Ala Thr Trp Gly Thr Leu Pro Pro Glu

195 200 205

Pro Asn Leu Val Pro Gly Gly Pro Pro Val Ser Leu Gln Thr Leu Thr

210 215 220

Phe Cys Trp Glu Ser Ser Pro Glu Glu Glu Glu Glu Ala Arg Glu Ser

225 230 235 240

Glu Ile Glu Asp Ser Asp Ala Gly Ser Trp Gly Ala Glu Ser Thr Gln

245 250 255

Arg Thr Glu Asp Arg Gly Arg Thr Leu Gly His Tyr Met Ala Arg

260 265 270

<210> 232

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IFNLR1 transcript variant 2 nm_173064_2

<400> 232

Lys Thr Leu Met Gly Asn Pro Trp Phe Gln Arg Ala Lys Met Pro Arg

1 5 10 15

Ala Leu Glu Leu Thr Arg Gly Val Arg Pro Thr Pro Arg Val Arg Ala

20 25 30

Pro Ala Thr Gln Gln Thr Arg Trp Lys Lys Asp Leu Ala Glu Asp Glu

35 40 45

Glu Glu Glu Asp Glu Glu Asp Thr Glu Asp Gly Val Ser Phe Gln Pro

50 55 60

Tyr Ile Glu Pro Pro Ser Phe Leu Gly Gln Glu His Gln Ala Pro Gly

65 70 75 80

His Ser Glu Ala Gly Gly Val Asp Ser Gly Arg Pro Arg Ala Pro Leu

85 90 95

Val Pro Ser Glu Gly Ser Ser Ala Trp Asp Ser Ser Asp Arg Ser Trp

100 105 110

Ala Ser Thr Val Asp Ser Ser Trp Asp Arg Ala Gly Ser Ser Gly Tyr

115 120 125

Leu Ala Glu Lys Gly Pro Gly Gln Gly Pro Gly Gly Asp Gly His Gln

130 135 140

Glu Ser Leu Pro Pro Pro Glu Phe Ser Lys Asp Ser Gly Phe Leu Glu

145 150 155 160

Glu Leu Pro Glu Asp Asn Leu Ser Ser Trp Ala Thr Trp Gly Thr Leu

165 170 175

Pro Pro Glu Pro Asn Leu Val Pro Gly Gly Pro Pro Val Ser Leu Gln

180 185 190

Thr Leu Thr Phe Cys Trp Glu Ser Ser Pro Glu Glu Glu Glu Glu Ala

195 200 205

Arg Glu Ser Glu Ile Glu Asp Ser Asp Ala Gly Ser Trp Gly Ala Glu

210 215 220

Ser Thr Gln Arg Thr Glu Asp Arg Gly Arg Thr Leu Gly His Tyr Met

225 230 235 240

Ala Arg

<210> 233

<211> 179

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1R1 transcript variant 2 NM_001288706_1

<400> 233

Lys Ile Asp Ile Val Leu Trp Tyr Arg Asp Ser Cys Tyr Asp Phe Leu

1 5 10 15

Pro Ile Lys Val Leu Pro Glu Val Leu Glu Lys Gln Cys Gly Tyr Lys

20 25 30

Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val Gly Glu Asp Ile Val Glu

35 40 45

Val Ile Asn Glu Asn Val Lys Lys Ser Arg Arg Leu Ile Ile Ile Leu

50 55 60

Val Arg Glu Thr Ser Gly Phe Ser Trp Leu Gly Gly Ser Ser Glu Glu

65 70 75 80

Gln Ile Ala Met Tyr Asn Ala Leu Val Gln Asp Gly Ile Lys Val Val

85 90 95

Leu Leu Glu Leu Glu Lys Ile Gln Asp Tyr Glu Lys Met Pro Glu Ser

100 105 110

Ile Lys Phe Ile Lys Gln Lys His Gly Ala Ile Arg Trp Ser Gly Asp

115 120 125

Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr Arg Phe Trp Lys Asn Val

130 135 140

Arg Tyr His Met Pro Val Gln Arg Arg Ser Pro Ser Ser Lys His Gln

145 150 155 160

Leu Leu Ser Pro Ala Thr Lys Glu Lys Leu Gln Arg Glu Ala His Val

165 170 175

Pro Leu Gly

<210> 234

<211> 210

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1R1 transcript variant 3NM_001320978_1

<400> 234

Lys Ile Asp Ile Val Leu Trp Tyr Arg Asp Ser Cys Tyr Asp Phe Leu

1 5 10 15

Pro Ile Lys Ala Ser Asp Gly Lys Thr Tyr Asp Ala Tyr Ile Leu Tyr

20 25 30

Pro Lys Thr Val Gly Glu Gly Ser Thr Ser Asp Cys Asp Ile Phe Val

35 40 45

Phe Lys Val Leu Pro Glu Val Leu Glu Lys Gln Cys Gly Tyr Lys Leu

50 55 60

Phe Ile Tyr Gly Arg Asp Asp Tyr Val Gly Glu Asp Ile Val Glu Val

65 70 75 80

Ile Asn Glu Asn Val Lys Lys Ser Arg Arg Leu Ile Ile Ile Leu Val

85 90 95

Arg Glu Thr Ser Gly Phe Ser Trp Leu Gly Gly Ser Ser Glu Glu Gln

100 105 110

Ile Ala Met Tyr Asn Ala Leu Val Gln Asp Gly Ile Lys Val Val Leu

115 120 125

Leu Glu Leu Glu Lys Ile Gln Asp Tyr Glu Lys Met Pro Glu Ser Ile

130 135 140

Lys Phe Ile Lys Gln Lys His Gly Ala Ile Arg Trp Ser Gly Asp Phe

145 150 155 160

Thr Gln Gly Pro Gln Ser Ala Lys Thr Arg Phe Trp Lys Asn Val Arg

165 170 175

Tyr His Met Pro Val Gln Arg Arg Ser Pro Ser Ser Lys His Gln Leu

180 185 190

Leu Ser Pro Ala Thr Lys Glu Lys Leu Gln Arg Glu Ala His Val Pro

195 200 205

Leu Gly

210

<210> 235

<211> 182

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RAP transcript variant 1 NM_002182_3

<400> 235

Tyr Arg Ala His Phe Gly Thr Asp Glu Thr Ile Leu Asp Gly Lys Glu

1 5 10 15

Tyr Asp Ile Tyr Val Ser Tyr Ala Arg Asn Ala Glu Glu Glu Glu Phe

20 25 30

Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu Phe Gly Tyr Lys

35 40 45

Leu Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly Ile Val Thr Asp

50 55 60

Glu Thr Leu Ser Phe Ile Gln Lys Ser Arg Arg Leu Leu Val Val Leu

65 70 75 80

Ser Pro Asn Tyr Val Leu Gln Gly Thr Gln Ala Leu Leu Glu Leu Lys

85 90 95

Ala Gly Leu Glu Asn Met Ala Ser Arg Gly Asn Ile Asn Val Ile Leu

100 105 110

Val Gln Tyr Lys Ala Val Lys Glu Thr Lys Val Lys Glu Leu Lys Arg

115 120 125

Ala Lys Thr Val Leu Thr Val Ile Lys Trp Lys Gly Glu Lys Ser Lys

130 135 140

Tyr Pro Gln Gly Arg Phe Trp Lys Gln Leu Gln Val Ala Met Pro Val

145 150 155 160

Lys Lys Ser Pro Arg Arg Ser Ser Ser Asp Glu Gln Gly Leu Ser Tyr

165 170 175

Ser Ser Leu Lys Asn Val

180

<210> 236

<211> 299

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RAP transcript variant 6 NM_001167931_1

<400> 236

Tyr Arg Ala His Phe Gly Thr Asp Glu Thr Ile Leu Asp Gly Lys Glu

1 5 10 15

Tyr Asp Ile Tyr Val Ser Tyr Ala Arg Asn Ala Glu Glu Glu Glu Phe

20 25 30

Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu Phe Gly Tyr Lys

35 40 45

Leu Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly Asn Thr Val Glu

50 55 60

Ala Val Phe Asp Phe Ile Gln Arg Ser Arg Arg Met Ile Val Val Leu

65 70 75 80

Ser Pro Asp Tyr Val Thr Glu Lys Ser Ile Ser Met Leu Glu Phe Lys

85 90 95

Leu Gly Val Met Cys Gln Asn Ser Ile Ala Thr Lys Leu Ile Val Val

100 105 110

Glu Tyr Arg Pro Leu Glu His Pro His Pro Gly Ile Leu Gln Leu Lys

115 120 125

Glu Ser Val Ser Phe Val Ser Trp Lys Gly Glu Lys Ser Lys His Ser

130 135 140

Gly Ser Lys Phe Trp Lys Ala Leu Arg Leu Ala Leu Pro Leu Arg Ser

145 150 155 160

Leu Ser Ala Ser Ser Gly Trp Asn Glu Ser Cys Ser Ser Gln Ser Asp

165 170 175

Ile Ser Leu Asp His Val Gln Arg Arg Arg Ser Arg Leu Lys Glu Pro

180 185 190

Pro Glu Leu Gln Ser Ser Glu Arg Ala Ala Gly Ser Pro Pro Ala Pro

195 200 205

Gly Thr Met Ser Lys His Arg Gly Lys Ser Ser Ala Thr Cys Arg Cys

210 215 220

Cys Val Thr Tyr Cys Glu Gly Glu Asn His Leu Arg Asn Lys Ser Arg

225 230 235 240

Ala Glu Ile His Asn Gln Pro Gln Trp Glu Thr His Leu Cys Lys Pro

245 250 255

Val Pro Gln Glu Ser Glu Thr Gln Trp Ile Gln Asn Gly Thr Arg Leu

260 265 270

Glu Pro Pro Ala Pro Gln Ile Ser Ala Leu Ala Leu His His Phe Thr

275 280 285

Asp Leu Ser Asn Asn Asn Asp Phe Tyr Ile Leu

290 295

<210> 237

<211> 207

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RL1 transcript variant 1 NM_016232.4

<400> 237

Leu Lys Met Phe Trp Ile Glu Ala Thr Leu Leu Trp Arg Asp Ile Ala

1 5 10 15

Lys Pro Tyr Lys Thr Arg Asn Asp Gly Lys Leu Tyr Asp Ala Tyr Val

20 25 30

Val Tyr Pro Arg Asn Tyr Lys Ser Ser Thr Asp Gly Ala Ser Arg Val

35 40 45

Glu His Phe Val His Gln Ile Leu Pro Asp Val Leu Glu Asn Lys Cys

50 55 60

Gly Tyr Thr Leu Cys Ile Tyr Gly Arg Asp Met Leu Pro Gly Glu Asp

65 70 75 80

Val Val Thr Ala Val Glu Thr Asn Ile Arg Lys Ser Arg Arg His Ile

85 90 95

Phe Ile Leu Thr Pro Gln Ile Thr His Asn Lys Glu Phe Ala Tyr Glu

100 105 110

Gln Glu Val Ala Leu His Cys Ala Leu Ile Gln Asn Asp Ala Lys Val

115 120 125

Ile Leu Ile Glu Met Glu Ala Leu Ser Glu Leu Asp Met Leu Gln Ala

130 135 140

Glu Ala Leu Gln Asp Ser Leu Gln His Leu Met Lys Val Gln Gly Thr

145 150 155 160

Ile Lys Trp Arg Glu Asp His Ile Ala Asn Lys Arg Ser Leu Asn Ser

165 170 175

Lys Phe Trp Lys His Val Arg Tyr Gln Met Pro Val Pro Ser Lys Ile

180 185 190

Pro Arg Lys Ala Ser Ser Leu Thr Pro Leu Ala Ala Gln Lys Gln

195 200 205

<210> 238

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL1RL2 NM-003854.2

<400> 238

Asn Ile Phe Lys Ile Asp Ile Val Leu Trp Tyr Arg Ser Ala Phe His

1 5 10 15

Ser Thr Glu Thr Ile Val Asp Gly Lys Leu Tyr Asp Ala Tyr Val Leu

20 25 30

Tyr Pro Lys Pro His Lys Glu Ser Gln Arg His Ala Val Asp Ala Leu

35 40 45

Val Leu Asn Ile Leu Pro Glu Val Leu Glu Arg Gln Cys Gly Tyr Lys

50 55 60

Leu Phe Ile Phe Gly Arg Asp Glu Phe Pro Gly Gln Ala Val Ala Asn

65 70 75 80

Val Ile Asp Glu Asn Val Lys Leu Cys Arg Arg Leu Ile Val Ile Val

85 90 95

Val Pro Glu Ser Leu Gly Phe Gly Leu Leu Lys Asn Leu Ser Glu Glu

100 105 110

Gln Ile Ala Val Tyr Ser Ala Leu Ile Gln Asp Gly Met Lys Val Ile

115 120 125

Leu Ile Glu Leu Glu Lys Ile Glu Asp Tyr Thr Val Met Pro Glu Ser

130 135 140

Ile Gln Tyr Ile Lys Gln Lys His Gly Ala Ile Arg Trp His Gly Asp

145 150 155 160

Phe Thr Glu Gln Ser Gln Cys Met Lys Thr Lys Phe Trp Lys Thr Val

165 170 175

Arg Tyr His Met Pro Pro Arg Arg Cys Arg Pro Phe Pro Pro Val Gln

180 185 190

Leu Leu Gln His Thr Pro Cys Tyr Arg Thr Ala Gly Pro Glu Leu Gly

195 200 205

Ser Arg Arg Lys Lys Cys Thr Leu Thr Thr Gly

210 215

<210> 239

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL2RA transcript variant 1 NM-000417-2

<400> 239

Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile

1 5 10

<210> 240

<211> 286

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL2RB transcript variant 1 NM-000878-4

<400> 240

Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn

1 5 10 15

Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly

20 25 30

Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe

35 40 45

Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu

50 55 60

Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu

65 70 75 80

Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn

85 90 95

Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala

100 105 110

Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp

115 120 125

Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln

130 135 140

Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp

145 150 155 160

Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro

165 170 175

Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro

180 185 190

Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly

195 200 205

Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro

210 215 220

Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro

225 230 235 240

Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu

245 250 255

Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu

260 265 270

Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val

275 280 285

<210> 241

<211> 86

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL2RG nm—000206_2

<400> 241

Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu

1 5 10 15

Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys

20 25 30

Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu

35 40 45

Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly

50 55 60

Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr

65 70 75 80

Thr Leu Lys Pro Glu Thr

85

<210> 242

<211> 53

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL3RA transcript variants 1 and 2NM_002183_3

<400> 242

Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro Arg Ile Pro His Met

1 5 10 15

Lys Asp Pro Ile Gly Asp Ser Phe Gln Asn Asp Lys Leu Val Val Trp

20 25 30

Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu Val Thr Glu Val Gln

35 40 45

Val Val Gln Lys Thr

50

<210> 243

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL4R transcript variant 1 NM-000418-3

<400> 243

Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser

1 5 10 15

Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu

20 25 30

Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn

35 40 45

Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg

50 55 60

Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser

65 70 75 80

Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp

85 90 95

Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro

100 105 110

Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe

115 120 125

Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu

130 135 140

Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly

145 150 155 160

Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu

165 170 175

Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe

180 185 190

Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro

195 200 205

Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp

210 215 220

Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala

225 230 235 240

Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu

245 250 255

Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val Glu Pro

260 265 270

Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln

275 280 285

Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln

290 295 300

His Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln

305 310 315 320

Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val

325 330 335

Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser

340 345 350

Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala

355 360 365

Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile Pro Gly

370 375 380

Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu Phe Thr Phe Gly

385 390 395 400

Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser

405 410 415

Ser Ser Pro Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp

420 425 430

Met Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val

435 440 445

Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu

450 455 460

Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly Gln Thr

465 470 475 480

Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser

485 490 495

Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro Gly Gly

500 505 510

Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala Pro Ser Gly

515 520 525

Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly

530 535 540

Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser

545 550 555 560

Val Gly Pro Thr Tyr Met Arg Val Ser

565

<210> 244

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL4R transcript variant 1 NM-000418-3

<400> 244

Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser

1 5 10 15

Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu

20 25 30

Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn

35 40 45

Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg

50 55 60

Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser

65 70 75 80

Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp

85 90 95

Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro

100 105 110

Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe

115 120 125

Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu

130 135 140

Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly

145 150 155 160

Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu

165 170 175

Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe

180 185 190

Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro

195 200 205

Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp

210 215 220

Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala

225 230 235 240

Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu

245 250 255

Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val Glu Pro

260 265 270

Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln

275 280 285

Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln

290 295 300

His Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln

305 310 315 320

Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val

325 330 335

Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser

340 345 350

Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala

355 360 365

Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile Pro Gly

370 375 380

Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu Phe Thr Phe Gly

385 390 395 400

Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser

405 410 415

Ser Ser Pro Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp

420 425 430

Met Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val

435 440 445

Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu

450 455 460

Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly Gln Thr

465 470 475 480

Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser

485 490 495

Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro Gly Gly

500 505 510

Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala Pro Ser Gly

515 520 525

Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly

530 535 540

Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser

545 550 555 560

Val Gly Pro Thr Tyr Met Arg Val Ser

565

<210> 245

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL5RA transcript variant 1 NM-000564-4

<400> 245

Lys Ile Cys His Leu Trp Ile Lys Leu Phe Pro Pro Ile Pro Ala Pro

1 5 10 15

Lys Ser Asn Ile Lys Asp Leu Phe Val Thr Thr Asn Tyr Glu Lys Ala

20 25 30

Gly Ser Ser Glu Thr Glu Ile Glu Val Ile Cys Tyr Ile Glu Lys Pro

35 40 45

Gly Val Glu Thr Leu Glu Asp Ser Val Phe

50 55

<210> 246

<211> 82

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL6R transcript variant 1 NM-000565-3

<400> 246

Arg Phe Lys Lys Thr Trp Lys Leu Arg Ala Leu Lys Glu Gly Lys Thr

1 5 10 15

Ser Met His Pro Pro Tyr Ser Leu Gly Gln Leu Val Pro Glu Arg Pro

20 25 30

Arg Pro Thr Pro Val Leu Val Pro Leu Ile Ser Pro Pro Val Ser Pro

35 40 45

Ser Ser Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro Asp Ala

50 55 60

Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr Asp Tyr Phe Phe

65 70 75 80

Pro Arg

<210> 247

<211> 277

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL6ST transcript variants 1 and 3NM_002184_3

<400> 247

Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro Asp

1 5 10 15

Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro Arg

20 25 30

His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe Thr

35 40 45

Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe Pro

50 55 60

Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn Thr

65 70 75 80

Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser Ser

85 90 95

Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn Thr

100 105 110

Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg His

115 120 125

Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln Pro

130 135 140

Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp His

145 150 155 160

Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys Gln

165 170 175

Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu Arg

180 185 190

Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu Lys

195 200 205

Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln Met

210 215 220

Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly Thr

225 230 235 240

Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala Thr

245 250 255

Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln Gly

260 265 270

Gly Tyr Met Pro Gln

275

<210> 248

<211> 196

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL7RA isoform 1 NM-002185.4

<400> 248

Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His

1 5 10 15

Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn

20 25 30

Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val

35 40 45

Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr

50 55 60

Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp

65 70 75 80

Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu

85 90 95

Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser

100 105 110

Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg

115 120 125

Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser

130 135 140

Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser

145 150 155 160

Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr

165 170 175

Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe

180 185 190

Tyr Gln Asn Gln

195

<210> 249

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL7RA isoform 3 (C-terminal deletion) (interleukin 7 receptor)

<400> 249

Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His

1 5 10 15

Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Val Ser Val

20 25 30

Phe Gly Ala

35

<210> 250

<211> 230

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL9R transcript variant 1 NM_002186_2

<400> 250

Lys Leu Ser Pro Arg Val Lys Arg Ile Phe Tyr Gln Asn Val Pro Ser

1 5 10 15

Pro Ala Met Phe Phe Gln Pro Leu Tyr Ser Val His Asn Gly Asn Phe

20 25 30

Gln Thr Trp Met Gly Ala His Gly Ala Gly Val Leu Leu Ser Gln Asp

35 40 45

Cys Ala Gly Thr Pro Gln Gly Ala Leu Glu Pro Cys Val Gln Glu Ala

50 55 60

Thr Ala Leu Leu Thr Cys Gly Pro Ala Arg Pro Trp Lys Ser Val Ala

65 70 75 80

Leu Glu Glu Glu Gln Glu Gly Pro Gly Thr Arg Leu Pro Gly Asn Leu

85 90 95

Ser Ser Glu Asp Val Leu Pro Ala Gly Cys Thr Glu Trp Arg Val Gln

100 105 110

Thr Leu Ala Tyr Leu Pro Gln Glu Asp Trp Ala Pro Thr Ser Leu Thr

115 120 125

Arg Pro Ala Pro Pro Asp Ser Glu Gly Ser Arg Ser Ser Ser Ser Ser

130 135 140

Ser Ser Ser Asn Asn Asn Asn Tyr Cys Ala Leu Gly Cys Tyr Gly Gly

145 150 155 160

Trp His Leu Ser Ala Leu Pro Gly Asn Thr Gln Ser Ser Gly Pro Ile

165 170 175

Pro Ala Leu Ala Cys Gly Leu Ser Cys Asp His Gln Gly Leu Glu Thr

180 185 190

Gln Gln Gly Val Ala Trp Val Leu Ala Gly His Cys Gln Arg Pro Gly

195 200 205

Leu His Glu Asp Leu Gln Gly Met Leu Leu Pro Ser Val Leu Ser Lys

210 215 220

Ala Arg Ser Trp Thr Phe

225 230

<210> 251

<211> 322

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL10RA transcript variant 1 NM_001558_3

<400> 251

Gln Leu Tyr Val Arg Arg Arg Lys Lys Leu Pro Ser Val Leu Leu Phe

1 5 10 15

Lys Lys Pro Ser Pro Phe Ile Phe Ile Ser Gln Arg Pro Ser Pro Glu

20 25 30

Thr Gln Asp Thr Ile His Pro Leu Asp Glu Glu Ala Phe Leu Lys Val

35 40 45

Ser Pro Glu Leu Lys Asn Leu Asp Leu His Gly Ser Thr Asp Ser Gly

50 55 60

Phe Gly Ser Thr Lys Pro Ser Leu Gln Thr Glu Glu Pro Gln Phe Leu

65 70 75 80

Leu Pro Asp Pro His Pro Gln Ala Asp Arg Thr Leu Gly Asn Arg Glu

85 90 95

Pro Pro Val Leu Gly Asp Ser Cys Ser Ser Gly Ser Ser Asn Ser Thr

100 105 110

Asp Ser Gly Ile Cys Leu Gln Glu Pro Ser Leu Ser Pro Ser Thr Gly

115 120 125

Pro Thr Trp Glu Gln Gln Val Gly Ser Asn Ser Arg Gly Gln Asp Asp

130 135 140

Ser Gly Ile Asp Leu Val Gln Asn Ser Glu Gly Arg Ala Gly Asp Thr

145 150 155 160

Gln Gly Gly Ser Ala Leu Gly His His Ser Pro Pro Glu Pro Glu Val

165 170 175

Pro Gly Glu Glu Asp Pro Ala Ala Val Ala Phe Gln Gly Tyr Leu Arg

180 185 190

Gln Thr Arg Cys Ala Glu Glu Lys Ala Thr Lys Thr Gly Cys Leu Glu

195 200 205

Glu Glu Ser Pro Leu Thr Asp Gly Leu Gly Pro Lys Phe Gly Arg Cys

210 215 220

Leu Val Asp Glu Ala Gly Leu His Pro Pro Ala Leu Ala Lys Gly Tyr

225 230 235 240

Leu Lys Gln Asp Pro Leu Glu Met Thr Leu Ala Ser Ser Gly Ala Pro

245 250 255

Thr Gly Gln Trp Asn Gln Pro Thr Glu Glu Trp Ser Leu Leu Ala Leu

260 265 270

Ser Ser Cys Ser Asp Leu Gly Ile Ser Asp Trp Ser Phe Ala His Asp

275 280 285

Leu Ala Pro Leu Gly Cys Val Ala Ala Pro Gly Gly Leu Leu Gly Ser

290 295 300

Phe Asn Ser Asp Leu Val Thr Leu Pro Leu Ile Ser Ser Leu Gln Ser

305 310 315 320

Ser Glu

<210> 252

<211> 83

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL10RB NM-000628_4

<400> 252

Ala Leu Leu Trp Cys Val Tyr Lys Lys Thr Lys Tyr Ala Phe Ser Pro

1 5 10 15

Arg Asn Ser Leu Pro Gln His Leu Lys Glu Phe Leu Gly His Pro His

20 25 30

His Asn Thr Leu Leu Phe Phe Ser Phe Pro Leu Ser Asp Glu Asn Asp

35 40 45

Val Phe Asp Lys Leu Ser Val Ile Ala Glu Asp Ser Glu Ser Gly Lys

50 55 60

Gln Asn Pro Gly Asp Ser Cys Ser Leu Gly Thr Pro Pro Gly Gln Gly

65 70 75 80

Pro Gln Ser

<210> 253

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL11RA NM-001142784_2

<400> 253

Arg Leu Arg Arg Gly Gly Lys Asp Gly Ser Pro Lys Pro Gly Phe Leu

1 5 10 15

Ala Ser Val Ile Pro Val Asp Arg Arg Pro Gly Ala Pro Asn Leu

20 25 30

<210> 254

<211> 92

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL12RB1 transcript variants 1 and 4NM_005535_2

<400> 254

Asn Arg Ala Ala Arg His Leu Cys Pro Pro Leu Pro Thr Pro Cys Ala

1 5 10 15

Ser Ser Ala Ile Glu Phe Pro Gly Gly Lys Glu Thr Trp Gln Trp Ile

20 25 30

Asn Pro Val Asp Phe Gln Glu Glu Ala Ser Leu Gln Glu Ala Leu Val

35 40 45

Val Glu Met Ser Trp Asp Lys Gly Glu Arg Thr Glu Pro Leu Glu Lys

50 55 60

Thr Glu Leu Pro Glu Gly Ala Pro Glu Leu Ala Leu Asp Thr Glu Leu

65 70 75 80

Ser Leu Glu Asp Gly Asp Arg Cys Lys Ala Lys Met

85 90

<210> 255

<211> 90

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL12RB1 transcript variant 3 NM_001290023_1

<400> 255

Asn Arg Ala Ala Arg His Leu Cys Pro Pro Leu Pro Thr Pro Cys Ala

1 5 10 15

Ser Ser Ala Ile Glu Phe Pro Gly Gly Lys Glu Thr Trp Gln Trp Ile

20 25 30

Asn Pro Val Asp Phe Gln Glu Glu Ala Ser Leu Gln Glu Ala Leu Val

35 40 45

Val Glu Met Ser Trp Asp Lys Gly Glu Arg Thr Glu Pro Leu Glu Lys

50 55 60

Thr Glu Leu Pro Glu Gly Ala Pro Glu Leu Ala Leu Asp Thr Glu Leu

65 70 75 80

Ser Leu Glu Asp Gly Asp Arg Cys Asp Arg

85 90

<210> 256

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL12RB2 transcript variants 1 and 3NM_001559_2

<400> 256

His Tyr Phe Gln Gln Lys Val Phe Val Leu Leu Ala Ala Leu Arg Pro

1 5 10 15

Gln Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser Thr Cys Ala

20 25 30

Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro Leu Asp Arg

35 40 45

Leu Leu Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu Pro Leu Val Ile

50 55 60

Ser Glu Val Leu His Gln Val Thr Pro Val Phe Arg His Pro Pro Cys

65 70 75 80

Ser Asn Trp Pro Gln Arg Glu Lys Gly Ile Gln Gly His Gln Ala Ser

85 90 95

Glu Lys Asp Met Met His Ser Ala Ser Ser Pro Pro Pro Pro Arg Ala

100 105 110

Leu Gln Ala Glu Ser Arg Gln Leu Val Asp Leu Tyr Lys Val Leu Glu

115 120 125

Ser Arg Gly Ser Asp Pro Lys Pro Glu Asn Pro Ala Cys Pro Trp Thr

130 135 140

Val Leu Pro Ala Gly Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser

145 150 155 160

Asn Ile Asp Asp Leu Pro Ser His Glu Ala Pro Leu Ala Asp Ser Leu

165 170 175

Glu Glu Leu Glu Pro Gln His Ile Ser Leu Ser Val Phe Pro Ser Ser

180 185 190

Ser Leu His Pro Leu Thr Phe Ser Cys Gly Asp Lys Leu Thr Leu Asp

195 200 205

Gln Leu Lys Met Arg Cys Asp Ser Leu Met Leu

210 215

<210> 257

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL13RA1 NM-001560-2

<400> 257

Lys Arg Leu Lys Ile Ile Ile Phe Pro Pro Ile Pro Asp Pro Gly Lys

1 5 10 15

Ile Phe Lys Glu Met Phe Gly Asp Gln Asn Asp Asp Thr Leu His Trp

20 25 30

Lys Lys Tyr Asp Ile Tyr Glu Lys Gln Thr Lys Glu Glu Thr Asp Ser

35 40 45

Val Val Leu Ile Glu Asn Leu Lys Lys Ala Ser Gln

50 55 60

<210> 258

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL13RA2 NM-000640-2

<400> 258

Arg Lys Pro Asn Thr Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp

1 5 10 15

Thr

<210> 259

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL15RA transcript variant 4 NM_001256765_1

<400> 259

Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu Met Glu Ala Met

1 5 10 15

Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg Asp Glu Asp Leu

20 25 30

Glu Asn Cys Ser His His Leu

35

<210> 260

<211> 525

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RA nm_014339_6

<400> 260

Cys Met Thr Trp Arg Leu Ala Gly Pro Gly Ser Glu Lys Tyr Ser Asp

1 5 10 15

Asp Thr Lys Tyr Thr Asp Gly Leu Pro Ala Ala Asp Leu Ile Pro Pro

20 25 30

Pro Leu Lys Pro Arg Lys Val Trp Ile Ile Tyr Ser Ala Asp His Pro

35 40 45

Leu Tyr Val Asp Val Val Leu Lys Phe Ala Gln Phe Leu Leu Thr Ala

50 55 60

Cys Gly Thr Glu Val Ala Leu Asp Leu Leu Glu Glu Gln Ala Ile Ser

65 70 75 80

Glu Ala Gly Val Met Thr Trp Val Gly Arg Gln Lys Gln Glu Met Val

85 90 95

Glu Ser Asn Ser Lys Ile Ile Val Leu Cys Ser Arg Gly Thr Arg Ala

100 105 110

Lys Trp Gln Ala Leu Leu Gly Arg Gly Ala Pro Val Arg Leu Arg Cys

115 120 125

Asp His Gly Lys Pro Val Gly Asp Leu Phe Thr Ala Ala Met Asn Met

130 135 140

Ile Leu Pro Asp Phe Lys Arg Pro Ala Cys Phe Gly Thr Tyr Val Val

145 150 155 160

Cys Tyr Phe Ser Glu Val Ser Cys Asp Gly Asp Val Pro Asp Leu Phe

165 170 175

Gly Ala Ala Pro Arg Tyr Pro Leu Met Asp Arg Phe Glu Glu Val Tyr

180 185 190

Phe Arg Ile Gln Asp Leu Glu Met Phe Gln Pro Gly Arg Met His Arg

195 200 205

Val Gly Glu Leu Ser Gly Asp Asn Tyr Leu Arg Ser Pro Gly Gly Arg

210 215 220

Gln Leu Arg Ala Ala Leu Asp Arg Phe Arg Asp Trp Gln Val Arg Cys

225 230 235 240

Pro Asp Trp Phe Glu Cys Glu Asn Leu Tyr Ser Ala Asp Asp Gln Asp

245 250 255

Ala Pro Ser Leu Asp Glu Glu Val Phe Glu Glu Pro Leu Leu Pro Pro

260 265 270

Gly Thr Gly Ile Val Lys Arg Ala Pro Leu Val Arg Glu Pro Gly Ser

275 280 285

Gln Ala Cys Leu Ala Ile Asp Pro Leu Val Gly Glu Glu Gly Gly Ala

290 295 300

Ala Val Ala Lys Leu Glu Pro His Leu Gln Pro Arg Gly Gln Pro Ala

305 310 315 320

Pro Gln Pro Leu His Thr Leu Val Leu Ala Ala Glu Glu Gly Ala Leu

325 330 335

Val Ala Ala Val Glu Pro Gly Pro Leu Ala Asp Gly Ala Ala Val Arg

340 345 350

Leu Ala Leu Ala Gly Glu Gly Glu Ala Cys Pro Leu Leu Gly Ser Pro

355 360 365

Gly Ala Gly Arg Asn Ser Val Leu Phe Leu Pro Val Asp Pro Glu Asp

370 375 380

Ser Pro Leu Gly Ser Ser Thr Pro Met Ala Ser Pro Asp Leu Leu Pro

385 390 395 400

Glu Asp Val Arg Glu His Leu Glu Gly Leu Met Leu Ser Leu Phe Glu

405 410 415

Gln Ser Leu Ser Cys Gln Ala Gln Gly Gly Cys Ser Arg Pro Ala Met

420 425 430

Val Leu Thr Asp Pro His Thr Pro Tyr Glu Glu Glu Gln Arg Gln Ser

435 440 445

Val Gln Ser Asp Gln Gly Tyr Ile Ser Arg Ser Ser Pro Gln Pro Pro

450 455 460

Glu Gly Leu Thr Glu Met Glu Glu Glu Glu Glu Glu Glu Gln Asp Pro

465 470 475 480

Gly Lys Pro Ala Leu Pro Leu Ser Pro Glu Asp Leu Glu Ser Leu Arg

485 490 495

Ser Leu Gln Arg Gln Leu Leu Phe Arg Gln Leu Gln Lys Asn Ser Gly

500 505 510

Trp Asp Thr Met Gly Ser Glu Ser Glu Gly Pro Ser Ala

515 520 525

<210> 261

<211> 189

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RB NM_018725_3

<400> 261

Arg His Glu Arg Ile Lys Lys Thr Ser Phe Ser Thr Thr Thr Leu Leu

1 5 10 15

Pro Pro Ile Lys Val Leu Val Val Tyr Pro Ser Glu Ile Cys Phe His

20 25 30

His Thr Ile Cys Tyr Phe Thr Glu Phe Leu Gln Asn His Cys Arg Ser

35 40 45

Glu Val Ile Leu Glu Lys Trp Gln Lys Lys Lys Ile Ala Glu Met Gly

50 55 60

Pro Val Gln Trp Leu Ala Thr Gln Lys Lys Ala Ala Asp Lys Val Val

65 70 75 80

Phe Leu Leu Ser Asn Asp Val Asn Ser Val Cys Asp Gly Thr Cys Gly

85 90 95

Lys Ser Glu Gly Ser Pro Ser Glu Asn Ser Gln Asp Leu Phe Pro Leu

100 105 110

Ala Phe Asn Leu Phe Cys Ser Asp Leu Arg Ser Gln Ile His Leu His

115 120 125

Lys Tyr Val Val Val Tyr Phe Arg Glu Ile Asp Thr Lys Asp Asp Tyr

130 135 140

Asn Ala Leu Ser Val Cys Pro Lys Tyr His Leu Met Lys Asp Ala Thr

145 150 155 160

Ala Phe Cys Ala Glu Leu Leu His Val Lys Gln Gln Val Ser Ala Gly

165 170 175

Lys Arg Ser Gln Ala Cys His Asp Gly Cys Cys Ser Leu

180 185

<210> 262

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RC transcript variant 1 NM_153460_3

<400> 262

Lys Lys Asp His Ala Lys Gly Trp Leu Arg Leu Leu Lys Gln Asp Val

1 5 10 15

Arg Ser Gly Ala Ala Ala Arg Gly Arg Ala Ala Leu Leu Leu Tyr Ser

20 25 30

Ala Asp Asp Ser Gly Phe Glu Arg Leu Val Gly Ala Leu Ala Ser Ala

35 40 45

Leu Cys Gln Leu Pro Leu Arg Val Ala Val Asp Leu Trp Ser Arg Arg

50 55 60

Glu Leu Ser Ala Gln Gly Pro Val Ala Trp Phe His Ala Gln Arg Arg

65 70 75 80

Gln Thr Leu Gln Glu Gly Gly Val Val Val Leu Leu Phe Ser Pro Gly

85 90 95

Ala Val Ala Leu Cys Ser Glu Trp Leu Gln Asp Gly Val Ser Gly Pro

100 105 110

Gly Ala His Gly Pro His Asp Ala Phe Arg Ala Ser Leu Ser Cys Val

115 120 125

Leu Pro Asp Phe Leu Gln Gly Arg Ala Pro Gly Ser Tyr Val Gly Ala

130 135 140

Cys Phe Asp Arg Leu Leu His Pro Asp Ala Val Pro Ala Leu Phe Arg

145 150 155 160

Thr Val Pro Val Phe Thr Leu Pro Ser Gln Leu Pro Asp Phe Leu Gly

165 170 175

Ala Leu Gln Gln Pro Arg Ala Pro Arg Ser Gly Arg Leu Gln Glu Arg

180 185 190

Ala Glu Gln Val Ser Arg Ala Leu Gln Pro Ala Leu Asp Ser Tyr Phe

195 200 205

His Pro Pro Gly Thr Pro Ala Pro Gly Arg Gly Val Gly Pro Gly Ala

210 215 220

Gly Pro Gly Ala Gly Asp Gly Thr

225 230

<210> 263

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RC transcript variant 4 NM_001203263_1

<400> 263

Lys Lys Asp His Ala Lys Ala Ala Ala Arg Gly Arg Ala Ala Leu Leu

1 5 10 15

Leu Tyr Ser Ala Asp Asp Ser Gly Phe Glu Arg Leu Val Gly Ala Leu

20 25 30

Ala Ser Ala Leu Cys Gln Leu Pro Leu Arg Val Ala Val Asp Leu Trp

35 40 45

Ser Arg Arg Glu Leu Ser Ala Gln Gly Pro Val Ala Trp Phe His Ala

50 55 60

Gln Arg Arg Gln Thr Leu Gln Glu Gly Gly Val Val Val Leu Leu Phe

65 70 75 80

Ser Pro Gly Ala Val Ala Leu Cys Ser Glu Trp Leu Gln Asp Gly Val

85 90 95

Ser Gly Pro Gly Ala His Gly Pro His Asp Ala Phe Arg Ala Ser Leu

100 105 110

Ser Cys Val Leu Pro Asp Phe Leu Gln Gly Arg Ala Pro Gly Ser Tyr

115 120 125

Val Gly Ala Cys Phe Asp Arg Leu Leu His Pro Asp Ala Val Pro Ala

130 135 140

Leu Phe Arg Thr Val Pro Val Phe Thr Leu Pro Ser Gln Leu Pro Asp

145 150 155 160

Phe Leu Gly Ala Leu Gln Gln Pro Arg Ala Pro Arg Ser Gly Arg Leu

165 170 175

Gln Glu Arg Ala Glu Gln Val Ser Arg Ala Leu Gln Pro Ala Leu Asp

180 185 190

Ser Tyr Phe His Pro Pro Gly Thr Pro Ala Pro Gly Arg Gly Val Gly

195 200 205

Pro Gly Ala Gly Pro Gly Ala Gly Asp Gly Thr

210 215

<210> 264

<211> 419

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RD transcript variant 2 NM_017563_4

<400> 264

Cys Arg Lys Lys Gln Gln Glu Asn Ile Tyr Ser His Leu Asp Glu Glu

1 5 10 15

Ser Ser Glu Ser Ser Thr Tyr Thr Ala Ala Leu Pro Arg Glu Arg Leu

20 25 30

Arg Pro Arg Pro Lys Val Phe Leu Cys Tyr Ser Ser Lys Asp Gly Gln

35 40 45

Asn His Met Asn Val Val Gln Cys Phe Ala Tyr Phe Leu Gln Asp Phe

50 55 60

Cys Gly Cys Glu Val Ala Leu Asp Leu Trp Glu Asp Phe Ser Leu Cys

65 70 75 80

Arg Glu Gly Gln Arg Glu Trp Val Ile Gln Lys Ile His Glu Ser Gln

85 90 95

Phe Ile Ile Val Val Cys Ser Lys Gly Met Lys Tyr Phe Val Asp Lys

100 105 110

Lys Asn Tyr Lys His Lys Gly Gly Gly Arg Gly Ser Gly Lys Gly Glu

115 120 125

Leu Phe Leu Val Ala Val Ser Ala Ile Ala Glu Lys Leu Arg Gln Ala

130 135 140

Lys Gln Ser Ser Ser Ala Ala Leu Ser Lys Phe Ile Ala Val Tyr Phe

145 150 155 160

Asp Tyr Ser Cys Glu Gly Asp Val Pro Gly Ile Leu Asp Leu Ser Thr

165 170 175

Lys Tyr Arg Leu Met Asp Asn Leu Pro Gln Leu Cys Ser His Leu His

180 185 190

Ser Arg Asp His Gly Leu Gln Glu Pro Gly Gln His Thr Arg Gln Gly

195 200 205

Ser Arg Arg Asn Tyr Phe Arg Ser Lys Ser Gly Arg Ser Leu Tyr Val

210 215 220

Ala Ile Cys Asn Met His Gln Phe Ile Asp Glu Glu Pro Asp Trp Phe

225 230 235 240

Glu Lys Gln Phe Val Pro Phe His Pro Pro Pro Leu Arg Tyr Arg Glu

245 250 255

Pro Val Leu Glu Lys Phe Asp Ser Gly Leu Val Leu Asn Asp Val Met

260 265 270

Cys Lys Pro Gly Pro Glu Ser Asp Phe Cys Leu Lys Val Glu Ala Ala

275 280 285

Val Leu Gly Ala Thr Gly Pro Ala Asp Ser Gln His Glu Ser Gln His

290 295 300

Gly Gly Leu Asp Gln Asp Gly Glu Ala Arg Pro Ala Leu Asp Gly Ser

305 310 315 320

Ala Ala Leu Gln Pro Leu Leu His Thr Val Lys Ala Gly Ser Pro Ser

325 330 335

Asp Met Pro Arg Asp Ser Gly Ile Tyr Asp Ser Ser Val Pro Ser Ser

340 345 350

Glu Leu Ser Leu Pro Leu Met Glu Gly Leu Ser Thr Asp Gln Thr Glu

355 360 365

Thr Ser Ser Leu Thr Glu Ser Val Ser Ser Ser Ser Gly Leu Gly Glu

370 375 380

Glu Glu Pro Pro Ala Leu Pro Ser Lys Leu Leu Ser Ser Gly Ser Cys

385 390 395 400

Lys Ala Asp Leu Gly Cys Arg Ser Tyr Thr Asp Glu Leu His Ala Val

405 410 415

Ala Pro Leu

<210> 265

<211> 192

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL17RE transcript variant 1 NM_153480_1

<400> 265

Thr Cys Arg Arg Pro Gln Ser Gly Pro Gly Pro Ala Arg Pro Val Leu

1 5 10 15

Leu Leu His Ala Ala Asp Ser Glu Ala Gln Arg Arg Leu Val Gly Ala

20 25 30

Leu Ala Glu Leu Leu Arg Ala Ala Leu Gly Gly Gly Arg Asp Val Ile

35 40 45

Val Asp Leu Trp Glu Gly Arg His Val Ala Arg Val Gly Pro Leu Pro

50 55 60

Trp Leu Trp Ala Ala Arg Thr Arg Val Ala Arg Glu Gln Gly Thr Val

65 70 75 80

Leu Leu Leu Trp Ser Gly Ala Asp Leu Arg Pro Val Ser Gly Pro Asp

85 90 95

Pro Arg Ala Ala Pro Leu Leu Ala Leu Leu His Ala Ala Pro Arg Pro

100 105 110

Leu Leu Leu Leu Ala Tyr Phe Ser Arg Leu Cys Ala Lys Gly Asp Ile

115 120 125

Pro Pro Pro Leu Arg Ala Leu Pro Arg Tyr Arg Leu Leu Arg Asp Leu

130 135 140

Pro Arg Leu Leu Arg Ala Leu Asp Ala Arg Pro Phe Ala Glu Ala Thr

145 150 155 160

Ser Trp Gly Arg Leu Gly Ala Arg Gln Arg Arg Gln Ser Arg Leu Glu

165 170 175

Leu Cys Ser Arg Leu Glu Arg Glu Ala Ala Arg Leu Ala Asp Leu Gly

180 185 190

<210> 266

<211> 191

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL18R1 transcript variant 1 NM-003855-3

<400> 266

Tyr Arg Val Asp Leu Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp

1 5 10 15

Glu Thr Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu

20 25 30

Lys Glu Cys Arg Pro Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu

35 40 45

Ile Leu Pro Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile

50 55 60

Phe Glu Arg Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile His

65 70 75 80

Ser Leu Ile Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser

85 90 95

Tyr Met Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu

100 105 110

Ala Leu Val Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro

115 120 125

Val Thr Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser

130 135 140

His Arg Val Leu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser

145 150 155 160

Arg Phe Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys

165 170 175

Pro Gly Arg Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser

180 185 190

<210> 267

<211> 222

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL18RAP NM-003853_3

<400> 267

Ser Ala Leu Leu Tyr Arg His Trp Ile Glu Ile Val Leu Leu Tyr Arg

1 5 10 15

Thr Tyr Gln Ser Lys Asp Gln Thr Leu Gly Asp Lys Lys Asp Phe Asp

20 25 30

Ala Phe Val Ser Tyr Ala Lys Trp Ser Ser Phe Pro Ser Glu Ala Thr

35 40 45

Ser Ser Leu Ser Glu Glu His Leu Ala Leu Ser Leu Phe Pro Asp Val

50 55 60

Leu Glu Asn Lys Tyr Gly Tyr Ser Leu Cys Leu Leu Glu Arg Asp Val

65 70 75 80

Ala Pro Gly Gly Val Tyr Ala Glu Asp Ile Val Ser Ile Ile Lys Arg

85 90 95

Ser Arg Arg Gly Ile Phe Ile Leu Ser Pro Asn Tyr Val Asn Gly Pro

100 105 110

Ser Ile Phe Glu Leu Gln Ala Ala Val Asn Leu Ala Leu Asp Asp Gln

115 120 125

Thr Leu Lys Leu Ile Leu Ile Lys Phe Cys Tyr Phe Gln Glu Pro Glu

130 135 140

Ser Leu Pro His Leu Val Lys Lys Ala Leu Arg Val Leu Pro Thr Val

145 150 155 160

Thr Trp Arg Gly Leu Lys Ser Val Pro Pro Asn Ser Arg Phe Trp Ala

165 170 175

Lys Met Arg Tyr His Met Pro Val Lys Asn Ser Gln Gly Phe Thr Trp

180 185 190

Asn Gln Leu Arg Ile Thr Ser Arg Ile Phe Gln Trp Lys Gly Leu Ser

195 200 205

Arg Thr Glu Thr Thr Gly Arg Ser Ser Gln Pro Lys Glu Trp

210 215 220

<210> 268

<211> 282

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL20RA transcript variant 1 NM_014432_3

<400> 268

Ser Ile Tyr Arg Tyr Ile His Val Gly Lys Glu Lys His Pro Ala Asn

1 5 10 15

Leu Ile Leu Ile Tyr Gly Asn Glu Phe Asp Lys Arg Phe Phe Val Pro

20 25 30

Ala Glu Lys Ile Val Ile Asn Phe Ile Thr Leu Asn Ile Ser Asp Asp

35 40 45

Ser Lys Ile Ser His Gln Asp Met Ser Leu Leu Gly Lys Ser Ser Asp

50 55 60

Val Ser Ser Leu Asn Asp Pro Gln Pro Ser Gly Asn Leu Arg Pro Pro

65 70 75 80

Gln Glu Glu Glu Glu Val Lys His Leu Gly Tyr Ala Ser His Leu Met

85 90 95

Glu Ile Phe Cys Asp Ser Glu Glu Asn Thr Glu Gly Thr Ser Leu Thr

100 105 110

Gln Gln Glu Ser Leu Ser Arg Thr Ile Pro Pro Asp Lys Thr Val Ile

115 120 125

Glu Tyr Glu Tyr Asp Val Arg Thr Thr Asp Ile Cys Ala Gly Pro Glu

130 135 140

Glu Gln Glu Leu Ser Leu Gln Glu Glu Val Ser Thr Gln Gly Thr Leu

145 150 155 160

Leu Glu Ser Gln Ala Ala Leu Ala Val Leu Gly Pro Gln Thr Leu Gln

165 170 175

Tyr Ser Tyr Thr Pro Gln Leu Gln Asp Leu Asp Pro Leu Ala Gln Glu

180 185 190

His Thr Asp Ser Glu Glu Gly Pro Glu Glu Glu Pro Ser Thr Thr Leu

195 200 205

Val Asp Trp Asp Pro Gln Thr Gly Arg Leu Cys Ile Pro Ser Leu Ser

210 215 220

Ser Phe Asp Gln Asp Ser Glu Gly Cys Glu Pro Ser Glu Gly Asp Gly

225 230 235 240

Leu Gly Glu Glu Gly Leu Leu Ser Arg Leu Tyr Glu Glu Pro Ala Pro

245 250 255

Asp Arg Pro Pro Gly Glu Asn Glu Thr Tyr Leu Met Gln Phe Met Glu

260 265 270

Glu Trp Gly Leu Tyr Val Gln Met Glu Asn

275 280

<210> 269

<211> 57

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL20RB NM-144717_3

<400> 269

Trp Lys Met Gly Arg Leu Leu Gln Tyr Ser Cys Cys Pro Val Val Val

1 5 10 15

Leu Pro Asp Thr Leu Lys Ile Thr Asn Ser Pro Gln Lys Leu Ile Ser

20 25 30

Cys Arg Arg Glu Glu Val Asp Ala Cys Ala Thr Ala Val Met Ser Pro

35 40 45

Glu Glu Leu Leu Arg Ala Trp Ile Ser

50 55

<210> 270

<211> 285

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL21R transcript variant 2 NM_181078_2

<400> 270

Ser Leu Lys Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala

1 5 10 15

Val Pro Ser Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser

20 25 30

Gly Asp Phe Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu

35 40 45

Glu Leu Gly Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr

50 55 60

Ser Cys His Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu

65 70 75 80

Leu Gln Glu Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro

85 90 95

Ser Phe Trp Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu

100 105 110

Glu Arg Asp Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val

115 120 125

Leu Asp Ala Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp

130 135 140

Gly Tyr Pro Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly

145 150 155 160

Leu Glu Asp Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly

165 170 175

Cys Val Ser Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu

180 185 190

Leu Asp Arg Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly

195 200 205

Gly Leu Pro Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu

210 215 220

Ala Gly Ser Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly

225 230 235 240

Phe Val Gly Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser

245 250 255

Pro Gly Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val

260 265 270

Ile Pro Pro Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser

275 280 285

<210> 271

<211> 325

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL22RA1 NM-021258_3

<400> 271

Ser Tyr Arg Tyr Val Thr Lys Pro Pro Ala Pro Pro Asn Ser Leu Asn

1 5 10 15

Val Gln Arg Val Leu Thr Phe Gln Pro Leu Arg Phe Ile Gln Glu His

20 25 30

Val Leu Ile Pro Val Phe Asp Leu Ser Gly Pro Ser Ser Leu Ala Gln

35 40 45

Pro Val Gln Tyr Ser Gln Ile Arg Val Ser Gly Pro Arg Glu Pro Ala

50 55 60

Gly Ala Pro Gln Arg His Ser Leu Ser Glu Ile Thr Tyr Leu Gly Gln

65 70 75 80

Pro Asp Ile Ser Ile Leu Gln Pro Ser Asn Val Pro Pro Pro Gln Ile

85 90 95

Leu Ser Pro Leu Ser Tyr Ala Pro Asn Ala Ala Pro Glu Val Gly Pro

100 105 110

Pro Ser Tyr Ala Pro Gln Val Thr Pro Glu Ala Gln Phe Pro Phe Tyr

115 120 125

Ala Pro Gln Ala Ile Ser Lys Val Gln Pro Ser Ser Tyr Ala Pro Gln

130 135 140

Ala Thr Pro Asp Ser Trp Pro Pro Ser Tyr Gly Val Cys Met Glu Gly

145 150 155 160

Ser Gly Lys Asp Ser Pro Thr Gly Thr Leu Ser Ser Pro Lys His Leu

165 170 175

Arg Pro Lys Gly Gln Leu Gln Lys Glu Pro Pro Ala Gly Ser Cys Met

180 185 190

Leu Gly Gly Leu Ser Leu Gln Glu Val Thr Ser Leu Ala Met Glu Glu

195 200 205

Ser Gln Glu Ala Lys Ser Leu His Gln Pro Leu Gly Ile Cys Thr Asp

210 215 220

Arg Thr Ser Asp Pro Asn Val Leu His Ser Gly Glu Glu Gly Thr Pro

225 230 235 240

Gln Tyr Leu Lys Gly Gln Leu Pro Leu Leu Ser Ser Val Gln Ile Glu

245 250 255

Gly His Pro Met Ser Leu Pro Leu Gln Pro Pro Ser Arg Pro Cys Ser

260 265 270

Pro Ser Asp Gln Gly Pro Ser Pro Trp Gly Leu Leu Glu Ser Leu Val

275 280 285

Cys Pro Lys Asp Glu Ala Lys Ser Pro Ala Pro Glu Thr Ser Asp Leu

290 295 300

Glu Gln Pro Thr Glu Leu Asp Ser Leu Phe Arg Gly Leu Ala Leu Thr

305 310 315 320

Val Gln Trp Glu Ser

325

<210> 272

<211> 253

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL23R nm_144701_2

<400> 272

Asn Arg Ser Phe Arg Thr Gly Ile Lys Arg Arg Ile Leu Leu Leu Ile

1 5 10 15

Pro Lys Trp Leu Tyr Glu Asp Ile Pro Asn Met Lys Asn Ser Asn Val

20 25 30

Val Lys Met Leu Gln Glu Asn Ser Glu Leu Met Asn Asn Asn Ser Ser

35 40 45

Glu Gln Val Leu Tyr Val Asp Pro Met Ile Thr Glu Ile Lys Glu Ile

50 55 60

Phe Ile Pro Glu His Lys Pro Thr Asp Tyr Lys Lys Glu Asn Thr Gly

65 70 75 80

Pro Leu Glu Thr Arg Asp Tyr Pro Gln Asn Ser Leu Phe Asp Asn Thr

85 90 95

Thr Val Val Tyr Ile Pro Asp Leu Asn Thr Gly Tyr Lys Pro Gln Ile

100 105 110

Ser Asn Phe Leu Pro Glu Gly Ser His Leu Ser Asn Asn Asn Glu Ile

115 120 125

Thr Ser Leu Thr Leu Lys Pro Pro Val Asp Ser Leu Asp Ser Gly Asn

130 135 140

Asn Pro Arg Leu Gln Lys His Pro Asn Phe Ala Phe Ser Val Ser Ser

145 150 155 160

Val Asn Ser Leu Ser Asn Thr Ile Phe Leu Gly Glu Leu Ser Leu Ile

165 170 175

Leu Asn Gln Gly Glu Cys Ser Ser Pro Asp Ile Gln Asn Ser Val Glu

180 185 190

Glu Glu Thr Thr Met Leu Leu Glu Asn Asp Ser Pro Ser Glu Thr Ile

195 200 205

Pro Glu Gln Thr Leu Leu Pro Asp Glu Phe Val Ser Cys Leu Gly Ile

210 215 220

Val Asn Glu Glu Leu Pro Ser Ile Asn Thr Tyr Phe Pro Gln Asn Ile

225 230 235 240

Leu Glu Ser His Phe Asn Arg Ile Ser Leu Leu Glu Lys

245 250

<210> 273

<211> 99

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL27RA NM-004843_3

<400> 273

Thr Ser Gly Arg Cys Tyr His Leu Arg His Lys Val Leu Pro Arg Trp

1 5 10 15

Val Trp Glu Lys Val Pro Asp Pro Ala Asn Ser Ser Ser Gly Gln Pro

20 25 30

His Met Glu Gln Val Pro Glu Ala Gln Pro Leu Gly Asp Leu Pro Ile

35 40 45

Leu Glu Val Glu Glu Met Glu Pro Pro Pro Val Met Glu Ser Ser Gln

50 55 60

Pro Ala Gln Ala Thr Ala Pro Leu Asp Ser Gly Tyr Glu Lys His Phe

65 70 75 80

Leu Pro Thr Pro Glu Glu Leu Gly Leu Leu Gly Pro Pro Arg Pro Gln

85 90 95

Val Leu Ala

<210> 274

<211> 86

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL27RA NM-004843_3

<400> 274

Thr Ser Trp Val Trp Glu Lys Val Pro Asp Pro Ala Asn Ser Ser Ser

1 5 10 15

Gly Gln Pro His Met Glu Gln Val Pro Glu Ala Gln Pro Leu Gly Asp

20 25 30

Leu Pro Ile Leu Glu Val Glu Glu Met Glu Pro Pro Pro Val Met Glu

35 40 45

Ser Ser Gln Pro Ala Gln Ala Thr Ala Pro Leu Asp Ser Gly Tyr Glu

50 55 60

Lys His Phe Leu Pro Thr Pro Glu Glu Leu Gly Leu Leu Gly Pro Pro

65 70 75 80

Arg Pro Gln Val Leu Ala

85

<210> 275

<211> 189

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL31RA transcript variant 1 NM_139017_5

<400> 275

Lys Lys Pro Asn Lys Leu Thr His Leu Cys Trp Pro Thr Val Pro Asn

1 5 10 15

Pro Ala Glu Ser Ser Ile Ala Thr Trp His Gly Asp Asp Phe Lys Asp

20 25 30

Lys Leu Asn Leu Lys Glu Ser Asp Asp Ser Val Asn Thr Glu Asp Arg

35 40 45

Ile Leu Lys Pro Cys Ser Thr Pro Ser Asp Lys Leu Val Ile Asp Lys

50 55 60

Leu Val Val Asn Phe Gly Asn Val Leu Gln Glu Ile Phe Thr Asp Glu

65 70 75 80

Ala Arg Thr Gly Gln Glu Asn Asn Leu Gly Gly Glu Lys Asn Gly Tyr

85 90 95

Val Thr Cys Pro Phe Arg Pro Asp Cys Pro Leu Gly Lys Ser Phe Glu

100 105 110

Glu Leu Pro Val Ser Pro Glu Ile Pro Pro Arg Lys Ser Gln Tyr Leu

115 120 125

Arg Ser Arg Met Pro Glu Gly Thr Arg Pro Glu Ala Lys Glu Gln Leu

130 135 140

Leu Phe Ser Gly Gln Ser Leu Val Pro Asp His Leu Cys Glu Glu Gly

145 150 155 160

Ala Pro Asn Pro Tyr Leu Lys Asn Ser Val Thr Ala Arg Glu Phe Leu

165 170 175

Val Ser Glu Lys Leu Pro Glu His Thr Lys Gly Glu Val

180 185

<210> 276

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: IL31RA transcript variant 4 NM_001242638_1

<400> 276

Lys Lys Pro Asn Lys Leu Thr His Leu Cys Trp Pro Thr Val Pro Asn

1 5 10 15

Pro Ala Glu Ser Ser Ile Ala Thr Trp His Gly Asp Asp Phe Lys Asp

20 25 30

Lys Leu Asn Leu Lys Glu Ser Asp Asp Ser Val Asn Thr Glu Asp Arg

35 40 45

Ile Leu Lys Pro Cys Ser Thr Pro Ser Asp Lys Leu Val Ile Asp Lys

50 55 60

Leu Val Val Asn Phe Gly Asn Val Leu Gln Glu Ile Phe Thr Asp Glu

65 70 75 80

Ala Arg Thr Gly Gln Glu Asn Asn Leu Gly Gly Glu Lys Asn Gly Thr

85 90 95

Arg Ile Leu Ser Ser Cys Pro Thr Ser Ile

100 105

<210> 277

<211> 303

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: LEPR transcript variant 1 nm_002303_5

<400> 277

Ser His Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro

1 5 10 15

Lys Asn Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Pro Glu Thr

20 25 30

Phe Glu His Leu Phe Ile Lys His Thr Ala Ser Val Thr Cys Gly Pro

35 40 45

Leu Leu Leu Glu Pro Glu Thr Ile Ser Glu Asp Ile Ser Val Asp Thr

50 55 60

Ser Trp Lys Asn Lys Asp Glu Met Met Pro Thr Thr Val Val Ser Leu

65 70 75 80

Leu Ser Thr Thr Asp Leu Glu Lys Gly Ser Val Cys Ile Ser Asp Gln

85 90 95

Phe Asn Ser Val Asn Phe Ser Glu Ala Glu Gly Thr Glu Val Thr Tyr

100 105 110

Glu Asp Glu Ser Gln Arg Gln Pro Phe Val Lys Tyr Ala Thr Leu Ile

115 120 125

Ser Asn Ser Lys Pro Ser Glu Thr Gly Glu Glu Gln Gly Leu Ile Asn

130 135 140

Ser Ser Val Thr Lys Cys Phe Ser Ser Lys Asn Ser Pro Leu Lys Asp

145 150 155 160

Ser Phe Ser Asn Ser Ser Trp Glu Ile Glu Ala Gln Ala Phe Phe Ile

165 170 175

Leu Ser Asp Gln His Pro Asn Ile Ile Ser Pro His Leu Thr Phe Ser

180 185 190

Glu Gly Leu Asp Glu Leu Leu Lys Leu Glu Gly Asn Phe Pro Glu Glu

195 200 205

Asn Asn Asp Lys Lys Ser Ile Tyr Tyr Leu Gly Val Thr Ser Ile Lys

210 215 220

Lys Arg Glu Ser Gly Val Leu Leu Thr Asp Lys Ser Arg Val Ser Cys

225 230 235 240

Pro Phe Pro Ala Pro Cys Leu Phe Thr Asp Ile Arg Val Leu Gln Asp

245 250 255

Ser Cys Ser His Phe Val Glu Asn Asn Ile Asn Leu Gly Thr Ser Ser

260 265 270

Lys Lys Thr Phe Ala Ser Tyr Met Pro Gln Phe Gln Thr Cys Ser Thr

275 280 285

Gln Thr His Lys Ile Met Glu Asn Lys Met Cys Asp Leu Thr Val

290 295 300

<210> 278

<211> 96

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: LEPR transcript variant 2 nm_001003680_3

<400> 278

Ser His Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro

1 5 10 15

Lys Asn Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Met Leu Glu

20 25 30

Gly Ser Met Phe Val Lys Ser His His His Ser Leu Ile Ser Ser Thr

35 40 45

Gln Gly His Lys His Cys Gly Arg Pro Gln Gly Pro Leu His Arg Lys

50 55 60

Thr Arg Asp Leu Cys Ser Leu Val Tyr Leu Leu Thr Leu Pro Pro Leu

65 70 75 80

Leu Ser Tyr Asp Pro Ala Lys Ser Pro Ser Val Arg Asn Thr Gln Glu

85 90 95

<210> 279

<211> 34

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: LEPR transcript variant 3nm_001003679_3

<400> 279

Ser His Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro

1 5 10 15

Lys Asn Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Arg Thr Asp

20 25 30

Ile Leu

<210> 280

<211> 44

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: variant LEPR transcript 5 nm_001198688_1

<400> 280

Ser His Gln Arg Met Lys Lys Leu Phe Trp Glu Asp Val Pro Asn Pro

1 5 10 15

Lys Asn Cys Ser Trp Ala Gln Gly Leu Asn Phe Gln Lys Lys Met Pro

20 25 30

Gly Thr Lys Glu Leu Leu Gly Gly Gly Trp Leu Thr

35 40

<210> 281

<211> 239

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: LIFR NM_001127671_1

<400> 281

Tyr Arg Lys Arg Glu Trp Ile Lys Glu Thr Phe Tyr Pro Asp Ile Pro

1 5 10 15

Asn Pro Glu Asn Cys Lys Ala Leu Gln Phe Gln Lys Ser Val Cys Glu

20 25 30

Gly Ser Ser Ala Leu Lys Thr Leu Glu Met Asn Pro Cys Thr Pro Asn

35 40 45

Asn Val Glu Val Leu Glu Thr Arg Ser Ala Phe Pro Lys Ile Glu Asp

50 55 60

Thr Glu Ile Ile Ser Pro Val Ala Glu Arg Pro Glu Asp Arg Ser Asp

65 70 75 80

Ala Glu Pro Glu Asn His Val Val Val Ser Tyr Cys Pro Pro Ile Ile

85 90 95

Glu Glu Glu Ile Pro Asn Pro Ala Ala Asp Glu Ala Gly Gly Thr Ala

100 105 110

Gln Val Ile Tyr Ile Asp Val Gln Ser Met Tyr Gln Pro Gln Ala Lys

115 120 125

Pro Glu Glu Glu Gln Glu Asn Asp Pro Val Gly Gly Ala Gly Tyr Lys

130 135 140

Pro Gln Met His Leu Pro Ile Asn Ser Thr Val Glu Asp Ile Ala Ala

145 150 155 160

Glu Glu Asp Leu Asp Lys Thr Ala Gly Tyr Arg Pro Gln Ala Asn Val

165 170 175

Asn Thr Trp Asn Leu Val Ser Pro Asp Ser Pro Arg Ser Ile Asp Ser

180 185 190

Asn Ser Glu Ile Val Ser Phe Gly Ser Pro Cys Ser Ile Asn Ser Arg

195 200 205

Gln Phe Leu Ile Pro Pro Lys Asp Glu Asp Ser Pro Lys Ser Asn Gly

210 215 220

Gly Gly Trp Ser Phe Thr Asn Phe Phe Gln Asn Lys Pro Asn Asp

225 230 235

<210> 282

<211> 202

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: lmp1nc_007505_1

<400> 282

Tyr Tyr His Gly Gln Arg His Ser Asp Glu His His His Asp Asp Ser

1 5 10 15

Leu Pro His Pro Gln Gln Ala Thr Asp Asp Ser Gly His Glu Ser Asp

20 25 30

Ser Asn Ser Asn Glu Gly Arg His His Leu Leu Val Ser Gly Ala Gly

35 40 45

Asp Gly Pro Pro Leu Cys Ser Gln Asn Leu Gly Ala Pro Gly Gly Gly

50 55 60

Pro Asp Asn Gly Pro Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro

65 70 75 80

Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro His Asp Pro Leu Pro

85 90 95

Gln Asp Pro Asp Asn Thr Asp Asp Asn Gly Pro Gln Asp Pro Asp Asn

100 105 110

Thr Asp Asp Asn Gly Pro His Asp Pro Leu Pro His Ser Pro Ser Asp

115 120 125

Ser Ala Gly Asn Asp Gly Gly Pro Pro Gln Leu Thr Glu Glu Val Glu

130 135 140

Asn Lys Gly Gly Asp Gln Gly Pro Pro Leu Met Thr Asp Gly Gly Gly

145 150 155 160

Gly His Ser His Asp Ser Gly His Gly Gly Gly Asp Pro His Leu Pro

165 170 175

Thr Leu Leu Leu Gly Ser Ser Gly Ser Gly Gly Asp Asp Asp Asp Pro

180 185 190

His Gly Pro Val Gln Leu Ser Tyr Tyr Asp

195 200

<210> 283

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: mplnm_005373_2

<400> 283

Arg Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu Trp

1 5 10 15

Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu Arg Asp

20 25 30

Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys Glu

35 40 45

Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys Ser Ser Glu Arg

50 55 60

Thr Pro Leu Pro Leu Cys Ser Ser Gln Ala Gln Met Asp Tyr Arg Arg

65 70 75 80

Leu Gln Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro Pro

85 90 95

Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser

100 105 110

Tyr Leu Pro Leu Ser Tyr Trp Gln Gln Pro

115 120

<210> 284

<211> 304

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 1 nm_001172567_1

<400> 284

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp

100 105 110

Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro

115 120 125

Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu

130 135 140

Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg

145 150 155 160

Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln

165 170 175

Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys

180 185 190

Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala

195 200 205

Ser Glu Leu Ile Glu Lys Arg Leu Ala Arg Arg Pro Arg Gly Gly Cys

210 215 220

Arg Arg Met Val Val Val Val Ser Asp Asp Tyr Leu Gln Ser Lys Glu

225 230 235 240

Cys Asp Phe Gln Thr Lys Phe Ala Leu Ser Leu Ser Pro Gly Ala His

245 250 255

Gln Lys Arg Leu Ile Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe

260 265 270

Pro Ser Ile Leu Arg Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys

275 280 285

Thr Lys Ser Trp Phe Trp Thr Arg Leu Ala Lys Ala Leu Ser Leu Pro

290 295 300

<210> 285

<211> 296

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 2 nm_002468_4

<400> 285

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp

100 105 110

Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro

115 120 125

Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu

130 135 140

Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg

145 150 155 160

Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln

165 170 175

Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys

180 185 190

Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala

195 200 205

Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser

210 215 220

Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala

225 230 235 240

Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Leu Ile Pro Ile Lys

245 250 255

Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr

260 265 270

Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg

275 280 285

Leu Ala Lys Ala Leu Ser Leu Pro

290 295

<210> 286

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 3 nm_001172568_1

<400> 286

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Gly His Met

100 105 110

Pro Glu Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln

115 120 125

Phe Val Gln Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu

130 135 140

Lys Leu Cys Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp

145 150 155 160

Ser Ile Ala Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val

165 170 175

Val Val Ser Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr

180 185 190

Lys Phe Ala Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Leu Ile

195 200 205

Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg

210 215 220

Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe

225 230 235 240

Trp Thr Arg Leu Ala Lys Ala Leu Ser Leu Pro

245 250

<210> 287

<211> 191

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 4 nm_001172569_1

<400> 287

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp

100 105 110

Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro

115 120 125

Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu

130 135 140

Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly Ala Ala Gly Trp Trp

145 150 155 160

Trp Leu Ser Leu Met Ile Thr Cys Arg Ala Arg Asn Val Thr Ser Arg

165 170 175

Pro Asn Leu His Ser Ala Ser Leu Gln Val Pro Ile Arg Ser Asp

180 185 190

<210> 288

<211> 146

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 5 nm_001172566_1

<400> 288

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Gly Ala Ala

100 105 110

Gly Trp Trp Trp Leu Ser Leu Met Ile Thr Cys Arg Ala Arg Asn Val

115 120 125

Thr Ser Arg Pro Asn Leu His Ser Ala Ser Leu Gln Val Pro Ile Arg

130 135 140

Ser Asp

145

<210> 289

<211> 172

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 1 nm_001172567_1

<400> 289

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp

100 105 110

Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro

115 120 125

Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu

130 135 140

Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg

145 150 155 160

Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile

165 170

<210> 290

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 3 nm_001172568_1

<400> 290

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Gly His Met

100 105 110

Pro Glu Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile

115 120 125

<210> 291

<211> 304

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 1 nm_001172567_1

<400> 291

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp

100 105 110

Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro

115 120 125

Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu

130 135 140

Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg

145 150 155 160

Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln

165 170 175

Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys

180 185 190

Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala

195 200 205

Ser Glu Leu Ile Glu Lys Arg Leu Ala Arg Arg Pro Arg Gly Gly Cys

210 215 220

Arg Arg Met Val Val Val Val Ser Asp Asp Tyr Leu Gln Ser Lys Glu

225 230 235 240

Cys Asp Phe Gln Thr Lys Phe Ala Leu Ser Leu Ser Pro Gly Ala His

245 250 255

Gln Lys Arg Pro Ile Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe

260 265 270

Pro Ser Ile Leu Arg Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys

275 280 285

Thr Lys Ser Trp Phe Trp Thr Arg Leu Ala Lys Ala Leu Ser Leu Pro

290 295 300

<210> 292

<211> 296

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 2 nm_002468_4

<400> 292

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp

100 105 110

Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro

115 120 125

Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu

130 135 140

Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg

145 150 155 160

Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln

165 170 175

Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys

180 185 190

Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala

195 200 205

Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser

210 215 220

Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala

225 230 235 240

Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Pro Ile Pro Ile Lys

245 250 255

Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr

260 265 270

Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg

275 280 285

Leu Ala Lys Ala Leu Ser Leu Pro

290 295

<210> 293

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MYD88 transcript variant 3 nm_001172568_1

<400> 293

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser

1 5 10 15

Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg

20 25 30

Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr

35 40 45

Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu

50 55 60

Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly

65 70 75 80

Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu

85 90 95

Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Gly His Met

100 105 110

Pro Glu Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln

115 120 125

Phe Val Gln Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu

130 135 140

Lys Leu Cys Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp

145 150 155 160

Ser Ile Ala Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val

165 170 175

Val Val Ser Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr

180 185 190

Lys Phe Ala Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Pro Ile

195 200 205

Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg

210 215 220

Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe

225 230 235 240

Trp Thr Arg Leu Ala Lys Ala Leu Ser Leu Pro

245 250

<210> 294

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: OSMR transcript variant 4 nm_001323505_1

<400> 294

Lys Ser Gln Trp Ile Lys Glu Thr Cys Tyr Pro Asp Ile Pro Asp Pro

1 5 10 15

Tyr Lys Ser Ser Ile Leu Ser Leu Ile Lys Phe Lys Glu Asn Pro His

20 25 30

Leu Ile Ile Met Asn Val Ser Asp Cys Ile Pro Asp Ala Ile Glu Val

35 40 45

Val Ser Lys Pro Glu Gly Thr Lys Ile Gln Phe Leu Gly Thr Arg Lys

50 55 60

Ser Leu Thr Glu Thr Glu Leu Thr Lys Pro Asn Tyr Leu Tyr Leu Leu

65 70 75 80

Pro Thr Glu Lys Asn His Ser Gly Pro Gly Pro Cys Ile Cys Phe Glu

85 90 95

Asn Leu Thr Tyr Asn Gln Ala Ala Ser Asp Ser Gly Ser Cys Gly His

100 105 110

Val Pro Val Ser Pro Lys Ala Pro Ser Met Leu Gly Leu Met Thr Ser

115 120 125

Pro Glu Asn Val Leu Lys Ala Leu Glu Lys Asn Tyr Met Asn Ser Leu

130 135 140

Gly Glu Ile Pro Ala Gly Glu Thr Ser Leu Asn Tyr Val Ser Gln Leu

145 150 155 160

Ala Ser Pro Met Phe Gly Asp Lys Asp Ser Leu Pro Thr Asn Pro Val

165 170 175

Glu Ala Pro His Cys Ser Glu Tyr Lys Met Gln Met Ala Val Ser Leu

180 185 190

Arg Leu Ala Leu Pro Pro Pro Thr Glu Asn Ser Ser Leu Ser Ser Ile

195 200 205

Thr Leu Leu Asp Pro Gly Glu His Tyr Cys

210 215

<210> 295

<211> 364

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: PRLR transcript variant 1 nm_000949_6

<400> 295

Lys Gly Tyr Ser Met Val Thr Cys Ile Phe Pro Pro Val Pro Gly Pro

1 5 10 15

Lys Ile Lys Gly Phe Asp Ala His Leu Leu Glu Lys Gly Lys Ser Glu

20 25 30

Glu Leu Leu Ser Ala Leu Gly Cys Gln Asp Phe Pro Pro Thr Ser Asp

35 40 45

Tyr Glu Asp Leu Leu Val Glu Tyr Leu Glu Val Asp Asp Ser Glu Asp

50 55 60

Gln His Leu Met Ser Val His Ser Lys Glu His Pro Ser Gln Gly Met

65 70 75 80

Lys Pro Thr Tyr Leu Asp Pro Asp Thr Asp Ser Gly Arg Gly Ser Cys

85 90 95

Asp Ser Pro Ser Leu Leu Ser Glu Lys Cys Glu Glu Pro Gln Ala Asn

100 105 110

Pro Ser Thr Phe Tyr Asp Pro Glu Val Ile Glu Lys Pro Glu Asn Pro

115 120 125

Glu Thr Thr His Thr Trp Asp Pro Gln Cys Ile Ser Met Glu Gly Lys

130 135 140

Ile Pro Tyr Phe His Ala Gly Gly Ser Lys Cys Ser Thr Trp Pro Leu

145 150 155 160

Pro Gln Pro Ser Gln His Asn Pro Arg Ser Ser Tyr His Asn Ile Thr

165 170 175

Asp Val Cys Glu Leu Ala Val Gly Pro Ala Gly Ala Pro Ala Thr Leu

180 185 190

Leu Asn Glu Ala Gly Lys Asp Ala Leu Lys Ser Ser Gln Thr Ile Lys

195 200 205

Ser Arg Glu Glu Gly Lys Ala Thr Gln Gln Arg Glu Val Glu Ser Phe

210 215 220

His Ser Glu Thr Asp Gln Asp Thr Pro Trp Leu Leu Pro Gln Glu Lys

225 230 235 240

Thr Pro Phe Gly Ser Ala Lys Pro Leu Asp Tyr Val Glu Ile His Lys

245 250 255

Val Asn Lys Asp Gly Ala Leu Ser Leu Leu Pro Lys Gln Arg Glu Asn

260 265 270

Ser Gly Lys Pro Lys Lys Pro Gly Thr Pro Glu Asn Asn Lys Glu Tyr

275 280 285

Ala Lys Val Ser Gly Val Met Asp Asn Asn Ile Leu Val Leu Val Pro

290 295 300

Asp Pro His Ala Lys Asn Val Ala Cys Phe Glu Glu Ser Ala Lys Glu

305 310 315 320

Ala Pro Pro Ser Leu Glu Gln Asn Gln Ala Glu Lys Ala Leu Ala Asn

325 330 335

Phe Thr Ala Thr Ser Ser Lys Cys Arg Leu Gln Leu Gly Gly Leu Asp

340 345 350

Tyr Leu Asp Pro Ala Cys Phe Thr His Ser Phe His

355 360

<210> 296

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: tnfrsf4nm_003327_3

<400> 296

Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His

1 5 10 15

Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln

20 25 30

Ala Asp Ala His Ser Thr Leu Ala Lys Ile

35 40

<210> 297

<211> 188

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF8 transcript variant 1 nm_001243_4

<400> 297

His Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys

1 5 10 15

Tyr Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg

20 25 30

Pro Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu

35 40 45

Pro Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr

50 55 60

Cys His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp

65 70 75 80

Ala Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro

85 90 95

Arg Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile

100 105 110

Met Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro

115 120 125

Glu Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu

130 135 140

Leu Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro

145 150 155 160

Pro Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly

165 170 175

Lys Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys

180 185

<210> 298

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: tnfrsf9nm_001561_5

<400> 298

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 299

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF14 transcript variant 1 nm_003820_3

<400> 299

Cys Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val

1 5 10 15

Ser Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile

20 25 30

Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu

35 40 45

Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His

50 55 60

<210> 300

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF18 transcript variant 1 nm_004195_2

<400> 300

Gln Leu Gly Leu His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro

1 5 10 15

Arg Glu Thr Gln Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala

20 25 30

Arg Ser Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu

35 40 45

Glu Lys Gly Arg Leu Gly Asp Leu Trp Val

50 55

<210> 301

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TNFRSF18 transcript variant 3_nm_148902_1

<400> 301

Gln Leu Gly Leu His Ile Trp Gln Leu Arg Lys Thr Gln Leu Leu Leu

1 5 10 15

Glu Val Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln Phe Pro Glu

20 25 30

Glu Glu Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg Leu Gly Asp

35 40 45

Leu Trp Val

50

<210> 302

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: connector

<400> 302

Gly Ser Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Ala Ala Thr

1 5 10 15

Ala Gly Ser Gly Ser Gly Ser

20

<210> 303

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TRAF1, TRAF2 and TRAF3 consensus binding sequences

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<400> 303

Pro Xaa Gln Xaa Thr

1 5

<210> 304

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TRAF2 consensus binding sequences

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 304

Ser Xaa Xaa Glu

1

<210> 305

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: TRAF6 consensus binding sequences

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<400> 305

Gln Xaa Pro Xaa Glu Xaa

1 5

<210> 306

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: box1 die body

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 306

Pro Xaa Xaa Pro

1

<210> 307

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: binding motif for binding Shc phosphotyrosine

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 307

Asn Xaa Xaa Tyr

1

<210> 308

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: STAT3 consensus binding sequences

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 308

Tyr Xaa Xaa Gln

1

<210> 309

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: STAT5 recruitment sequence

<400> 309

Tyr Leu Pro Leu

1

<210> 310

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: STAT5 consensus recruitment sequences

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa is phosphorylated tyrosine

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 310

Xaa Leu Xaa Leu

1

<210> 311

<211> 570

<212> PRT

<213> influenza Virus

<220>

<221> misc_feature

<222> (1)..(570)

<223> influenza A HA from H1N1

<400> 311

Met Lys Ala Asn Leu Leu Val Leu Leu Cys Ala Leu Ala Ala Ala Asp

1 5 10 15

Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr

20 25 30

Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn

35 40 45

Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile

50 55 60

Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly

65 70 75 80

Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile

85 90 95

Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe

100 105 110

Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe

115 120 125

Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn

130 135 140

Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe

145 150 155 160

Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys

165 170 175

Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu

180 185 190

Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Leu Tyr

195 200 205

Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg

210 215 220

Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala

225 230 235 240

Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile

245 250 255

Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala

260 265 270

Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met

275 280 285

His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser

290 295 300

Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro

305 310 315 320

Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn

325 330 335

Ile Pro Ser Ile Gln Ser Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly

340 345 350

Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly

355 360 365

Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala

370 375 380

Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val

385 390 395 400

Asn Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val Gly Lys

405 410 415

Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val

420 425 430

Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val

435 440 445

Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys

450 455 460

Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu

465 470 475 480

Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys

485 490 495

Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu

500 505 510

Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu Glu Ser

515 520 525

Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser

530 535 540

Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser

545 550 555 560

Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

565 570

<210> 312

<211> 470

<212> PRT

<213> influenza Virus

<220>

<221> misc_feature

<222> (1)..(470)

<223> influenza A NA from H10N7

<400> 312

Met Asn Pro Asn Gln Lys Leu Phe Ala Leu Ser Gly Val Ala Ile Ala

1 5 10 15

Leu Ser Ile Leu Asn Leu Leu Ile Gly Ile Ser Asn Val Gly Leu Asn

20 25 30

Val Ser Leu His Leu Lys Gly Ser Ser Asp Gln Asp Lys Asn Trp Thr

35 40 45

Cys Thr Ser Val Thr Gln Asn Asn Thr Thr Leu Ile Glu Asn Thr Tyr

50 55 60

Val Asn Asn Thr Thr Val Ile Asn Lys Gly Thr Gly Thr Thr Lys Gln

65 70 75 80

Asn Tyr Leu Met Leu Asn Lys Ser Leu Cys Lys Val Glu Gly Trp Val

85 90 95

Val Val Ala Lys Asp Asn Ala Ile Arg Phe Gly Glu Ser Glu Gln Ile

100 105 110

Ile Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Leu Gly Cys Lys

115 120 125

Met Tyr Ala Leu His Gln Gly Thr Thr Ile Arg Asn Lys His Ser Asn

130 135 140

Gly Thr Ile His Asp Arg Thr Ala Phe Arg Gly Leu Ile Ser Thr Pro

145 150 155 160

Leu Gly Ser Pro Pro Val Val Ser Asn Ser Asp Phe Leu Cys Val Gly

165 170 175

Trp Ser Ser Thr Ser Cys His Asp Gly Ile Gly Arg Met Thr Ile Cys

180 185 190

Val Gln Gly Asn Asn Asn Asn Ala Thr Ala Thr Val Tyr Tyr Asp Arg

195 200 205

Arg Leu Thr Thr Thr Ile Lys Thr Trp Ala Gly Asn Ile Leu Arg Thr

210 215 220

Gln Glu Ser Glu Cys Val Cys His Asn Gly Thr Cys Val Val Ile Met

225 230 235 240

Thr Asp Gly Ser Ala Ser Ser Gln Ala His Thr Lys Val Leu Tyr Phe

245 250 255

His Lys Gly Leu Val Ile Lys Glu Glu Ala Leu Lys Gly Ser Ala Arg

260 265 270

His Ile Glu Glu Cys Ser Cys Tyr Gly His Asn Ser Lys Val Thr Cys

275 280 285

Val Cys Arg Asp Asn Trp Gln Gly Ala Asn Arg Pro Val Ile Glu Ile

290 295 300

Asp Met Asn Ala Met Glu His Thr Ser Gln Tyr Leu Cys Thr Gly Val

305 310 315 320

Leu Thr Asp Thr Ser Arg Pro Ser Asp Lys Ser Met Gly Asp Cys Asn

325 330 335

Asn Pro Ile Thr Gly Ser Pro Gly Ala Pro Gly Val Lys Gly Phe Gly

340 345 350

Phe Leu Asp Ser Asp Asn Thr Trp Leu Gly Arg Thr Ile Ser Pro Arg

355 360 365

Ser Arg Ser Gly Phe Glu Met Leu Lys Ile Pro Asn Ala Gly Thr Asp

370 375 380

Pro Asn Ser Arg Ile Thr Glu Arg Gln Glu Ile Val Asp Asn Asn Asn

385 390 395 400

Trp Ser Gly Tyr Ser Gly Ser Phe Ile Asp Tyr Trp Asp Glu Ser Ser

405 410 415

Val Cys Tyr Asn Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro

420 425 430

Glu Glu Ala Lys Tyr Val Trp Trp Thr Ser Asn Ser Leu Val Ala Leu

435 440 445

Cys Gly Ser Pro Ile Ser Val Gly Ser Gly Ser Phe Pro Asp Gly Ala

450 455 460

Gln Ile Gln Tyr Phe Ser

465 470

<210> 313

<211> 523

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MV (ed) -F-delta-30

<400> 313

Met Ser Ile Met Gly Leu Lys Val Asn Val Ser Ala Ile Phe Met Ala

1 5 10 15

Val Leu Leu Thr Leu Gln Thr Pro Thr Gly Gln Ile His Trp Gly Asn

20 25 30

Leu Ser Lys Ile Gly Val Val Gly Ile Gly Ser Ala Ser Tyr Lys Val

35 40 45

Met Thr Arg Ser Ser His Gln Ser Leu Val Ile Lys Leu Met Pro Asn

50 55 60

Ile Thr Leu Leu Asn Asn Cys Thr Arg Val Glu Ile Ala Glu Tyr Arg

65 70 75 80

Arg Leu Leu Arg Thr Val Leu Glu Pro Ile Arg Asp Ala Leu Asn Ala

85 90 95

Met Thr Gln Asn Ile Arg Pro Val Gln Ser Val Ala Ser Ser Arg Arg

100 105 110

His Lys Arg Phe Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val

115 120 125

Ala Thr Ala Ala Gln Ile Thr Ala Gly Ile Ala Leu His Gln Ser Met

130 135 140

Leu Asn Ser Gln Ala Ile Asp Asn Leu Arg Ala Ser Leu Glu Thr Thr

145 150 155 160

Asn Gln Ala Ile Glu Ala Ile Arg Gln Ala Gly Gln Glu Met Ile Leu

165 170 175

Ala Val Gln Gly Val Gln Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser

180 185 190

Met Asn Gln Leu Ser Cys Asp Leu Ile Gly Gln Lys Leu Gly Leu Lys

195 200 205

Leu Leu Arg Tyr Tyr Thr Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu

210 215 220

Arg Asp Pro Ile Ser Ala Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala

225 230 235 240

Leu Gly Gly Asp Ile Asn Lys Val Leu Glu Lys Leu Gly Tyr Ser Gly

245 250 255

Gly Asp Leu Leu Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile

260 265 270

Thr His Val Asp Thr Glu Ser Tyr Phe Ile Val Leu Ser Ile Ala Tyr

275 280 285

Pro Thr Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly

290 295 300

Val Ser Tyr Asn Ile Gly Ser Gln Glu Trp Tyr Thr Thr Val Pro Lys

305 310 315 320

Tyr Val Ala Thr Gln Gly Tyr Leu Ile Ser Asn Phe Asp Glu Ser Ser

325 330 335

Cys Thr Phe Met Pro Glu Gly Thr Val Cys Ser Gln Asn Ala Leu Tyr

340 345 350

Pro Met Ser Pro Leu Leu Gln Glu Cys Leu Arg Gly Ser Thr Lys Ser

355 360 365

Cys Ala Arg Thr Leu Val Ser Gly Ser Phe Gly Asn Arg Phe Ile Leu

370 375 380

Ser Gln Gly Asn Leu Ile Ala Asn Cys Ala Ser Ile Leu Cys Lys Cys

385 390 395 400

Tyr Thr Thr Gly Thr Ile Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr

405 410 415

Tyr Ile Ala Ala Asp His Cys Pro Val Val Glu Val Asn Gly Val Thr

420 425 430

Ile Gln Val Gly Ser Arg Arg Tyr Pro Asp Ala Val Tyr Leu His Arg

435 440 445

Ile Asp Leu Gly Pro Pro Ile Ser Leu Glu Arg Leu Asp Val Gly Thr

450 455 460

Asn Leu Gly Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu

465 470 475 480

Glu Ser Ser Asp Gln Ile Leu Arg Ser Met Lys Gly Leu Ser Ser Thr

485 490 495

Ser Ile Val Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly

500 505 510

Ile Pro Ala Leu Ile Cys Cys Cys Arg Gly Arg

515 520

<210> 314

<211> 599

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MV (ed) -H-delta-18

<400> 314

Met Gly Ser Arg Ile Val Ile Asn Arg Glu His Leu Met Ile Asp Arg

1 5 10 15

Pro Tyr Val Leu Leu Ala Val Leu Phe Val Met Ser Leu Ser Leu Ile

20 25 30

Gly Leu Leu Ala Ile Ala Gly Ile Arg Leu His Arg Ala Ala Ile Tyr

35 40 45

Thr Ala Glu Ile His Lys Ser Leu Ser Thr Asn Leu Asp Val Thr Asn

50 55 60

Ser Ile Glu His Gln Val Lys Asp Val Leu Thr Pro Leu Phe Lys Ile

65 70 75 80

Ile Gly Asp Glu Val Gly Leu Arg Thr Pro Gln Arg Phe Thr Asp Leu

85 90 95

Val Lys Phe Ile Ser Asp Lys Ile Lys Phe Leu Asn Pro Asp Arg Glu

100 105 110

Tyr Asp Phe Arg Asp Leu Thr Trp Cys Ile Asn Pro Pro Glu Arg Ile

115 120 125

Lys Leu Asp Tyr Asp Gln Tyr Cys Ala Asp Val Ala Ala Glu Glu Leu

130 135 140

Met Asn Ala Leu Val Asn Ser Thr Leu Leu Glu Thr Arg Thr Thr Asn

145 150 155 160

Gln Phe Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr Thr Ile

165 170 175

Arg Gly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu Tyr Leu

180 185 190

Ser Arg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser Gln Gly

195 200 205

Met Tyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser Ser Lys

210 215 220

Arg Ser Glu Leu Ser Gln Leu Ser Met Tyr Arg Val Phe Glu Val Gly

225 230 235 240

Val Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met Thr Asn

245 250 255

Tyr Leu Glu Gln Pro Val Ser Asn Asp Leu Ser Asn Cys Met Val Ala

260 265 270

Leu Gly Glu Leu Lys Leu Ala Ala Leu Cys His Gly Glu Asp Ser Ile

275 280 285

Thr Ile Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser Phe Gln Leu Val

290 295 300

Lys Leu Gly Val Trp Lys Ser Pro Thr Asp Met Gln Ser Trp Val Pro

305 310 315 320

Leu Ser Thr Asp Asp Pro Val Ile Asp Arg Leu Tyr Leu Ser Ser His

325 330 335

Arg Gly Val Ile Ala Asp Asn Gln Ala Lys Trp Ala Val Pro Thr Thr

340 345 350

Arg Thr Asp Asp Lys Leu Arg Met Glu Thr Cys Phe Gln Gln Ala Cys

355 360 365

Lys Gly Lys Ile Gln Ala Leu Cys Glu Asn Pro Glu Trp Ala Pro Leu

370 375 380

Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val Leu Ser Val Asp Leu Ser

385 390 395 400

Leu Thr Val Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly Pro Leu

405 410 415

Ile Thr His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His Asn Asn

420 425 430

Val Tyr Trp Leu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu Gly Val

435 440 445

Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro Asn Leu

450 455 460

Phe Thr Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala Pro Thr

465 470 475 480

Tyr Leu Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser Asn Leu

485 490 495

Val Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr Tyr Asp

500 505 510

Thr Ser Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser Pro Gly

515 520 525

Arg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys Gly Val

530 535 540

Pro Ile Glu Leu Gln Val Glu Cys Phe Thr Trp Asp Gln Lys Leu Trp

545 550 555 560

Cys Arg His Phe Cys Val Leu Ala Asp Ser Glu Ser Gly Gly His Ile

565 570 575

Thr His Ser Gly Met Val Gly Met Gly Val Ser Cys Thr Val Thr Arg

580 585 590

Glu Asp Gly Thr Asn Arg Arg

595

<210> 315

<211> 593

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: MV (ed) -H-delta-24

<400> 315

Met Asn Arg Glu His Leu Met Ile Asp Arg Pro Tyr Val Leu Leu Ala

1 5 10 15

Val Leu Phe Val Met Ser Leu Ser Leu Ile Gly Leu Leu Ala Ile Ala

20 25 30

Gly Ile Arg Leu His Arg Ala Ala Ile Tyr Thr Ala Glu Ile His Lys

35 40 45

Ser Leu Ser Thr Asn Leu Asp Val Thr Asn Ser Ile Glu His Gln Val

50 55 60

Lys Asp Val Leu Thr Pro Leu Phe Lys Ile Ile Gly Asp Glu Val Gly

65 70 75 80

Leu Arg Thr Pro Gln Arg Phe Thr Asp Leu Val Lys Phe Ile Ser Asp

85 90 95

Lys Ile Lys Phe Leu Asn Pro Asp Arg Glu Tyr Asp Phe Arg Asp Leu

100 105 110

Thr Trp Cys Ile Asn Pro Pro Glu Arg Ile Lys Leu Asp Tyr Asp Gln

115 120 125

Tyr Cys Ala Asp Val Ala Ala Glu Glu Leu Met Asn Ala Leu Val Asn

130 135 140

Ser Thr Leu Leu Glu Thr Arg Thr Thr Asn Gln Phe Leu Ala Val Ser

145 150 155 160

Lys Gly Asn Cys Ser Gly Pro Thr Thr Ile Arg Gly Gln Phe Ser Asn

165 170 175

Met Ser Leu Ser Leu Leu Asp Leu Tyr Leu Ser Arg Gly Tyr Asn Val

180 185 190

Ser Ser Ile Val Thr Met Thr Ser Gln Gly Met Tyr Gly Gly Thr Tyr

195 200 205

Leu Val Glu Lys Pro Asn Leu Ser Ser Lys Arg Ser Glu Leu Ser Gln

210 215 220

Leu Ser Met Tyr Arg Val Phe Glu Val Gly Val Ile Arg Asn Pro Gly

225 230 235 240

Leu Gly Ala Pro Val Phe His Met Thr Asn Tyr Leu Glu Gln Pro Val

245 250 255

Ser Asn Asp Leu Ser Asn Cys Met Val Ala Leu Gly Glu Leu Lys Leu

260 265 270

Ala Ala Leu Cys His Gly Glu Asp Ser Ile Thr Ile Pro Tyr Gln Gly

275 280 285

Ser Gly Lys Gly Val Ser Phe Gln Leu Val Lys Leu Gly Val Trp Lys

290 295 300

Ser Pro Thr Asp Met Gln Ser Trp Val Pro Leu Ser Thr Asp Asp Pro

305 310 315 320

Val Ile Asp Arg Leu Tyr Leu Ser Ser His Arg Gly Val Ile Ala Asp

325 330 335

Asn Gln Ala Lys Trp Ala Val Pro Thr Thr Arg Thr Asp Asp Lys Leu

340 345 350

Arg Met Glu Thr Cys Phe Gln Gln Ala Cys Lys Gly Lys Ile Gln Ala

355 360 365

Leu Cys Glu Asn Pro Glu Trp Ala Pro Leu Lys Asp Asn Arg Ile Pro

370 375 380

Ser Tyr Gly Val Leu Ser Val Asp Leu Ser Leu Thr Val Glu Leu Lys

385 390 395 400

Ile Lys Ile Ala Ser Gly Phe Gly Pro Leu Ile Thr His Gly Ser Gly

405 410 415

Met Asp Leu Tyr Lys Ser Asn His Asn Asn Val Tyr Trp Leu Thr Ile

420 425 430

Pro Pro Met Lys Asn Leu Ala Leu Gly Val Ile Asn Thr Leu Glu Trp

435 440 445

Ile Pro Arg Phe Lys Val Ser Pro Asn Leu Phe Thr Val Pro Ile Lys

450 455 460

Glu Ala Gly Glu Asp Cys His Ala Pro Thr Tyr Leu Pro Ala Glu Val

465 470 475 480

Asp Gly Asp Val Lys Leu Ser Ser Asn Leu Val Ile Leu Pro Gly Gln

485 490 495

Asp Leu Gln Tyr Val Leu Ala Thr Tyr Asp Thr Ser Arg Val Glu His

500 505 510

Ala Val Val Tyr Tyr Val Tyr Ser Pro Gly Arg Ser Phe Ser Tyr Phe

515 520 525

Tyr Pro Phe Arg Leu Pro Ile Lys Gly Val Pro Ile Glu Leu Gln Val

530 535 540

Glu Cys Phe Thr Trp Asp Gln Lys Leu Trp Cys Arg His Phe Cys Val

545 550 555 560

Leu Ala Asp Ser Glu Ser Gly Gly His Ile Thr His Ser Gly Met Val

565 570 575

Gly Met Gly Val Ser Cys Thr Val Thr Arg Glu Asp Gly Thr Asn Arg

580 585 590

Arg

<210> 316

<211> 477

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: hGH PolyA

<400> 316

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180

acctgtaggg cctgcggggt ctgttgggaa ccaagctgga gtgcagtggc acaatcttgg 240

ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300

ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360

ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420

tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtcctt 477

<210> 317

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: SPA1

<400> 317

aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtg 49

<210> 318

<211> 120

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: SPA2

<400> 318

aataaaatat ctcagagctc tagacatctg tgtgttggtt ttttgtgtgt agtaatgagg 60

atctggagat attgaagtat cttccggacg actaacagct gtcattggcg gatcttaata 120

<210> 319

<211> 295

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: b-globulin poly-A spacer B

<400> 319

atctcaagag tggcagcggt cttgagtggc agcggcggta tacggcagcg gcatgtaact 60

agctcctcag tggcagcgat gaggaggcaa taaaggaaat tgattttcat tgcaatagtg 120

tgttggaatt ttttgtgtct ctcaaggttc tgttaagtaa ctgaacccaa tgtcgttagt 180

gacgcttagc tcttaagagg tcactgacct aacaatctca agagtggcag cggtcttgag 240

tggcagcggc ggtatacggc agcgctatct aagtagtaac aagtagcgtg gggca 295

<210> 320

<211> 512

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: b-globulin poly-A spacer A

<400> 320

acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60

ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120

cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180

gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttaa taaaggaaat 240

tgattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcacacgta gtgggccatc 300

gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttcg atagtggact 360

cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 420

gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 480

gaattttaac aaaatattaa cgcttagaat tt 512

<210> 321

<211> 243

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 250 cHS4 insulator v1

<400> 321

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagcc tgcagacacg tggggggata cggggaaaag 240

ctt 243

<210> 322

<211> 243

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 250 cHS4 insulator v2

<400> 322

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagcg tgcagacacg tggggggata cggggaaaag 240

ctt 243

<210> 323

<211> 650

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 650 cHS4 insulator

<400> 323

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagca tgcagacaca tggggggata cggggaaaaa 240

gctttaggct ctgcatgttt gatggtgtat ggatgcaagc agaaggggtg gaagagcttg 300

cctggagaga tacagctggg tcagtaggac tgggacaggc agctggagaa ttgccatgta 360

gatgttcata caatcgtcaa atcatgaagg ctggaaaagc cctccaagat ccccaagacc 420

aaccccaacc cacccagcgt gcccactggc catgtccctc agtgccacat ccccacagtt 480

cttcatcacc tccagggacg gtgacccccc cacctccgtg ggcagctgtg ccactgcagc 540

accgctcttt ggagaagata aatcttgcta aatccagccc gaccctcccc tggcacaaca 600

taaggccatt atctctcatc caactccagg acggagtcag tgagaatatt 650

<210> 324

<211> 420

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 400 cHS4 insulator

<400> 324

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagca tgcagacaca tggggggata cggggaaaaa 240

gctttaggct gaaagagaga tttagaatga cagaatcata gaacggcctg ggttgcaaag 300

gagcacagtg ctcatccaga tccaaccccc tgctatgtgc agggtcatca accagcagcc 360

caggctgccc agagccacat ccagcctggc cttgaatgcc tgcagggatg gggcatccac 420

<210> 325

<211> 949

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 650 cHS4 spacer and B-globulin poly-A spacer B

<400> 325

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagca tgcagacaca tggggggata cggggaaaaa 240

gctttaggct ctgcatgttt gatggtgtat ggatgcaagc agaaggggtg gaagagcttg 300

cctggagaga tacagctggg tcagtaggac tgggacaggc agctggagaa ttgccatgta 360

gatgttcata caatcgtcaa atcatgaagg ctggaaaagc cctccaagat ccccaagacc 420

aaccccaacc cacccagcgt gcccactggc catgtccctc agtgccacat ccccacagtt 480

cttcatcacc tccagggacg gtgacccccc cacctccgtg ggcagctgtg ccactgcagc 540

accgctcttt ggagaagata aatcttgcta aatccagccc gaccctcccc tggcacaaca 600

taaggccatt atctctcatc caactccagg acggagtcag tgagaatatt gcgatgcccc 660

acgctacttg ttactactta gatagcgctg ccgtataccg ccgctgccac tcaagaccgc 720

tgccactctt gagattgtta ggtcagtgac ctcttaagag ctaagcgtca ctaacgacat 780

tgggttcagt tacttaacag aaccttgaga gacacaaaaa attccaacac actattgcaa 840

tgaaaatcaa tttcctttat tgcctcctca tcgctgccac tgaggagcta gttacatgcc 900

gctgccgtat accgccgctg ccactcaaga ccgctgccac tcttgagat 949

<210> 326

<211> 949

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: b-globulin poly-A spacer B and 650 cHS4 spacers

<400> 326

atctcaagag tggcagcggt cttgagtggc agcggcggta tacggcagcg gcatgtaact 60

agctcctcag tggcagcgat gaggaggcaa taaaggaaat tgattttcat tgcaatagtg 120

tgttggaatt ttttgtgtct ctcaaggttc tgttaagtaa ctgaacccaa tgtcgttagt 180

gacgcttagc tcttaagagg tcactgacct aacaatctca agagtggcag cggtcttgag 240

tggcagcggc ggtatacggc agcgctatct aagtagtaac aagtagcgtg gggcatcgcg 300

agctcacggg gacagccccc ccccaaagcc cccagggatg gtcgtacgtc cctcccccgc 360

tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc 420

cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc 480

tctgaacgct tctcgctgct ctttgagcat gcagacacat ggggggatac ggggaaaaag 540

ctttaggctc tgcatgtttg atggtgtatg gatgcaagca gaaggggtgg aagagcttgc 600

ctggagagat acagctgggt cagtaggact gggacaggca gctggagaat tgccatgtag 660

atgttcatac aatcgtcaaa tcatgaaggc tggaaaagcc ctccaagatc cccaagacca 720

accccaaccc acccagcgtg cccactggcc atgtccctca gtgccacatc cccacagttc 780

ttcatcacct ccagggacgg tgaccccccc acctccgtgg gcagctgtgc cactgcagca 840

ccgctctttg gagaagataa atcttgctaa atccagcccg accctcccct ggcacaacat 900

aaggccatta tctctcatcc aactccagga cggagtcagt gagaatatt 949

<210> 327

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence

<220>

<221> misc_feature

<222> (1)..(3)

<223> nnn, if present, is GCC

<220>

<221> misc_feature

<222> (10)..(10)

<223> n is A or G

<400> 327

nnngccgccn ccatg 15

<210> 328

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is T or U

<220>

<221> misc_feature

<222> (9)..(9)

<223> n if present is G

<400> 328

ccaccangn 9

<210> 329

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence 2

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is T or U

<220>

<221> misc_feature

<222> (9)..(9)

<223> n if present is G

<400> 329

ccgccangn 9

<210> 330

<211> 13

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence 3

<220>

<221> misc_feature

<222> (11)..(11)

<223> n is T or U

<220>

<221> misc_feature

<222> (13)..(13)

<223> n if present is G

<400> 330

gccgccgcca ngn 13

<210> 331

<211> 13

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence

<220>

<221> misc_feature

<222> (11)..(11)

<223> n is T or U

<220>

<221> misc_feature

<222> (13)..(13)

<223> n if present is G

<400> 331

gccgccacca ngn 13

<210> 332

<211> 12

<212> RNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence

<400> 332

gccgccacca ug 12

<210> 333

<211> 28

<212> DNA

<213> mice

<220>

<221> misc_feature

<222> (1)..(28)

<223> SIBR (synthetic inhibitory BIC-derived RNA)

<400> 333

ctggaggctt gctgaaggct gtatgctg 28

<210> 334

<211> 45

<212> DNA

<213> mice

<220>

<221> misc_feature

<222> (1)..(45)

<223> 3 microRNA flanking sequences of miR-155

<400> 334

caggacacaa ggcctgttac tagcactcac atggaacaaa tggcc 45

<210> 335

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: synthetic DNA encoding stems

<400> 335

gttttggcca ctgactgac 19

<210> 336

<211> 511

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: VSV-G envelope protein

<400> 336

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn

20 25 30

Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp

35 40 45

His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met Pro Lys Ser

50 55 60

His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp

65 70 75 80

Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His

85 90 95

Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile

100 105 110

Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln

115 120 125

Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val Ile Val Gln

130 135 140

Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val

145 150 155 160

Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile Cys Pro Thr

165 170 175

Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu

180 185 190

Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp

195 200 205

Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn

210 215 220

Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met Gln Tyr Cys

225 230 235 240

Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala

245 250 255

Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly

260 265 270

Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile

275 280 285

Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp

290 295 300

Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr

305 310 315 320

Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn

325 330 335

Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala

340 345 350

Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr

355 360 365

Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile

370 375 380

Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu

385 390 395 400

Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser

405 410 415

Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala Ser Gln

420 425 430

Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys

435 440 445

Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser

450 455 460

Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu

465 470 475 480

Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys

485 490 495

Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys

500 505 510

<210> 337

<211> 563

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: baboon retrovirus envelope glycoprotein

<400> 337

Met Gly Phe Thr Thr Lys Ile Ile Phe Leu Tyr Asn Leu Val Leu Val

1 5 10 15

Tyr Ala Gly Phe Asp Asp Pro Arg Lys Ala Ile Glu Leu Val Gln Lys

20 25 30

Arg Tyr Gly Arg Pro Cys Asp Cys Ser Gly Gly Gln Val Ser Glu Pro

35 40 45

Pro Ser Asp Arg Val Ser Gln Val Thr Cys Ser Gly Lys Thr Ala Tyr

50 55 60

Leu Met Pro Asp Gln Arg Trp Lys Cys Lys Ser Ile Pro Lys Asp Thr

65 70 75 80

Ser Pro Ser Gly Pro Leu Gln Glu Cys Pro Cys Asn Ser Tyr Gln Ser

85 90 95

Ser Val His Ser Ser Cys Tyr Thr Ser Tyr Gln Gln Cys Arg Ser Gly

100 105 110

Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Thr Gln Thr Gly Gly

115 120 125

Thr Ser Asp Val Gln Val Leu Gly Ser Thr Asn Lys Leu Ile Gln Ser

130 135 140

Pro Cys Asn Gly Ile Lys Gly Gln Ser Ile Cys Trp Ser Thr Thr Ala

145 150 155 160

Pro Ile His Val Ser Asp Gly Gly Gly Pro Leu Asp Thr Thr Arg Ile

165 170 175

Lys Ser Val Gln Arg Lys Leu Glu Glu Ile His Lys Ala Leu Tyr Pro

180 185 190

Glu Leu Gln Tyr His Pro Leu Ala Ile Pro Lys Val Arg Asp Asn Leu

195 200 205

Met Val Asp Ala Gln Thr Leu Asn Ile Leu Asn Ala Thr Tyr Asn Leu

210 215 220

Leu Leu Met Ser Asn Thr Ser Leu Val Asp Asp Cys Trp Leu Cys Leu

225 230 235 240

Lys Leu Gly Pro Pro Thr Pro Leu Ala Ile Pro Asn Phe Leu Leu Ser

245 250 255

Tyr Val Thr Arg Ser Ser Asp Asn Ile Ser Cys Leu Ile Ile Pro Pro

260 265 270

Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Phe Ser

275 280 285

Pro Ser Tyr Asn Ser Thr Glu Glu Ile Asp Leu Gly His Val Ala Phe

290 295 300

Ser Asn Cys Thr Ser Ile Thr Asn Val Thr Gly Pro Ile Cys Ala Val

305 310 315 320

Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr Leu

325 330 335

Pro Thr Asn Trp Thr Gly Leu Cys Val Leu Ala Thr Leu Leu Pro Asp

340 345 350

Ile Asp Ile Ile Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile Asp

355 360 365

His Phe Ile Tyr Arg Pro Lys Arg Ala Ile Gln Phe Ile Pro Leu Leu

370 375 380

Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly Leu

385 390 395 400

Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser Asn Gln Leu Ile Ser

405 410 415

Asp Val Gln Ile Leu Ser Ser Thr Ile Gln Asp Leu Gln Asp Gln Val

420 425 430

Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu

435 440 445

Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala Leu Gln Glu Lys Cys

450 455 460

Cys Phe Tyr Val Asn Lys Ser Gly Ile Val Arg Asp Lys Ile Lys Thr

465 470 475 480

Leu Gln Glu Glu Leu Glu Arg Arg Arg Lys Asp Leu Ala Ser Asn Pro

485 490 495

Leu Trp Thr Gly Leu Gln Gly Leu Leu Pro Tyr Leu Leu Pro Phe Leu

500 505 510

Gly Pro Leu Leu Thr Leu Leu Leu Leu Leu Thr Ile Gly Pro Cys Ile

515 520 525

Phe Asn Arg Leu Thr Ala Phe Ile Asn Asp Lys Leu Asn Ile Ile His

530 535 540

Ala Met Val Leu Thr Gln Gln Tyr Gln Val Leu Arg Thr Asp Glu Glu

545 550 555 560

Ala Gln Asp

<210> 338

<211> 654

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: muLV envelope protein

<400> 338

Met Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn Pro

1 5 10 15

Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Met Ala

20 25 30

Glu Ser Pro His Gln Val Phe Asn Val Thr Trp Arg Val Thr Asn Leu

35 40 45

Met Thr Gly Arg Thr Ala Asn Ala Thr Ser Leu Leu Gly Thr Val Gln

50 55 60

Asp Ala Phe Pro Lys Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly Glu

65 70 75 80

Glu Trp Asp Pro Ser Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys Lys

85 90 95

Tyr Pro Ala Gly Arg Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val Cys

100 105 110

Pro Gly His Thr Val Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly Tyr

115 120 125

Cys Gly Lys Trp Gly Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys Pro

130 135 140

Thr Ser Ser Trp Asp Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro Trp

145 150 155 160

Asp Thr Gly Cys Ser Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu Ser

165 170 175

Lys Val Ser Asn Ser Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys Asn

180 185 190

Pro Leu Val Leu Glu Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp Asp

195 200 205

Gly Pro Lys Ser Trp Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp Pro

210 215 220

Ile Thr Met Phe Ser Leu Thr Arg Gln Val Leu Asn Val Gly Pro Arg

225 230 235 240

Val Pro Ile Gly Pro Asn Pro Val Leu Pro Asp Gln Arg Leu Pro Ser

245 250 255

Ser Pro Ile Glu Ile Val Pro Ala Pro Gln Pro Pro Ser Pro Leu Asn

260 265 270

Thr Ser Tyr Pro Pro Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro Thr

275 280 285

Ser Pro Ser Val Pro Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu Leu

290 295 300

Ala Leu Val Lys Gly Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro Asp

305 310 315 320

Lys Thr Gln Glu Cys Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr Tyr

325 330 335

Glu Gly Val Ala Val Val Gly Thr Tyr Thr Asn His Ser Thr Ala Pro

340 345 350

Ala Asn Cys Thr Ala Thr Ser Gln His Lys Leu Thr Leu Ser Glu Val

355 360 365

Thr Gly Gln Gly Leu Cys Met Gly Ala Val Pro Lys Thr His Gln Ala

370 375 380

Leu Cys Asn Thr Thr Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu Ala

385 390 395 400

Ala Pro Ala Gly Thr Met Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys

405 410 415

Leu Ser Thr Thr Val Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu Val

420 425 430

Glu Leu Trp Pro Arg Val Ile Tyr His Ser Pro Asp Tyr Met Tyr Gly

435 440 445

Gln Leu Glu Gln Arg Thr Lys Tyr Lys Arg Glu Pro Val Ser Leu Thr

450 455 460

Leu Ala Leu Leu Leu Gly Gly Leu Thr Met Gly Gly Ile Ala Ala Gly

465 470 475 480

Ile Gly Thr Gly Thr Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu Gln

485 490 495

Leu His Ala Ala Ile Gln Thr Asp Leu Asn Glu Val Glu Lys Ser Ile

500 505 510

Thr Asn Leu Glu Lys Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln

515 520 525

Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys

530 535 540

Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp His Thr Gly Leu

545 550 555 560

Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln

565 570 575

Lys Leu Phe Glu Thr Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg

580 585 590

Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile

595 600 605

Val Leu Leu Leu Ile Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu

610 615 620

Val Gln Phe Val Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu

625 630 635 640

Thr Gln Gln Tyr His Gln Leu Lys Pro Ile Glu Tyr Glu Pro

645 650

<210> 339

<211> 545

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: baboon retrovirus envelope glycoprotein-delta-R (HA)

<400> 339

Met Gly Phe Thr Thr Lys Ile Ile Phe Leu Tyr Asn Leu Val Leu Val

1 5 10 15

Tyr Ala Gly Phe Asp Asp Pro Arg Lys Ala Ile Glu Leu Val Gln Lys

20 25 30

Arg Tyr Gly Arg Pro Cys Asp Cys Ser Gly Gly Gln Val Ser Glu Pro

35 40 45

Pro Ser Asp Arg Val Ser Gln Val Thr Cys Ser Gly Lys Thr Ala Tyr

50 55 60

Leu Met Pro Asp Gln Arg Trp Lys Cys Lys Ser Ile Pro Lys Asp Thr

65 70 75 80

Ser Pro Ser Gly Pro Leu Gln Glu Cys Pro Cys Asn Ser Tyr Gln Ser

85 90 95

Ser Val His Ser Ser Cys Tyr Thr Ser Tyr Gln Gln Cys Arg Ser Gly

100 105 110

Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Thr Gln Thr Gly Gly

115 120 125

Thr Ser Asp Val Gln Val Leu Gly Ser Thr Asn Lys Leu Ile Gln Ser

130 135 140

Pro Cys Asn Gly Ile Lys Gly Gln Ser Ile Cys Trp Ser Thr Thr Ala

145 150 155 160

Pro Ile His Val Ser Asp Gly Gly Gly Pro Leu Asp Thr Thr Arg Ile

165 170 175

Lys Ser Val Gln Arg Lys Leu Glu Glu Ile His Lys Ala Leu Tyr Pro

180 185 190

Glu Leu Gln Tyr His Pro Leu Ala Ile Pro Lys Val Arg Asp Asn Leu

195 200 205

Met Val Asp Ala Gln Thr Leu Asn Ile Leu Asn Ala Thr Tyr Asn Leu

210 215 220

Leu Leu Met Ser Asn Thr Ser Leu Val Asp Asp Cys Trp Leu Cys Leu

225 230 235 240

Lys Leu Gly Pro Pro Thr Pro Leu Ala Ile Pro Asn Phe Leu Leu Ser

245 250 255

Tyr Val Thr Arg Ser Ser Asp Asn Ile Ser Cys Leu Ile Ile Pro Pro

260 265 270

Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Phe Ser

275 280 285

Pro Ser Tyr Asn Ser Thr Glu Glu Ile Asp Leu Gly His Val Ala Phe

290 295 300

Ser Asn Cys Thr Ser Ile Thr Asn Val Thr Gly Pro Ile Cys Ala Val

305 310 315 320

Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr Leu

325 330 335

Pro Thr Asn Trp Thr Gly Leu Cys Val Leu Ala Thr Leu Leu Pro Asp

340 345 350

Ile Asp Ile Ile Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile Asp

355 360 365

His Phe Ile Tyr Arg Pro Lys Arg Ala Ile Gln Phe Ile Pro Leu Leu

370 375 380

Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly Leu

385 390 395 400

Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser Asn Gln Leu Ile Ser

405 410 415

Asp Val Gln Ile Leu Ser Ser Thr Ile Gln Asp Leu Gln Asp Gln Val

420 425 430

Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu

435 440 445

Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala Leu Gln Glu Lys Cys

450 455 460

Cys Phe Tyr Val Asn Lys Ser Gly Ile Val Arg Asp Lys Ile Lys Thr

465 470 475 480

Leu Gln Glu Glu Leu Glu Arg Arg Arg Lys Asp Leu Ala Ser Asn Pro

485 490 495

Leu Trp Thr Gly Leu Gln Gly Leu Leu Pro Tyr Leu Leu Pro Phe Leu

500 505 510

Gly Pro Leu Leu Thr Leu Leu Leu Leu Leu Thr Ile Gly Pro Cys Ile

515 520 525

Phe Asn Arg Leu Thr Ala Phe Ile Asn Asp Lys Leu Asn Ile Ile His

530 535 540

Ala

545

<210> 340

<211> 546

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: baboon retrovirus envelope glycoprotein-delta-R (HAM)

<400> 340

Met Gly Phe Thr Thr Lys Ile Ile Phe Leu Tyr Asn Leu Val Leu Val

1 5 10 15

Tyr Ala Gly Phe Asp Asp Pro Arg Lys Ala Ile Glu Leu Val Gln Lys

20 25 30

Arg Tyr Gly Arg Pro Cys Asp Cys Ser Gly Gly Gln Val Ser Glu Pro

35 40 45

Pro Ser Asp Arg Val Ser Gln Val Thr Cys Ser Gly Lys Thr Ala Tyr

50 55 60

Leu Met Pro Asp Gln Arg Trp Lys Cys Lys Ser Ile Pro Lys Asp Thr

65 70 75 80

Ser Pro Ser Gly Pro Leu Gln Glu Cys Pro Cys Asn Ser Tyr Gln Ser

85 90 95

Ser Val His Ser Ser Cys Tyr Thr Ser Tyr Gln Gln Cys Arg Ser Gly

100 105 110

Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Thr Gln Thr Gly Gly

115 120 125

Thr Ser Asp Val Gln Val Leu Gly Ser Thr Asn Lys Leu Ile Gln Ser

130 135 140

Pro Cys Asn Gly Ile Lys Gly Gln Ser Ile Cys Trp Ser Thr Thr Ala

145 150 155 160

Pro Ile His Val Ser Asp Gly Gly Gly Pro Leu Asp Thr Thr Arg Ile

165 170 175

Lys Ser Val Gln Arg Lys Leu Glu Glu Ile His Lys Ala Leu Tyr Pro

180 185 190

Glu Leu Gln Tyr His Pro Leu Ala Ile Pro Lys Val Arg Asp Asn Leu

195 200 205

Met Val Asp Ala Gln Thr Leu Asn Ile Leu Asn Ala Thr Tyr Asn Leu

210 215 220

Leu Leu Met Ser Asn Thr Ser Leu Val Asp Asp Cys Trp Leu Cys Leu

225 230 235 240

Lys Leu Gly Pro Pro Thr Pro Leu Ala Ile Pro Asn Phe Leu Leu Ser

245 250 255

Tyr Val Thr Arg Ser Ser Asp Asn Ile Ser Cys Leu Ile Ile Pro Pro

260 265 270

Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Phe Ser

275 280 285

Pro Ser Tyr Asn Ser Thr Glu Glu Ile Asp Leu Gly His Val Ala Phe

290 295 300

Ser Asn Cys Thr Ser Ile Thr Asn Val Thr Gly Pro Ile Cys Ala Val

305 310 315 320

Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr Leu

325 330 335

Pro Thr Asn Trp Thr Gly Leu Cys Val Leu Ala Thr Leu Leu Pro Asp

340 345 350

Ile Asp Ile Ile Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile Asp

355 360 365

His Phe Ile Tyr Arg Pro Lys Arg Ala Ile Gln Phe Ile Pro Leu Leu

370 375 380

Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly Leu

385 390 395 400

Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser Asn Gln Leu Ile Ser

405 410 415

Asp Val Gln Ile Leu Ser Ser Thr Ile Gln Asp Leu Gln Asp Gln Val

420 425 430

Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu

435 440 445

Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala Leu Gln Glu Lys Cys

450 455 460

Cys Phe Tyr Val Asn Lys Ser Gly Ile Val Arg Asp Lys Ile Lys Thr

465 470 475 480

Leu Gln Glu Glu Leu Glu Arg Arg Arg Lys Asp Leu Ala Ser Asn Pro

485 490 495

Leu Trp Thr Gly Leu Gln Gly Leu Leu Pro Tyr Leu Leu Pro Phe Leu

500 505 510

Gly Pro Leu Leu Thr Leu Leu Leu Leu Leu Thr Ile Gly Pro Cys Ile

515 520 525

Phe Asn Arg Leu Thr Ala Phe Ile Asn Asp Lys Leu Asn Ile Ile His

530 535 540

Ala Met

545

<210> 341

<211> 905

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: fusion of anti-CD 3 scFV from UCHT1 with MuLV envelope protein

<400> 341

Met Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn Pro

1 5 10 15

Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Asp Ile

20 25 30

Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

35 40 45

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn

50 55 60

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr

65 70 75 80

Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

85 90 95

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

100 105 110

Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe

115 120 125

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly

195 200 205

Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val

210 215 220

Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp

245 250 255

Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

260 265 270

Ser Ser Ala Ala Ala Ile Glu Gly Arg Met Ala Glu Ser Pro His Gln

275 280 285

Val Phe Asn Val Thr Trp Arg Val Thr Asn Leu Met Thr Gly Arg Thr

290 295 300

Ala Asn Ala Thr Ser Leu Leu Gly Thr Val Gln Asp Ala Phe Pro Lys

305 310 315 320

Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly Glu Glu Trp Asp Pro Ser

325 330 335

Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys Lys Tyr Pro Ala Gly Arg

340 345 350

Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val Cys Pro Gly His Thr Val

355 360 365

Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly Tyr Cys Gly Lys Trp Gly

370 375 380

Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys Pro Thr Ser Ser Trp Asp

385 390 395 400

Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro Trp Asp Thr Gly Cys Ser

405 410 415

Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu Ser Lys Val Ser Asn Ser

420 425 430

Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys Asn Pro Leu Val Leu Glu

435 440 445

Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp Asp Gly Pro Lys Ser Trp

450 455 460

Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp Pro Ile Thr Met Phe Ser

465 470 475 480

Leu Thr Arg Gln Val Leu Asn Val Gly Pro Arg Val Pro Ile Gly Pro

485 490 495

Asn Pro Val Leu Pro Asp Gln Arg Leu Pro Ser Ser Pro Ile Glu Ile

500 505 510

Val Pro Ala Pro Gln Pro Pro Ser Pro Leu Asn Thr Ser Tyr Pro Pro

515 520 525

Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro Thr Ser Pro Ser Val Pro

530 535 540

Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu Leu Ala Leu Val Lys Gly

545 550 555 560

Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro Asp Lys Thr Gln Glu Cys

565 570 575

Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr Tyr Glu Gly Val Ala Val

580 585 590

Val Gly Thr Tyr Thr Asn His Ser Thr Ala Pro Ala Asn Cys Thr Ala

595 600 605

Thr Ser Gln His Lys Leu Thr Leu Ser Glu Val Thr Gly Gln Gly Leu

610 615 620

Cys Met Gly Ala Val Pro Lys Thr His Gln Ala Leu Cys Asn Thr Thr

625 630 635 640

Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu Ala Ala Pro Ala Gly Thr

645 650 655

Met Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Leu Ser Thr Thr Val

660 665 670

Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu Val Glu Leu Trp Pro Arg

675 680 685

Val Ile Tyr His Ser Pro Asp Tyr Met Tyr Gly Gln Leu Glu Gln Arg

690 695 700

Thr Lys Tyr Lys Arg Glu Pro Val Ser Leu Thr Leu Ala Leu Leu Leu

705 710 715 720

Gly Gly Leu Thr Met Gly Gly Ile Ala Ala Gly Ile Gly Thr Gly Thr

725 730 735

Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu Gln Leu His Ala Ala Ile

740 745 750

Gln Thr Asp Leu Asn Glu Val Glu Lys Ser Ile Thr Asn Leu Glu Lys

755 760 765

Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln Asn Arg Arg Gly Leu

770 775 780

Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys Ala Ala Leu Lys Glu

785 790 795 800

Glu Cys Cys Phe Tyr Ala Asp His Thr Gly Leu Val Arg Asp Ser Met

805 810 815

Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln Lys Leu Phe Glu Thr

820 825 830

Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg Ser Pro Trp Phe Thr

835 840 845

Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile Val Leu Leu Leu Ile

850 855 860

Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu Val Gln Phe Val Lys

865 870 875 880

Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu Thr Gln Gln Tyr His

885 890 895

Gln Leu Lys Pro Ile Glu Tyr Glu Pro

900 905

<210> 342

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: kozak sequence

<400> 342

gccgccacc 9

<210> 343

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: triple termination sequences

<400> 343

taatagtga 9

<210> 344

<211> 191

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: WPRE (Wireless power supply)

<400> 344

gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg 60

ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 120

gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 180

ctccccgcct g 191

<210> 345

<211> 654

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: muLVSUx

<400> 345

Met Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn Pro

1 5 10 15

Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Met Ala

20 25 30

Glu Ser Pro His Gln Val Phe Asn Val Thr Trp Arg Val Thr Asn Leu

35 40 45

Met Thr Gly Arg Thr Ala Asn Ala Thr Ser Leu Leu Gly Thr Val Gln

50 55 60

Asp Ala Phe Pro Lys Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly Glu

65 70 75 80

Glu Trp Asp Pro Ser Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys Lys

85 90 95

Tyr Pro Ala Gly Arg Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val Cys

100 105 110

Pro Gly His Thr Val Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly Tyr

115 120 125

Cys Gly Lys Trp Gly Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys Pro

130 135 140

Thr Ser Ser Trp Asp Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro Trp

145 150 155 160

Asp Thr Gly Cys Ser Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu Ser

165 170 175

Lys Val Ser Asn Ser Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys Asn

180 185 190

Pro Leu Val Leu Glu Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp Asp

195 200 205

Gly Pro Lys Ser Trp Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp Pro

210 215 220

Ile Thr Met Phe Ser Leu Thr Arg Gln Val Leu Asn Val Gly Pro Arg

225 230 235 240

Val Pro Ile Gly Pro Asn Pro Val Leu Pro Asp Gln Arg Leu Pro Ser

245 250 255

Ser Pro Ile Glu Ile Val Pro Ala Pro Gln Pro Pro Ser Pro Leu Asn

260 265 270

Thr Ser Tyr Pro Pro Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro Thr

275 280 285

Ser Pro Ser Val Pro Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu Leu

290 295 300

Ala Leu Val Lys Gly Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro Asp

305 310 315 320

Lys Thr Gln Glu Cys Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr Tyr

325 330 335

Glu Gly Val Ala Val Val Gly Thr Tyr Thr Asn His Ser Thr Ala Pro

340 345 350

Ala Asn Cys Thr Ala Thr Ser Gln His Lys Leu Thr Leu Ser Glu Val

355 360 365

Thr Gly Gln Gly Leu Cys Met Gly Ala Val Pro Lys Thr His Gln Ala

370 375 380

Leu Cys Asn Thr Thr Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu Ala

385 390 395 400

Ala Pro Ala Gly Thr Met Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys

405 410 415

Leu Ser Thr Thr Val Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu Val

420 425 430

Glu Leu Trp Pro Arg Val Ile Tyr His Ser Pro Asp Tyr Met Tyr Gly

435 440 445

Gln Leu Glu Gln Arg Thr Ile Glu Gly Arg Glu Pro Val Ser Leu Thr

450 455 460

Leu Ala Leu Leu Leu Gly Gly Leu Thr Met Gly Gly Ile Ala Ala Gly

465 470 475 480

Ile Gly Thr Gly Thr Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu Gln

485 490 495

Leu His Ala Ala Ile Gln Thr Asp Leu Asn Glu Val Glu Lys Ser Ile

500 505 510

Thr Asn Leu Glu Lys Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln

515 520 525

Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys

530 535 540

Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp His Thr Gly Leu

545 550 555 560

Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln

565 570 575

Lys Leu Phe Glu Thr Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg

580 585 590

Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile

595 600 605

Val Leu Leu Leu Ile Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu

610 615 620

Val Gln Phe Val Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu

625 630 635 640

Thr Gln Gln Tyr His Gln Leu Lys Pro Ile Glu Tyr Glu Pro

645 650

<210> 346

<211> 905

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: UMuLVSUx

<400> 346

Met Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn Pro

1 5 10 15

Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Asp Ile

20 25 30

Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

35 40 45

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn

50 55 60

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr

65 70 75 80

Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

85 90 95

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

100 105 110

Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe

115 120 125

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly

195 200 205

Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val

210 215 220

Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp

245 250 255

Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

260 265 270

Ser Ser Ala Ala Ala Ile Glu Gly Arg Met Ala Glu Ser Pro His Gln

275 280 285

Val Phe Asn Val Thr Trp Arg Val Thr Asn Leu Met Thr Gly Arg Thr

290 295 300

Ala Asn Ala Thr Ser Leu Leu Gly Thr Val Gln Asp Ala Phe Pro Lys

305 310 315 320

Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly Glu Glu Trp Asp Pro Ser

325 330 335

Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys Lys Tyr Pro Ala Gly Arg

340 345 350

Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val Cys Pro Gly His Thr Val

355 360 365

Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly Tyr Cys Gly Lys Trp Gly

370 375 380

Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys Pro Thr Ser Ser Trp Asp

385 390 395 400

Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro Trp Asp Thr Gly Cys Ser

405 410 415

Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu Ser Lys Val Ser Asn Ser

420 425 430

Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys Asn Pro Leu Val Leu Glu

435 440 445

Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp Asp Gly Pro Lys Ser Trp

450 455 460

Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp Pro Ile Thr Met Phe Ser

465 470 475 480

Leu Thr Arg Gln Val Leu Asn Val Gly Pro Arg Val Pro Ile Gly Pro

485 490 495

Asn Pro Val Leu Pro Asp Gln Arg Leu Pro Ser Ser Pro Ile Glu Ile

500 505 510

Val Pro Ala Pro Gln Pro Pro Ser Pro Leu Asn Thr Ser Tyr Pro Pro

515 520 525

Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro Thr Ser Pro Ser Val Pro

530 535 540

Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu Leu Ala Leu Val Lys Gly

545 550 555 560

Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro Asp Lys Thr Gln Glu Cys

565 570 575

Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr Tyr Glu Gly Val Ala Val

580 585 590

Val Gly Thr Tyr Thr Asn His Ser Thr Ala Pro Ala Asn Cys Thr Ala

595 600 605

Thr Ser Gln His Lys Leu Thr Leu Ser Glu Val Thr Gly Gln Gly Leu

610 615 620

Cys Met Gly Ala Val Pro Lys Thr His Gln Ala Leu Cys Asn Thr Thr

625 630 635 640

Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu Ala Ala Pro Ala Gly Thr

645 650 655

Met Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Leu Ser Thr Thr Val

660 665 670

Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu Val Glu Leu Trp Pro Arg

675 680 685

Val Ile Tyr His Ser Pro Asp Tyr Met Tyr Gly Gln Leu Glu Gln Arg

690 695 700

Thr Ile Glu Gly Arg Glu Pro Val Ser Leu Thr Leu Ala Leu Leu Leu

705 710 715 720

Gly Gly Leu Thr Met Gly Gly Ile Ala Ala Gly Ile Gly Thr Gly Thr

725 730 735

Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu Gln Leu His Ala Ala Ile

740 745 750

Gln Thr Asp Leu Asn Glu Val Glu Lys Ser Ile Thr Asn Leu Glu Lys

755 760 765

Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln Asn Arg Arg Gly Leu

770 775 780

Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys Ala Ala Leu Lys Glu

785 790 795 800

Glu Cys Cys Phe Tyr Ala Asp His Thr Gly Leu Val Arg Asp Ser Met

805 810 815

Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln Lys Leu Phe Glu Thr

820 825 830

Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg Ser Pro Trp Phe Thr

835 840 845

Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile Val Leu Leu Leu Ile

850 855 860

Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu Val Gln Phe Val Lys

865 870 875 880

Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu Thr Gln Gln Tyr His

885 890 895

Gln Leu Lys Pro Ile Glu Tyr Glu Pro

900 905

<210> 347

<211> 770

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: UCHT1- (G4S) 3-VSVG

<400> 347

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

20 25 30

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

35 40 45

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

50 55 60

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

100 105 110

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr

180 185 190

Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile

195 200 205

Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr

225 230 235 240

Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

260 265 270

Gly Gly Ser Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn

275 280 285

Trp Lys Asn Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp

290 295 300

Leu Asn Trp His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met

305 310 315 320

Pro Lys Ser His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala

325 330 335

Ser Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr

340 345 350

Ile Thr His Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys

355 360 365

Glu Ser Ile Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe

370 375 380

Pro Pro Gln Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val

385 390 395 400

Ile Val Gln Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly

405 410 415

Glu Trp Val Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile

420 425 430

Cys Pro Thr Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val

435 440 445

Lys Gly Leu Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe

450 455 460

Ser Glu Asp Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe

465 470 475 480

Arg Ser Asn Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met

485 490 495

Gln Tyr Cys Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe

500 505 510

Glu Met Ala Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys

515 520 525

Pro Glu Gly Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val

530 535 540

Ser Leu Ile Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln

545 550 555 560

Glu Thr Trp Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp

565 570 575

Leu Ser Tyr Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr

580 585 590

Ile Ile Asn Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val

595 600 605

Asp Ile Ala Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly

610 615 620

Thr Thr Thr Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp

625 630 635 640

Val Glu Ile Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys

645 650 655

Phe Pro Leu Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His

660 665 670

Leu Ser Ser Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala

675 680 685

Ala Ser Gln Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly

690 695 700

Leu Ser Lys Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp

705 710 715 720

Lys Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly

725 730 735

Leu Phe Leu Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys

740 745 750

His Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu

755 760 765

Gly Lys

770

<210> 348

<211> 767

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: UCHT 1-hinge-VSVG

<400> 348

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

20 25 30

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

35 40 45

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

50 55 60

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

100 105 110

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr

180 185 190

Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile

195 200 205

Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr

225 230 235 240

Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

260 265 270

Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn

275 280 285

Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp

290 295 300

His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met Pro Lys Ser

305 310 315 320

His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp

325 330 335

Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His

340 345 350

Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile

355 360 365

Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln

370 375 380

Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val Ile Val Gln

385 390 395 400

Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val

405 410 415

Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile Cys Pro Thr

420 425 430

Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu

435 440 445

Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp

450 455 460

Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn

465 470 475 480

Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met Gln Tyr Cys

485 490 495

Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala

500 505 510

Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly

515 520 525

Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile

530 535 540

Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp

545 550 555 560

Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr

565 570 575

Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn

580 585 590

Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala

595 600 605

Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr

610 615 620

Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile

625 630 635 640

Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu

645 650 655

Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser

660 665 670

Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala Ser Gln

675 680 685

Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys

690 695 700

Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser

705 710 715 720

Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu

725 730 735

Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys

740 745 750

Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys

755 760 765

<210> 349

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> connector

<400> 349

Gly Ser Thr Ser Gly Ser

1 5

<210> 350

<211> 1179

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: EF1a

<400> 350

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360

gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420

cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480

ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540

caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600

gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660

gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720

ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780

gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840

acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900

tcctcagccg tcgcttcatg tgactccact gagtaccggg cgccgtccag gcacctcgat 960

tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020

gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080

ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140

gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1179

<210> 351

<211> 511

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: PGK

<400> 351

ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60

tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120

cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180

tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240

ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300

gcgccgaccg cgatgggctg tggccaatag cggctgctca gcggggcgcg ccgagagcag 360

cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420

gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480

cgttgaccga atcaccgacc tctctcccca g 511

<210> 352

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 1x NFAT

<400> 352

ggaggaaaaa ctgtttcata cagaaggcgt 30

<210> 353

<211> 372

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 6X NFAT

<400> 353

ataagcttga tatcgaatta ggaggaaaaa ctgtttcata cagaaggcgt caattaggag 60

gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa aactgtttca tacagaaggc 120

gtcaattggt cccatcgaat taggaggaaa aactgtttca tacagaaggc gtcaattagg 180

aggaaaaact gtttcataca gaaggcgtca attaggagga aaaactgttt catacagaag 240

gcgtcaattg gtcccgggac attttgacac ccccataata tttttccaga attaacagta 300

taaattgcat ctcttgttca agagttccct atcactctct taaatcacta ctcatagtaa 360

cctcaactcc tg 372

<210> 354

<211> 114

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: minIL2P

<400> 354

cattttgaca cccccataat atttttccag aattaacagt ataaattgca tctcttgttc 60

aagagttccc tatcactctc ttaaatcact actcatagta acctcaactc ctga 114

<210> 355

<211> 373

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 6X NFAT-minIL2P

<400> 355

ataagcttga tatcgaatta ggaggaaaaa ctgtttcata cagaaggcgt caattaggag 60

gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa aactgtttca tacagaaggc 120

gtcaattggt cccatcgaat taggaggaaa aactgtttca tacagaaggc gtcaattagg 180

aggaaaaact gtttcataca gaaggcgtca attaggagga aaaactgttt catacagaag 240

gcgtcaattg gtcccgggac attttgacac ccccataata tttttccaga attaacagta 300

taaattgcat ctcttgttca agagttccct atcactctct taaatcacta ctcatagtaa 360

cctcaactcc tga 373

<210> 356

<211> 295

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: b-globulin poly-A spacer B

<400> 356

atctcaagag tggcagcggt cttgagtggc agcggcggta tacggcagcg gcatgtaact 60

agctcctcag tggcagcgat gaggaggcaa taaaggaaat tgattttcat tgcaatagtg 120

tgttggaatt ttttgtgtct ctcaaggttc tgttaagtaa ctgaacccaa tgtcgttagt 180

gacgcttagc tcttaagagg tcactgacct aacaatctca agagtggcag cggtcttgag 240

tggcagcggc ggtatacggc agcgctatct aagtagtaac aagtagcgtg gggca 295

<210> 357

<211> 512

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: b-globulin poly-A spacer A

<400> 357

acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60

ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120

cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180

gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttaa taaaggaaat 240

tgattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcacacgta gtgggccatc 300

gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttcg atagtggact 360

cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 420

gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 480

gaattttaac aaaatattaa cgcttagaat tt 512

<210> 358

<211> 243

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 250 cHS4 insulator v1

<400> 358

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagcc tgcagacacg tggggggata cggggaaaag 240

ctt 243

<210> 359

<211> 243

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 250 cHS4 insulator v2

<400> 359

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagcg tgcagacacg tggggggata cggggaaaag 240

ctt 243

<210> 360

<211> 650

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 650 cHS4 insulator

<400> 360

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagca tgcagacaca tggggggata cggggaaaaa 240

gctttaggct ctgcatgttt gatggtgtat ggatgcaagc agaaggggtg gaagagcttg 300

cctggagaga tacagctggg tcagtaggac tgggacaggc agctggagaa ttgccatgta 360

gatgttcata caatcgtcaa atcatgaagg ctggaaaagc cctccaagat ccccaagacc 420

aaccccaacc cacccagcgt gcccactggc catgtccctc agtgccacat ccccacagtt 480

cttcatcacc tccagggacg gtgacccccc cacctccgtg ggcagctgtg ccactgcagc 540

accgctcttt ggagaagata aatcttgcta aatccagccc gaccctcccc tggcacaaca 600

taaggccatt atctctcatc caactccagg acggagtcag tgagaatatt 650

<210> 361

<211> 420

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 400 cHS4 insulator

<400> 361

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagca tgcagacaca tggggggata cggggaaaaa 240

gctttaggct gaaagagaga tttagaatga cagaatcata gaacggcctg ggttgcaaag 300

gagcacagtg ctcatccaga tccaaccccc tgctatgtgc agggtcatca accagcagcc 360

caggctgccc agagccacat ccagcctggc cttgaatgcc tgcagggatg gggcatccac 420

<210> 362

<211> 949

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: 650 cHS4 spacer and B-globulin poly-A spacer B

<400> 362

gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg 60

ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc 120

ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 180

ctctgaacgc ttctcgctgc tctttgagca tgcagacaca tggggggata cggggaaaaa 240

gctttaggct ctgcatgttt gatggtgtat ggatgcaagc agaaggggtg gaagagcttg 300

cctggagaga tacagctggg tcagtaggac tgggacaggc agctggagaa ttgccatgta 360

gatgttcata caatcgtcaa atcatgaagg ctggaaaagc cctccaagat ccccaagacc 420

aaccccaacc cacccagcgt gcccactggc catgtccctc agtgccacat ccccacagtt 480

cttcatcacc tccagggacg gtgacccccc cacctccgtg ggcagctgtg ccactgcagc 540

accgctcttt ggagaagata aatcttgcta aatccagccc gaccctcccc tggcacaaca 600

taaggccatt atctctcatc caactccagg acggagtcag tgagaatatt gcgatgcccc 660

acgctacttg ttactactta gatagcgctg ccgtataccg ccgctgccac tcaagaccgc 720

tgccactctt gagattgtta ggtcagtgac ctcttaagag ctaagcgtca ctaacgacat 780

tgggttcagt tacttaacag aaccttgaga gacacaaaaa attccaacac actattgcaa 840

tgaaaatcaa tttcctttat tgcctcctca tcgctgccac tgaggagcta gttacatgcc 900

gctgccgtat accgccgctg ccactcaaga ccgctgccac tcttgagat 949

<210> 363

<211> 949

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: b-globulin poly-A spacer B and 650 cHS4 spacers

<400> 363

atctcaagag tggcagcggt cttgagtggc agcggcggta tacggcagcg gcatgtaact 60

agctcctcag tggcagcgat gaggaggcaa taaaggaaat tgattttcat tgcaatagtg 120

tgttggaatt ttttgtgtct ctcaaggttc tgttaagtaa ctgaacccaa tgtcgttagt 180

gacgcttagc tcttaagagg tcactgacct aacaatctca agagtggcag cggtcttgag 240

tggcagcggc ggtatacggc agcgctatct aagtagtaac aagtagcgtg gggcatcgcg 300

agctcacggg gacagccccc ccccaaagcc cccagggatg gtcgtacgtc cctcccccgc 360

tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc 420

cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc 480

tctgaacgct tctcgctgct ctttgagcat gcagacacat ggggggatac ggggaaaaag 540

ctttaggctc tgcatgtttg atggtgtatg gatgcaagca gaaggggtgg aagagcttgc 600

ctggagagat acagctgggt cagtaggact gggacaggca gctggagaat tgccatgtag 660

atgttcatac aatcgtcaaa tcatgaaggc tggaaaagcc ctccaagatc cccaagacca 720

accccaaccc acccagcgtg cccactggcc atgtccctca gtgccacatc cccacagttc 780

ttcatcacct ccagggacgg tgaccccccc acctccgtgg gcagctgtgc cactgcagca 840

ccgctctttg gagaagataa atcttgctaa atccagcccg accctcccct ggcacaacat 900

aaggccatta tctctcatcc aactccagga cggagtcagt gagaatatt 949

<210> 364

<211> 1761

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: E006-T016-S186-S050 eTag

<400> 364

atggctctgc ctgtgacagc tctgctgctg cctctggctc tgcttctgca tgctgctaga 60

cctagaaaag tgtgcaacgg catcggcatc ggagagttca aggacagcct gagcatcaac 120

gccaccaaca tcaagcactt caagaactgc accagcatca gcggcgacct gcacattctg 180

cctgtggcct ttagaggcga cagcttcacc cacacacctc cactggatcc ccaagagctg 240

gacatcctga aaaccgtgaa agagatcacc ggatttctgt tgatccaggc ttggcccgag 300

aaccggacag atctgcacgc cttcgagaac ctggaaatca tcagaggccg gaccaagcag 360

cacggccagt tttctctggc tgtggtgtcc ctgaacatca ccagcctggg cctgagaagc 420

ctgaaagaaa tcagcgacgg cgacgtgatc atctccggca acaagaacct gtgctacgcc 480

aacaccatca actggaagaa gctgttcggc accagcggcc agaaaacaaa gatcatcagc 540

aaccggggcg agaacagttg caaggctaca ggccaagtgt gccacgctct gtgtagccct 600

gaaggctgtt ggggacccga gcctagagat tgcgtgtcct gcagaaacgt gtcccggggc 660

agagaatgcg tggacaagtg caatctgctg gaaggcgagc cccgcgagtt cgtggaaaac 720

agcgagtgca tccagtgtca ccccgagtgt ctgccccagg ccatgaacat tacatgtacc 780

ggcagaggcc ccgacaactg cattcagtgc gcccactaca tcgacggccc tcactgcgtg 840

aaaacatgtc ctgctggcgt gatgggagag aacaacaccc tcgtgtggaa gtatgccgac 900

gccggacacg tgtgccacct gtgtcaccct aattgcacct atggctgtac cggccctggc 960

ctggaaggct gtccaacaaa cggcctggaa cggatcgccc ggctggaaga gaaagtgaaa 1020

acactgaagg cccagaacag cgagctggcc tccacagcca acatgctgag agaacaggtg 1080

gcccagctga agcagaaagt cggcggctct aatctgggca gcgtgtacat ctacgtgctg 1140

ctgatcgtgg gcacactcgt gtgcggaatc gtgctgggct ttctgtttgg cggcagcaga 1200

tggcagttcc ccgctcacta tcggagactg agacacgccc tgtggccatc tctgcccgat 1260

ctgcaccggg tgctgggcca gtatctgaga gataccgccg ctctgtctcc acctaaggcc 1320

accgtgtccg atacatgcga ggaagtggaa cccagcctgc tggaaatcct gcccaagagc 1380

agcgagagaa cccctctgcc tctgtgttct agccaggctc agatggacta ccgcagactg 1440

cagcctagct gcctgggaac aatgcccctg tctgtgtgtc ctcccatggc cgagagcggc 1500

agctgctgca caacccacat tgccaaccac agctacctgc ctctgagcta ctggcagcaa 1560

cctggcggat caaagaaggt ggccaagaag cccaccaaca aggcccctca tcctaagcaa 1620

gagccccaag agatcaactt ccccgacgat ctgcccggca gcaatactgc tgctcccgtg 1680

caagaaaccc tgcacggttg tcagcccgtg acacaagagg acggcaaaga aagccggatc 1740

agcgtccaag aacggcagta a 1761

<210> 365

<211> 239

<212> DNA

<213> artificial sequence

<220>

<223> synthesis: spacer C without spacer

<400> 365

atctcaagag tggcagcggt cttgagtggc agcggcggta tacggcagcg gcatgtaact 60

agctcctcag tggcagcgat gaggaggcag gttctgttaa gtaactgaac ccaatgtcgt 120

tagtgacgct tagctcttaa gaggtcactg acctaacaat ctcaagagtg gcagcggtct 180

tgagtggcag cggcggtata cggcagcgct atctaagtag taacaagtag cgtggggca 239

<210> 366

<211> 905

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: UMuLVSUx

<400> 366

Met Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn Pro

1 5 10 15

Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Asp Ile

20 25 30

Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

35 40 45

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn

50 55 60

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr

65 70 75 80

Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

85 90 95

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

100 105 110

Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe

115 120 125

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly

195 200 205

Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val

210 215 220

Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp

245 250 255

Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

260 265 270

Ser Ser Ala Ala Ala Ile Glu Gly Arg Met Ala Glu Ser Pro His Gln

275 280 285

Val Phe Asn Val Thr Trp Arg Val Thr Asn Leu Met Thr Gly Arg Thr

290 295 300

Ala Asn Ala Thr Ser Leu Leu Gly Thr Val Gln Asp Ala Phe Pro Lys

305 310 315 320

Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly Glu Glu Trp Asp Pro Ser

325 330 335

Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys Lys Tyr Pro Ala Gly Arg

340 345 350

Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val Cys Pro Gly His Thr Val

355 360 365

Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly Tyr Cys Gly Lys Trp Gly

370 375 380

Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys Pro Thr Ser Ser Trp Asp

385 390 395 400

Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro Trp Asp Thr Gly Cys Ser

405 410 415

Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu Ser Lys Val Ser Asn Ser

420 425 430

Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys Asn Pro Leu Val Leu Glu

435 440 445

Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp Asp Gly Pro Lys Ser Trp

450 455 460

Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp Pro Ile Thr Met Phe Ser

465 470 475 480

Leu Thr Arg Gln Val Leu Asn Val Gly Pro Arg Val Pro Ile Gly Pro

485 490 495

Asn Pro Val Leu Pro Asp Gln Arg Leu Pro Ser Ser Pro Ile Glu Ile

500 505 510

Val Pro Ala Pro Gln Pro Pro Ser Pro Leu Asn Thr Ser Tyr Pro Pro

515 520 525

Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro Thr Ser Pro Ser Val Pro

530 535 540

Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu Leu Ala Leu Val Lys Gly

545 550 555 560

Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro Asp Lys Thr Gln Glu Cys

565 570 575

Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr Tyr Glu Gly Val Ala Val

580 585 590

Val Gly Thr Tyr Thr Asn His Ser Thr Ala Pro Ala Asn Cys Thr Ala

595 600 605

Thr Ser Gln His Lys Leu Thr Leu Ser Glu Val Thr Gly Gln Gly Leu

610 615 620

Cys Met Gly Ala Val Pro Lys Thr His Gln Ala Leu Cys Asn Thr Thr

625 630 635 640

Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu Ala Ala Pro Ala Gly Thr

645 650 655

Met Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Leu Ser Thr Thr Val

660 665 670

Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu Val Glu Leu Trp Pro Arg

675 680 685

Val Ile Tyr His Ser Pro Asp Tyr Met Tyr Gly Gln Leu Glu Gln Arg

690 695 700

Thr Ile Glu Gly Arg Glu Pro Val Ser Leu Thr Leu Ala Leu Leu Leu

705 710 715 720

Gly Gly Leu Thr Met Gly Gly Ile Ala Ala Gly Ile Gly Thr Gly Thr

725 730 735

Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu Gln Leu His Ala Ala Ile

740 745 750

Gln Thr Asp Leu Asn Glu Val Glu Lys Ser Ile Thr Asn Leu Glu Lys

755 760 765

Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln Asn Arg Arg Gly Leu

770 775 780

Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys Ala Ala Leu Lys Glu

785 790 795 800

Glu Cys Cys Phe Tyr Ala Asp His Thr Gly Leu Val Arg Asp Ser Met

805 810 815

Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln Lys Leu Phe Glu Thr

820 825 830

Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg Ser Pro Trp Phe Thr

835 840 845

Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile Val Leu Leu Leu Ile

850 855 860

Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu Val Gln Phe Val Lys

865 870 875 880

Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu Thr Gln Gln Tyr His

885 890 895

Gln Leu Lys Pro Ile Glu Tyr Glu Pro

900 905

<210> 367

<211> 770

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: UCHT1- (G4S) 3-VSVG

<400> 367

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

20 25 30

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

35 40 45

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

50 55 60

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

65 70 75 80

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

85 90 95

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

100 105 110

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr

180 185 190

Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile

195 200 205

Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr

225 230 235 240

Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

260 265 270

Gly Gly Ser Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn

275 280 285

Trp Lys Asn Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp

290 295 300

Leu Asn Trp His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met

305 310 315 320

Pro Lys Ser His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala

325 330 335

Ser Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr

340 345 350

Ile Thr His Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys

355 360 365

Glu Ser Ile Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe

370 375 380

Pro Pro Gln Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val

385 390 395 400

Ile Val Gln Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly

405 410 415

Glu Trp Val Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile

420 425 430

Cys Pro Thr Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val

435 440 445

Lys Gly Leu Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe

450 455 460

Ser Glu Asp Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe

465 470 475 480

Arg Ser Asn Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met

485 490 495

Gln Tyr Cys Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe

500 505 510

Glu Met Ala Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys

515 520 525

Pro Glu Gly Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val

530 535 540

Ser Leu Ile Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln

545 550 555 560

Glu Thr Trp Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp

565 570 575

Leu Ser Tyr Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr

580 585 590

Ile Ile Asn Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val

595 600 605

Asp Ile Ala Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly

610 615 620

Thr Thr Thr Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp

625 630 635 640

Val Glu Ile Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys

645 650 655

Phe Pro Leu Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His

660 665 670

Leu Ser Ser Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala

675 680 685

Ala Ser Gln Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly

690 695 700

Leu Ser Lys Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp

705 710 715 720

Lys Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly

725 730 735

Leu Phe Leu Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys

740 745 750

His Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu

755 760 765

Gly Lys

770

<210> 368

<211> 376

<212> PRT

<213> herpes simplex virus 7

<400> 368

Met Ala Ser Tyr Pro Cys His Gln His Ala Ser Ala Phe Asp Gln Ala

1 5 10 15

Ala Arg Ser Arg Gly His Ser Asn Arg Arg Thr Ala Leu Arg Pro Arg

20 25 30

Arg Gln Gln Glu Ala Thr Glu Val Arg Leu Glu Gln Lys Met Pro Thr

35 40 45

Leu Leu Arg Val Tyr Ile Asp Gly Pro His Gly Met Gly Lys Thr Thr

50 55 60

Thr Thr Gln Leu Leu Val Ala Leu Gly Ser Arg Asp Asp Ile Val Tyr

65 70 75 80

Val Pro Glu Pro Met Thr Tyr Trp Gln Val Leu Gly Ala Ser Glu Thr

85 90 95

Ile Ala Asn Ile Tyr Thr Thr Gln His Arg Leu Asp Gln Gly Glu Ile

100 105 110

Ser Ala Gly Asp Ala Ala Val Val Met Thr Ser Ala Gln Ile Thr Met

115 120 125

Gly Met Pro Tyr Ala Val Thr Asp Ala Val Leu Ala Pro His Ile Gly

130 135 140

Gly Glu Ala Gly Ser Ser His Ala Pro Pro Pro Ala Leu Thr Leu Ile

145 150 155 160

Phe Asp Arg His Pro Ile Ala Ala Leu Leu Cys Tyr Pro Ala Ala Arg

165 170 175

Tyr Leu Met Gly Ser Met Thr Pro Gln Ala Val Leu Ala Phe Val Ala

180 185 190

Leu Ile Pro Pro Thr Leu Pro Gly Thr Asn Ile Val Leu Gly Ala Leu

195 200 205

Pro Glu Asp Arg His Ile Asp Arg Leu Ala Lys Arg Gln Arg Pro Gly

210 215 220

Glu Arg Leu Asp Leu Ala Met Leu Ala Ala Ile Arg Arg Val Tyr Gly

225 230 235 240

Leu Leu Ala Asn Thr Val Arg Tyr Leu Gln Gly Gly Gly Ser Trp Arg

245 250 255

Glu Asp Trp Gly Gln Leu Ser Gly Thr Ala Val Pro Pro Gln Gly Ala

260 265 270

Glu Pro Gln Ser Asn Ala Gly Pro Arg Pro His Ile Gly Asp Thr Leu

275 280 285

Phe Thr Leu Phe Arg Ala Pro Glu Leu Leu Ala Pro Asn Gly Asp Leu

290 295 300

Tyr Asn Val Phe Ala Trp Ala Leu Asp Val Leu Ala Lys Arg Leu Arg

305 310 315 320

Pro Met His Val Phe Val Leu Asp Tyr Asp Gln Ser Pro Ala Gly Cys

325 330 335

Arg Asp Ala Leu Leu Gln Leu Thr Ser Gly Met Val Gln Thr His Val

340 345 350

Thr Thr Pro Gly Ser Ile Pro Thr Ile Cys Asp Leu Ala Arg Thr Phe

355 360 365

Ala Arg Glu Met Gly Glu Ala Asn

370 375

<210> 369

<211> 427

<212> PRT

<213> Chile person

<400> 369

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys

20 25 30

Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn

35 40 45

Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys

50 55 60

Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr

65 70 75 80

Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser

85 90 95

Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly

100 105 110

Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys

115 120 125

Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr

130 135 140

Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His

145 150 155 160

Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln

165 170 175

Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro

180 185 190

Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr

195 200 205

Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile

210 215 220

Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ser Ser Gln

225 230 235 240

Pro Val Val Thr Arg Gly Thr Thr Asp Asn Leu Ile Pro Val Tyr Cys

245 250 255

Ser Ile Leu Ala Ala Val Val Val Gly Leu Val Ala Tyr Ile Ala Phe

260 265 270

Lys Arg Trp Asn Ser Cys Lys Gln Asn Lys Gln Gly Ala Asn Ser Arg

275 280 285

Pro Val Asn Gln Thr Pro Pro Pro Glu Gly Glu Lys Leu His Ser Asp

290 295 300

Ser Gly Ile Ser Val Asp Ser Gln Ser Leu His Asp Gln Gln Pro His

305 310 315 320

Thr Gln Thr Ala Ser Gly Gln Ala Leu Lys Gly Asp Gly Gly Leu Tyr

325 330 335

Ser Ser Leu Pro Pro Ala Lys Arg Glu Glu Val Glu Lys Leu Leu Asn

340 345 350

Gly Ser Ala Gly Asp Thr Trp Arg His Leu Ala Gly Glu Leu Gly Tyr

355 360 365

Gln Pro Glu His Ile Asp Ser Phe Thr His Glu Ala Cys Pro Val Arg

370 375 380

Ala Leu Leu Ala Ser Trp Ala Thr Gln Asp Ser Ala Thr Leu Asp Ala

385 390 395 400

Leu Leu Ala Ala Leu Arg Arg Ile Gln Arg Ala Asp Leu Val Glu Ser

405 410 415

Leu Cys Ser Glu Ser Thr Ala Thr Ser Pro Val

420 425

<210> 370

<211> 297

<212> PRT

<213> Chile person

<400> 370

Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro

1 5 10 15

Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg

20 25 30

Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu

35 40 45

Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile

50 55 60

Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile

65 70 75 80

Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile

85 90 95

Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu

100 105 110

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile

115 120 125

Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser

130 135 140

His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro

145 150 155 160

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn

165 170 175

Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly

180 185 190

Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile

195 200 205

Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys

210 215 220

Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile

225 230 235 240

Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro

245 250 255

Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu

260 265 270

Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser

275 280 285

Ser Pro Ile Glu Asn Asp Ser Ser Pro

290 295

<210> 371

<211> 12

<212> PRT

<213> Chile person

<400> 371

Gly Gln Asn Asp Thr Ser Gln Thr Ser Ser Pro Ser

1 5 10

<210> 372

<211> 654

<212> PRT

<213> artificial sequence

<220>

<223> synthesis: muLVSUx

<400> 372

Met Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn Pro

1 5 10 15

Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Met Ala

20 25 30

Glu Ser Pro His Gln Val Phe Asn Val Thr Trp Arg Val Thr Asn Leu

35 40 45

Met Thr Gly Arg Thr Ala Asn Ala Thr Ser Leu Leu Gly Thr Val Gln

50 55 60

Asp Ala Phe Pro Lys Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly Glu

65 70 75 80

Glu Trp Asp Pro Ser Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys Lys

85 90 95

Tyr Pro Ala Gly Arg Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val Cys

100 105 110

Pro Gly His Thr Val Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly Tyr

115 120 125

Cys Gly Lys Trp Gly Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys Pro

130 135 140

Thr Ser Ser Trp Asp Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro Trp

145 150 155 160

Asp Thr Gly Cys Ser Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu Ser

165 170 175

Lys Val Ser Asn Ser Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys Asn

180 185 190

Pro Leu Val Leu Glu Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp Asp

195 200 205

Gly Pro Lys Ser Trp Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp Pro

210 215 220

Ile Thr Met Phe Ser Leu Thr Arg Gln Val Leu Asn Val Gly Pro Arg

225 230 235 240

Val Pro Ile Gly Pro Asn Pro Val Leu Pro Asp Gln Arg Leu Pro Ser

245 250 255

Ser Pro Ile Glu Ile Val Pro Ala Pro Gln Pro Pro Ser Pro Leu Asn

260 265 270

Thr Ser Tyr Pro Pro Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro Thr

275 280 285

Ser Pro Ser Val Pro Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu Leu

290 295 300

Ala Leu Val Lys Gly Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro Asp

305 310 315 320

Lys Thr Gln Glu Cys Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr Tyr

325 330 335

Glu Gly Val Ala Val Val Gly Thr Tyr Thr Asn His Ser Thr Ala Pro

340 345 350

Ala Asn Cys Thr Ala Thr Ser Gln His Lys Leu Thr Leu Ser Glu Val

355 360 365

Thr Gly Gln Gly Leu Cys Met Gly Ala Val Pro Lys Thr His Gln Ala

370 375 380

Leu Cys Asn Thr Thr Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu Ala

385 390 395 400

Ala Pro Ala Gly Thr Met Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys

405 410 415

Leu Ser Thr Thr Val Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu Val

420 425 430

Glu Leu Trp Pro Arg Val Ile Tyr His Ser Pro Asp Tyr Met Tyr Gly

435 440 445

Gln Leu Glu Gln Arg Thr Ile Glu Gly Arg Glu Pro Val Ser Leu Thr

450 455 460

Leu Ala Leu Leu Leu Gly Gly Leu Thr Met Gly Gly Ile Ala Ala Gly

465 470 475 480

Ile Gly Thr Gly Thr Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu Gln

485 490 495

Leu His Ala Ala Ile Gln Thr Asp Leu Asn Glu Val Glu Lys Ser Ile

500 505 510

Thr Asn Leu Glu Lys Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln

515 520 525

Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys

530 535 540

Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp His Thr Gly Leu

545 550 555 560

Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg Gln

565 570 575

Lys Leu Phe Glu Thr Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn Arg

580 585 590

Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met Gly Pro Leu Ile

595 600 605

Val Leu Leu Leu Ile Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu

610 615 620

Val Gln Phe Val Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu

625 630 635 640

Thr Gln Gln Tyr His Gln Leu Lys Pro Ile Glu Tyr Glu Pro

645 650

<210> 373

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 373

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 374

<211> 546

<212> PRT

<213> Nipah Henry Pav virus

<400> 374

Met Val Val Ile Leu Asp Lys Arg Cys Tyr Cys Asn Leu Leu Ile Leu

1 5 10 15

Ile Leu Met Ile Ser Glu Cys Ser Val Gly Ile Leu His Tyr Glu Lys

20 25 30

Leu Ser Lys Ile Gly Leu Val Lys Gly Val Thr Arg Lys Tyr Lys Ile

35 40 45

Lys Ser Asn Pro Leu Thr Lys Asp Ile Val Ile Lys Met Ile Pro Asn

50 55 60

Val Ser Asn Met Ser Gln Cys Thr Gly Ser Val Met Glu Asn Tyr Lys

65 70 75 80

Thr Arg Leu Asn Gly Ile Leu Thr Pro Ile Lys Gly Ala Leu Glu Ile

85 90 95

Tyr Lys Asn Asn Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly

100 105 110

Val Ile Met Ala Gly Val Ala Ile Gly Ile Ala Thr Ala Ala Gln Ile

115 120 125

Thr Ala Gly Val Ala Leu Tyr Glu Ala Met Lys Asn Ala Asp Asn Ile

130 135 140

Asn Lys Leu Lys Ser Ser Ile Glu Ser Thr Asn Glu Ala Val Val Lys

145 150 155 160

Leu Gln Glu Thr Ala Glu Lys Thr Val Tyr Val Leu Thr Ala Leu Gln

165 170 175

Asp Tyr Ile Asn Thr Asn Leu Val Pro Thr Ile Asp Lys Ile Ser Cys

180 185 190

Lys Gln Thr Glu Leu Ser Leu Asp Leu Ala Leu Ser Lys Tyr Leu Ser

195 200 205

Asp Leu Leu Phe Val Phe Gly Pro Asn Leu Gln Asp Pro Val Ser Asn

210 215 220

Ser Met Thr Ile Gln Ala Ile Ser Gln Ala Phe Gly Gly Asn Tyr Glu

225 230 235 240

Thr Leu Leu Arg Thr Leu Gly Tyr Ala Thr Glu Asp Phe Asp Asp Leu

245 250 255

Leu Glu Ser Asp Ser Ile Thr Gly Gln Ile Ile Tyr Val Asp Leu Ser

260 265 270

Ser Tyr Tyr Ile Ile Val Arg Val Tyr Phe Pro Ile Leu Thr Glu Ile

275 280 285

Gln Gln Ala Tyr Ile Gln Glu Leu Leu Pro Val Ser Phe Asn Asn Asp

290 295 300

Asn Ser Glu Trp Ile Ser Ile Val Pro Asn Phe Ile Leu Val Arg Asn

305 310 315 320

Thr Leu Ile Ser Asn Ile Glu Ile Gly Phe Cys Leu Ile Thr Lys Arg

325 330 335

Ser Val Ile Cys Asn Gln Asp Tyr Ala Thr Pro Met Thr Asn Asn Met

340 345 350

Arg Glu Cys Leu Thr Gly Ser Thr Glu Lys Cys Pro Arg Glu Leu Val

355 360 365

Val Ser Ser His Val Pro Arg Phe Ala Leu Ser Asn Gly Val Leu Phe

370 375 380

Ala Asn Cys Ile Ser Val Thr Cys Gln Cys Gln Thr Thr Gly Arg Ala

385 390 395 400

Ile Ser Gln Ser Gly Glu Gln Thr Leu Leu Met Ile Asp Asn Thr Thr

405 410 415

Cys Pro Thr Ala Val Leu Gly Asn Val Ile Ile Ser Leu Gly Lys Tyr

420 425 430

Leu Gly Ser Val Asn Tyr Asn Ser Glu Gly Ile Ala Ile Gly Pro Pro

435 440 445

Val Phe Thr Asp Lys Val Asp Ile Ser Ser Gln Ile Ser Ser Met Asn

450 455 460

Gln Ser Leu Gln Gln Ser Lys Asp Tyr Ile Lys Glu Ala Gln Arg Leu

465 470 475 480

Leu Asp Thr Val Asn Pro Ser Leu Ile Ser Met Leu Ser Met Ile Ile

485 490 495

Leu Tyr Val Leu Ser Ile Ala Ser Leu Cys Ile Gly Leu Ile Thr Phe

500 505 510

Ile Ser Phe Ile Ile Val Glu Lys Lys Arg Asn Thr Tyr Ser Arg Leu

515 520 525

Glu Asp Arg Arg Val Arg Pro Thr Ser Ser Gly Asp Leu Tyr Tyr Ile

530 535 540

Gly Thr

545

<210> 375

<211> 602

<212> PRT

<213> Nipah Henry Pav virus

<400> 375

Met Pro Ala Glu Asn Lys Lys Val Arg Phe Glu Asn Thr Thr Ser Asp

1 5 10 15

Lys Gly Lys Ile Pro Ser Lys Val Ile Lys Ser Tyr Tyr Gly Thr Met

20 25 30

Asp Ile Lys Lys Ile Asn Glu Gly Leu Leu Asp Ser Lys Ile Leu Ser

35 40 45

Ala Phe Asn Thr Val Ile Ala Leu Leu Gly Ser Ile Val Ile Ile Val

50 55 60

Met Asn Ile Met Ile Ile Gln Asn Tyr Thr Arg Ser Thr Asp Asn Gln

65 70 75 80

Ala Val Ile Lys Asp Ala Leu Gln Gly Ile Gln Gln Gln Ile Lys Gly

85 90 95

Leu Ala Asp Lys Ile Gly Thr Glu Ile Gly Pro Lys Val Ser Leu Ile

100 105 110

Asp Thr Ser Ser Thr Ile Thr Ile Pro Ala Asn Ile Gly Leu Leu Gly

115 120 125

Ser Lys Ile Ser Gln Ser Thr Ala Ser Ile Asn Glu Asn Val Asn Glu

130 135 140

Lys Cys Lys Phe Thr Leu Pro Pro Leu Lys Ile His Glu Cys Asn Ile

145 150 155 160

Ser Cys Pro Asn Pro Leu Pro Phe Arg Glu Tyr Arg Pro Gln Thr Glu

165 170 175

Gly Val Ser Asn Leu Val Gly Leu Pro Asn Asn Ile Cys Leu Gln Lys

180 185 190

Thr Ser Asn Gln Ile Leu Lys Pro Lys Leu Ile Ser Tyr Thr Leu Pro

195 200 205

Val Val Gly Gln Ser Gly Thr Cys Ile Thr Asp Pro Leu Leu Ala Met

210 215 220

Asp Glu Gly Tyr Phe Ala Tyr Ser His Leu Glu Arg Ile Gly Ser Cys

225 230 235 240

Ser Arg Gly Val Ser Lys Gln Arg Ile Ile Gly Val Gly Glu Val Leu

245 250 255

Asp Arg Gly Asp Glu Val Pro Ser Leu Phe Met Thr Asn Val Trp Thr

260 265 270

Pro Pro Asn Pro Asn Thr Val Tyr His Cys Ser Ala Val Tyr Asn Asn

275 280 285

Glu Phe Tyr Tyr Val Leu Cys Ala Val Ser Thr Val Gly Asp Pro Ile

290 295 300

Leu Asn Ser Thr Tyr Trp Ser Gly Ser Leu Met Met Thr Arg Leu Ala

305 310 315 320

Val Lys Pro Lys Ser Asn Gly Gly Gly Tyr Asn Gln His Gln Leu Ala

325 330 335

Leu Arg Ser Ile Glu Lys Gly Arg Tyr Asp Lys Val Met Pro Tyr Gly

340 345 350

Pro Ser Gly Ile Lys Gln Gly Asp Thr Leu Tyr Phe Pro Ala Val Gly

355 360 365

Phe Leu Val Arg Thr Glu Phe Lys Tyr Asn Asp Ser Asn Cys Pro Ile

370 375 380

Thr Lys Cys Gln Tyr Ser Lys Pro Glu Asn Cys Arg Leu Ser Met Gly

385 390 395 400

Ile Arg Pro Asn Ser His Tyr Ile Leu Arg Ser Gly Leu Leu Lys Tyr

405 410 415

Asn Leu Ser Asp Gly Glu Asn Pro Lys Val Val Phe Ile Glu Ile Ser

420 425 430

Asp Gln Arg Leu Ser Ile Gly Ser Pro Ser Lys Ile Tyr Asp Ser Leu

435 440 445

Gly Gln Pro Val Phe Tyr Gln Ala Ser Phe Ser Trp Asp Thr Met Ile

450 455 460

Lys Phe Gly Asp Val Leu Thr Val Asn Pro Leu Val Val Asn Trp Arg

465 470 475 480

Asn Asn Thr Val Ile Ser Arg Pro Gly Gln Ser Gln Cys Pro Arg Phe

485 490 495

Asn Thr Cys Pro Glu Ile Cys Trp Glu Gly Val Tyr Asn Asp Ala Phe

500 505 510

Leu Ile Asp Arg Ile Asn Trp Ile Ser Ala Gly Val Phe Leu Asp Ser

515 520 525

Asn Gln Thr Ala Glu Asn Pro Val Phe Thr Val Phe Lys Asp Asn Glu

530 535 540

Ile Leu Tyr Arg Ala Gln Leu Ala Ser Glu Asp Thr Asn Ala Gln Lys

545 550 555 560

Thr Ile Thr Asn Cys Phe Leu Leu Lys Asn Lys Ile Trp Cys Ile Ser

565 570 575

Leu Val Glu Ile Tyr Asp Thr Gly Asp Asn Val Ile Arg Pro Lys Leu

580 585 590

Phe Ala Val Lys Ile Pro Glu Gln Cys Thr

595 600

<210> 376

<211> 774

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 376

gatgttgtga tgactcagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaagc tggagatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtgcagctgc aggagtctgg gggaggcctg 420

gtccagcctg gggggtccct gagactctcg tgtgcagcct ctggattcac cttcagtagc 480

tatagcatga actgggtccg ccaggctcca gggaaggggc tggagtgggt ctcatccatt 540

agtagtcgta gtaatgacat atactacgca gactcagtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 660

gctgtgtatt actgtgcgag accctattac tttgatagta gtggttatgt caattactac 720

tcctactacg gtttggacgt ctggggccaa gggaccacgg tcaccgtctc ctca 774

<210> 377

<211> 750

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 377

gaaattgtgc tgactcagtc tccatcctcc ctctctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc aggtatttaa attggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcagcag agatacagta ttccctacga tttcggccct 300

gggacacgac tggagattaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgt tgcagtctgg agcagaggtg 420

aaaaagcccg gggagtctct gaagatctcc tgtaaggttt ctggatacag cttttccagc 480

tactggatcg gctgggtgcg ccagatgccc gggaaaggcc tggaatggat ggggatcatc 540

tatcctggtg actccaatac cagatacagc ccgtccttcc aaggccaggt ctccttctcg 600

gccgacaagt ccatcagcac cgcctacctg cagtggagca gcctgaaggc ctcggacacc 660

gccatgtatt actgtgcgag gcttatctat ggtgactacg gtgggggtct tgactactgg 720

ggccagggaa ccctggtcac cgtctcctca 750

<210> 378

<211> 744

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 378

gaaattgtgc tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccgaccgtg gacgttcggc 300

caagggacca agctggagat caaacgtgga ggcggtggct ccggaggcgg gggtagtgga 360

ggcggaggga gcggaggggg aggaagcgcc gaggtgcagc tgttgcagtc tggggctgag 420

gtgaagaagc ctgggtcctc ggtgaaggtc tcctgcaagg cttctggagg caccttcagc 480

agctatgcta tcagctgggt gcgacaggcc cctggacaag ggcttgagtg gatgggaggg 540

atcatcccta tctttggtac agcaaactac gcacagaagt tccagggcag agtcacgatt 600

accgcggacg aatccacgag cacagcctac atggagctga gcagcctgag atctgaggac 660

acggccgtgt attactgtgc gagggtgtat agcagtggct ggtttgacta ctggggccag 720

ggcaccctgg tcaccgtctc ctca 744

<210> 379

<211> 753

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 379

gacatcgtga tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccaatca gggcattagc agttttttag cctggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatcaatgct gcatccactt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacacaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag catagtagtt accctctcac tttcggcgga 300

gggaccaagg tggagatcaa acgtggaggc ggtggctccg gtggcggcgg atccggaggc 360

gggggtagtg gaggcggagg gagcggaggg ggaggaagcg ccgaggtgca gctgttgcag 420

tctggagcag aggtgaaaaa gcccggggag tctctgagga tctcctgtaa gggttctgga 480

tacagcttta ccagctactg gatcgcctgg gtgcgccaga tgcccgggaa aggcctggag 540

tggatgggga tcatctatcc tggtgagtct gatatcagat acagcccgtc gttccaaggc 600

caggtcacca tctcagccga caagtccatc agcaccgcct acctgcagtg gagcagcctg 660

aaggcctcgg acaccgccat gtattactgt gcgagacaga ggtactggta cttcgatctc 720

tggggccgtg gaaccctggt caccgtctcc tca 753

<210> 380

<211> 729

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 380

gacatccaga tgacccagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaacttcc 300

acgtacagtt ttggccaggg gaccaagctg gagatcaaac gtggaggcag ctcccggtca 360

agcgcccagg tgcagctggt gcagtctggc ccaggactgg tgaagccttc cgagaccctg 420

tccctcacct gcgctgtctc tggtgactcc atccgtggtc actattggag ctggatccgg 480

cagaccgccg ggaagggcct ggagtggatt ggttacacat ctgatactgg ggataccaag 540

tacaatccct ccctcgggag tcgcgtcacc atgtcactgg acacgtccaa gaaccagttg 600

tccctgaacc tgaggtctgt aaccgccacg gacacggccc tctattactg tgcaagagtt 660

gatcccgaag atccctcagg agcactgttg gacctctggg gccagggcac cctggtcacc 720

gtctcctca 729

<210> 381

<211> 741

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 381

gacatccaga tgacccagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaagc tggagatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtgcagctgg tgcagtctgg ggctgaggtg 420

aagaagcctg gggcctcagt gaaggtttcc tgcaaggcat ctggatacac cttcaccagc 480

tactatatgc actgggtgcg acaggcccct ggacaagggc ttgagtggat gggaataatc 540

aaccctagtg gtggtagcac aagctacgca cagaagttcc agggcagagt caccatgacc 600

agggacacgt ccacgagcac agtctacatg gagctgagca gcctgagatc tgaggacacg 660

gccgtgtatt actgtgcgag gtctaagggc aaagggccct ttgactactg gggccaggga 720

accctggtca ccgtctcctc a 741

<210> 382

<211> 780

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 382

gatgttgtga tgactcagtc tccactctcc ttgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300

tacacttttg gccaggggac caagctggag atcaaacgtg gaggcggtgg ctccggcggt 360

ggtgggtccg gtggcggcgg atccggaggc gggggtagtg gaggcggagg gagcggaggg 420

ggaggaagcg cccaggtgca gctgcaggag tcgggcccag gactggtgaa gccttcggag 480

accctgtccc tcacctgcac tgtctctggt tactccatca ccagtcgttc ctactggggc 540

tgggtccggc agtccccagg gaaggggctg gaatggcttg ggagtttgtc ctggactgga 600

agtactcaat acaacccgtc cctcaggggt cgagtcacaa tatcactgga caggtccaac 660

agtcaattct ccctgaggtt gacctctgtg tccgccgccg acacggccac ttactactgt 720

gtgagagata atagagccct tgactcctgg ggccagggaa ccctggtcac cgtctcctca 780

<210> 383

<211> 774

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 383

gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccagg ggtgtacact 300

tttggccagg ggaccaaagt ggatatcaaa cgtggaggcg gtggctccgg cggtggtggg 360

tccggtggcg gcggatccgg aggcgggggt agtggaggcg gagggagcgg agggggagga 420

agcgcccagg tgcagctgca ggagtcgggc ccaggactgg tgaagccttc ggagaccctg 480

tccctcacct gcactgtctc tggttactcc atcaccagtc gttcctactg gggctgggtc 540

cggcagtccc cagggaaggg gctggaatgg cttgggagtt tgtcctggac tggaagtact 600

caatacaacc cgtccctcag gggtcgagtc acaatatcac tggacaggtc caacagtcaa 660

ttctccctga ggttgacctc tgtgtccgcc gccgacacgg ccacttacta ctgtgtgaga 720

gataatagag cccttgactc ctggggccag ggaaccctgg tcaccgtctc ctca 774

<210> 384

<211> 765

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 384

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaacca 120

gggaaagctc ctaagctcct gatctatgat gcctccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacatta cccctcacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtcaacttaa gggagtctgg gggaggcttg 420

gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac ctttagtagc 480

tattggatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ggccaacata 540

aagcaagatg gaagtgagaa atactatgtg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 660

gctgtgtatt actgtgcgag agcggtgggc gatagcagtg gctggtacca agactactac 720

ggtatggacg tctggggcca agggacaatg gtcaccgtct cttca 765

<210> 385

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 385

gatgttgtga tgactcagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgctcac tttcggcgga 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggagg gagcggaggg 360

ggaggaagcg ccgaggtgca gctggtggag tctgggggag gcttggtaca gcctgggggg 420

tccttgagac tctcctgtgc agcctctgga ttcacctttg cccacgatgc catgagctgg 480

gtccgccagg ctccagggaa ggggctggaa tgggtctcaa ctattactta tggtggtggt 540

ggcacatact acgcagactc cgtgaagggc cggttcacca tctccagaga caattccaag 600

aacacgctat ttctgcaaat gaacggcctg agagccgagg acacggccgt atattcctgt 660

gcgagaggcg gcgtgggggc gtttgactcc tggggccagg gaaccctggt caccgtctcc 720

tca 723

<210> 386

<211> 798

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 386

gacatcgtga tgacccagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctt catagtaatg gatacaacta tttggattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgc aggctgagga tgttggggtt tatttctgca tgcaagctct acaaactcct 300

ccaactttcg gcggagggac caagctggag atcaaacgtg gaggcggtgg ctccggcggt 360

ggtgggtccg gtggcggcgg atccggaggc gggggtagtg gaggcggagg gagcggaggg 420

ggaggaagcg cccaggtgca gctgcaggag tcggggggag gcgtggtcca gcctgggagg 480

tccctgacac tctcctgtgc agcctctgga ttcaccttca gtagttatcc tatgcactgg 540

gtccgccagg ctccaggcaa ggggctggag tgggtggcag ttatatcaca tgatgaaagc 600

aataaatact acgcagactc cgtgaagggc cgattcacca tctccagaga caattccaag 660

aacacgctgt atctgcaaat gaacagcctg agagctgagg acacggctgt gtattactgt 720

gcgagggagc gggggggagg gtacagttat gggatagggg actactgggg ccagggcacc 780

ctggtcaccg tctcctca 798

<210> 387

<211> 741

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 387

gaaattgtgt tgacgcagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctataag gcatctactt tagaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caagttatta ctgccaacag tatcatactt attggacttt cggccctggg 300

accaaagtgg atatcaaacg tggaggcggt ggctccggag gcgggggtag tggaggcgga 360

gggagcggag ggggaggaag cgccgaggtg cagctggtgg agtcgggccc aggactggtg 420

aagccttcac agaccctgtc cctcacctgc actgtctctg gtggctccat cagcagtggt 480

ggttactact ggagctggat ccgccagcac ccagggaagg gcctggagtg gattgggtac 540

atctattaca gtgggagcac ctactacaac ccgtccctca agagtcgagt taccatatca 600

gtagacacgt ctaagaacca gttctccctg aagctgagct ctgtgactgc cgcggacacg 660

gccgtgtatt actgtgcgag agatcgtagt gaaagggctt ttgatatctg gggccaaggg 720

acaatggtca ccgtctcttc a 741

<210> 388

<211> 726

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 388

gatgttgtga tgactcagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc 60

atcacttgcc gggcaagtca gactattcgc aactatttaa attggtatca gcacaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccccatcac cttcggccga 300

gggacacgac tggagattaa acgtggaggc ggtggctccg gaggcggagg gagcggaggg 360

ggaggaagcg ccgaggtgca gctgttgcag tctggggctg aggtgaaaga gcctggggcc 420

tcagtgaagg tctcctgcaa ggcttctgga tacaccttca ccgggaatta tattcactgg 480

gtgcgacagg cccctggaca agggcttcag tggatgggat ggatcaaccc taacactgat 540

ggcacaagat acagtccgaa ctttcagggc aggctcacct tcaccaggga cacgtccatc 600

aacacagcct acttggaact gaacaggctg acatctgacg acacggccgt gtactactgt 660

gcgagagata cgactggcaa tgcttttcat atctggggcc aagggacaat ggtcaccgtc 720

tcttca 726

<210> 389

<211> 735

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 389

gccgaggtgc agctgttgca gtctggagga ggcttgatcc agcctggggg gtccctgaga 60

ctctcctgtg cagcctctgg gttcaccgtc agtagcaact acatgagctg ggtccgccag 120

gctccaggga aggggctgga gtgggtctca gttatttata gcggtggtag cacatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

cttcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatcag 300

cccggcgcca ggttcgaccc ctggggccag ggaaccctgg tcaccgtctc ctcaggaggc 360

ggtggctccg gaggcggagg gagcggaggg ggaggaagcg aaattgtgct gactcagtct 420

ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcgcttgccg ggcaagtcag 480

agcattagcg gctatttaaa ttggtatcag cagaaaccag ggaaagcccc taagctcctg 540

atctatgctg catccagttt gcaaagtggg gtcccatcaa ggttcagtgg cagtggatct 600

gggacagatt tcactctcac catcagcagt ctgcaacctg aagattttgc aacttactac 660

tgtcaacaga gttacagtag tcctcagcta ccaatgtaca cttttggcca ggggaccaag 720

ctggaaatca aacgt 735

<210> 390

<211> 759

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 390

gatgttgtga tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc ggcagctact tagcctggta ccagcagaaa 120

cctggccggg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cattatggta gctcaccgct cactttcggc 300

ggagggacca aggtggagat caaacgtgga ggcggtggct ccggtggcgg cggatccgga 360

ggcgggggta gtggaggcgg agggagcgga gggggaggaa gcgccgaggt gcagctggtg 420

gagtctgggg gaggcttggt acagcctggg gggtccctga gactctcctg tgcagcctct 480

ggattcacct ttagcagcta tgccatgagc tgggtccgcc aggctccagg gaaggggctg 540

gagtgggtct cagctattag tggtagtggt ggtagcacat actacgcaga ctccgtgaag 600

ggccggttca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc 660

ctgagagccg aggacacggc cgtatattac tgtgcgaaag acccagagat ggctacaatt 720

cactactggg gccagggaac cctggtcacc gtctcctca 759

<210> 391

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 391

gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccatc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatcttt ggtgcatcca ccagggccac tggcgtccca 180

gacaggttca gtggcagtgg gtccgggaca gacttcactc tcaccatcag caggctggag 240

cctgaagatc ttgcagtgta ttactgtcag cagtatggta gctcacccat gtacactttt 300

ggccagggga ccaagctgga gatcaaacgt ggaggcggtg gctccggagg cgggggtagt 360

ggaggcggag ggagcggagg gggaggaagc gccgaggtgc agctggtgga gtctggggga 420

ggcgtggtcc agcctgggag gtccctgaga ctctcctgtg cagcctctgg attcaccttc 480

agtagctatg ctatgcactg ggtccgccag gctccaggca aggggctaga gtgggtggca 540

gttatatcat atgatggaag taataaatac tacgcagact ccgtgaaggg ccgattcacc 600

atctccagag acaattccaa gaacacgctg tatctgcaaa tgaacagcct gagagctgag 660

gacacggctg tgtattactg tgcgagaagt gacggataca gctatcccag acactttgac 720

tactggggcc agggaaccct ggtcaccgtc tcctca 756

<210> 392

<211> 774

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 392

gaaattgtgt tgacgcagtc tccatcctcc ctgtctgcat ctgtaggaga cagggtcacc 60

atcacttgcc gggcaagtcc gcgcattgga aatgatttag gctggtatca gcagaagcca 120

gggaaagccc ctgagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataataatt acccgtacac ttttggccag 300

gggaccaaag tggatatcaa acgtggaggc ggtggctccg gcggtggtgg gtccggtggc 360

ggcggatccg gaggcggggg tagtggaggc ggagggagcg gagggggagg aagcgccgag 420

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 480

tgtaagggtt ctggatacag ctttaccagc tactggatcg gctgggtgcg ccagatgccc 540

gggaaaggcc tggagtggat ggggatcatc tatcctggtg actctgatac cagatacagc 600

ccgtccttcc aaggccaggt caccatctca gccgacaagt ccatcagcac cgcctacctg 660

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag acagccgggg 720

tgggggggcc cgttcgaccc ctggggccag ggaaccctgg tcaccgtctc ctca 774

<210> 393

<211> 714

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 393

gacatcgtga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgtacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gagggggagg aagcgccgag 360

gtgcagctgt tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 420

tgtaagggtt ctggatacag ctttaccagc tactggatcg gctgggtgcg ccagatgccc 480

gggaaaggcc tggagtggat ggggatcatc tatcctggtg actctgatac cagatacagc 540

ccgtccttcc aaggccaggt caccatctca gccgacaagt ccatcagcac cgcctacctg 600

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag acagccgggg 660

tgggggggcc cgttcgaccc ctggggccag ggaaccctgg tcaccgtctc ctca 714

<210> 394

<211> 774

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 394

gaaattgtgt tgacgcagtc tccatcctcc ctgtctgcat ctgtaggaga cagggtcacc 60

atcacttgcc gggcaagtcc gggcattgga aatgatttag gctggtatca gcagaagcca 120

gggaaagccc ctgagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataataatt acccgtacac ttttggccag 300

gggaccaaag tggatatcaa acgtggaggc ggtggctccg gcggtggtgg gtccggtggc 360

ggcggatccg gaggcggggg tagtggaggc ggagggagcg gagggggagg aagcgccgag 420

gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 480

tgtaagggtt ctggatacag ctttaccagc tactggatcg gctgggtgcg ccagatgccc 540

gggaaaggcc tggagtggat ggggatcatc tatcctggtg actctgatac cagatacagc 600

ccgtccttcc aaggccaggt caccatctca gccgacaagt ccatcagcac cgcctacctg 660

cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag acagccgggg 720

tgggggggcc cgttcgaccc ctggggccag ggaaccctgg tcaccgtctc ctca 774

<210> 395

<211> 777

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 395

gacatcgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tataatagtt acccctacac ttttggccag 300

gggaccaagg tggagatcaa acgtggaggc ggtggctccg gcggtggtgg gtccggtggc 360

ggcggatccg gaggcggggg tagtggaggc ggagggagcg gagggggagg aagcgccgag 420

gtacagctgc agcagtcggg cccaggactg gtgaagcctt cacagaccct gtccgtcacc 480

tgcactttct ctggtggctc catcagcagt agtggttact actggaactg gatccgccag 540

cacccaggga agggcctgga gtggattggg tacatctatc acagtgggag cacctactac 600

aacccgtccc tcaagcgtcg acttaccatg tcagtagaca cgtctaagaa ccagttctcc 660

ctgaggttga gctctgtgac tgccgcggac acggccgtgt attattgtgc gagaagtctg 720

cggctctacg gtttctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 777

<210> 396

<211> 747

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 396

gatgttgtga tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccgtatgta cacttttggc 300

caggggacca aggtggagat caaacgtgga ggcggtggct ccggaggcgg gggtagtgga 360

ggcggaggga gcggaggggg aggaagcgcc gaggtgcagc tggtggagtc tgggggaggc 420

ttggtacagc ctggcaggtc cctgagactc tcctgtgcag cctctggatt cacctttgat 480

gattatgcca tgcactgggt ccggcaagct ccagggaagg gcctggagtg ggtctcaggt 540

attagttgga atagtggtag cataggctat gcggactctg tgaagggccg attcaccatc 600

tccagagaca acgccaagaa ctccctgtat ctgcaaatga acagtctgag agctgaggac 660

acggccttgt attactgtgc aaaagatttc acaggggata atgcttttga tatctggggc 720

caagggacaa tggtcaccgt ctcttca 747

<210> 397

<211> 750

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 397

gacatcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttactt ttgtcaacag gcttacagtt tccccccgta cacttttggc 300

caggggacca agctggagat caaacgtgga ggcggtggct ccggaggcgg gggtagtgga 360

ggcggaggga gcggaggggg aggaagcgcc gaggtacagc tgcagcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gagactggct atggttcagg gagccccagc ggactactgg 720

ggccagggca ccctggtcac cgtctcctca 750

<210> 398

<211> 735

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 398

gccgaggtac agctgcagca gtctggggga ggcttggtca agcctggagg gtccctgaga 60

ctgtcctgtg cagcctctgg attcactttc agcgactcct atatgacttg gattcggcag 120

actccggggc agagtctcga gtgggtctcc tacatttcta atagtggtga tgccatttac 180

tacgcaggct ctgtgagggg ccgattcatc gtctccaggg acaactccaa gaatttattg 240

tttctgcaaa tgaacaacct gagagccgac gactcggcca catattactg cacgactgga 300

aaacgctcct atcgcgtctg gggccagggc accctggtca ccgtctcctc aggaggcggt 360

ggctccggag gcgggggtag tggaggcgga gggagcggag ggggaggaag cgaaattgtg 420

ctgactcagt ctccatcctc cctgtctgca tctgtaggag acagagtcac catcacttgc 480

cgggcaagtc agagcattag cagctattta aattggtatc agcagaaacc agggaaagcc 540

cctaagctcc tgatctatgc tgcatccagt ttgcaaagtg gggtcccatc aaggttcagt 600

ggcagtggat ctgggacaga tttcactctc accatcagca gtctgcaacc tgaagatttt 660

gcaacttact actgtcaaca gagttacagt acccctcaaa cttttggcca ggggacacga 720

ctggagatta aacgt 735

<210> 399

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 399

gacatccaga tgacccagtc tccttccacc ctgtcagcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt acctggttgg cctggtatca acaaaaacca 120

gggaaagccc ctaacctcct gatctataag gcgtctactt taaaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tatgatagtt ccccgtacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgg tggagtctgg gggaggcttg 420

gtacagcctg gcaggtccct gagactctcc tgtgcagcct ctggattcac ctttgatgat 480

tatgccatgc actgggtccg gcaagctcca gggaagggcc tggagtgggt ctcaggtatt 540

agttggaata gtggtagcat aggctatgcg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc cctgtatctg caaatgaaca gtctgagagc tgaggacacg 660

gccttgtatt actgtgcaaa agatatctcc cctgactgga gcaggggggg atggttcgac 720

ccctggggcc agggcaccct ggtcaccgtc tcctca 756

<210> 400

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 400

gacatcgtga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccttacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtacagctgc agcagtctgg gggaggcttg 420

gtacagcctg gcaggtccct gagactctcc tgtgcagcct ctggattcac ctttgatgat 480

tatgccatgc actgggtccg gcaagctcca gggaagggcc tggagtgggt ctcaggtatt 540

agttggaata gtggtagcat aggctatgcg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc cctgtatctg caaatgaaca gtctgagagc tgaggacacg 660

gccttgtatt actgtgcaaa agatatctcc cctgactgga gcaggggggg atggttcgac 720

ccctggggcc agggaaccct ggtcaccgtc tcctca 756

<210> 401

<211> 720

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 401

gccgaggtgc agctgttgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 60

atctcctgta gggattctgg acacaccttt ggcgactact ggatcggctg ggtgcgccag 120

atgcccggga aaggcctgga gtggatgggg atcatttatc ctgatgactc tgatactaga 180

tatagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 240

tacctacagt ggaacagcct gaaggcctcg gacagcgcca tatattactg tgcggcccgg 300

ggcagtggct ggtacagtga tgcttttgat gcctggggcc aagggaccac ggtcaccgtc 360

tcctcaggag gcggtggctc cggaggggga ggaagcgaca tccagatgac ccagtctcca 420

tcctccctgt ctgcatctgt aggagacaga gtcaccatca cttgccgggc aagtcagagc 480

attagcacct atttaaattg gtatcagcag aaaccaggga aagcccctaa gctcctgatc 540

tatgctgcat ccagtttgca aagtggggtc ccatcaaggt tcagtggcag tggatctggg 600

acagatttca ccctcaccat cagcagtctg caacctgaag attttgcaac ttactcctgt 660

caacagagtt acagttaccc catcaccttc ggccaaggga cacgactgga gattaaacgt 720

<210> 402

<211> 735

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 402

gaaattgtgc tgactcagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggagg gagcggaggg 360

ggaggaagcg ccgaggtgca gctggtggag tctgggggag gcttggtcca gccggggggg 420

tccctgagac tctcctgttc agcctctgga ttcaccttca atgactatgc tatgcactgg 480

gtccgccagg ctccagggaa gggactggaa tttgtttcag ctattaacag taatgggggt 540

agcacatatt acgcagactc agtgaagggc agattcacca tctccagaga caattccaag 600

aacacgctct atatccaaat gagcagtctg agacctgagg acacggctgt gtattactgt 660

gtgaaagtgg gggggagcag cagctggtac gggtttgact actggggcca gggaaccctg 720

gtcaccgtct cctca 735

<210> 403

<211> 744

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 403

gatgttgtga tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctcg gacttttggc 300

caggggacca aggtggagat caaacgtgga ggcggtggct ccggaggcgg agggagcgga 360

gggggaggaa gcgccgaggt gcagctggtg cagtctgggg gaggcttggt aaagcctggg 420

gggtccctta gactctcctg tgcagcctct ggattcactt tcagtaacgc ctggatgagc 480

tgggtccgcc aggctccagg gaaggggctg gagtgggttg gccgtattaa aagcaaaact 540

gatggtggga caacagacta cgctgcaccc gtgaaaggca gattcaccat ctcaagagat 600

gattcaaaaa acacgctgta tctgcaaatg aacagcctga aaaccgagga cacagccgtg 660

tattactgta ccacagagcc gggtatagca gtggctggcc ttgggggcta ctggggccag 720

ggaaccctgg tcaccgtctc ctca 744

<210> 404

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 404

gccgaggtgc agctggtgga gtctggggga ggcttggtaa agcctggggg gtcccttaga 60

ctctcctgtg cagcctctgg attcactttc agtaacgcct ggatgagctg ggtccgccag 120

gctccaggga aggggctgga gtgggttggc cgtattaaaa gcaaaactga tggtgggaca 180

acagactacg ctgcacccgt gaaaggcaga ttcaccatct caagagatga ttcaaaaaac 240

acgctgtatc tgcaaatgaa cagcctgaaa accgaggaca cagccgtgta ttactgtacc 300

acagttactg gtgttctccc caagggcggc tactggggcc agggaaccct ggtcaccgtc 360

tcctcaggag gcggtggctc cggaggcggg ggtagtggag gcggagggag cggaggggga 420

ggaagcgaaa cgacactcac gcagtctcca ggcaccctgt ctttgtctcc aggggaagga 480

gccaccctct cctgcagggc cagtcagagt gttagtagtg gcaacttagc ctggtatcag 540

cagaaacctg gccagcctcc caggctcctc atctttggtg catccagcag ggccactggc 600

atcccggaca ggttcagtgg cagtgggtct gggctagact tcactctcac aatcagcaga 660

ctggagcctg aggattttgc agtgtattac tgccaacaat atggcagttc acccatgtac 720

agttttggcc aggggaccaa ggtggagatc aaacgt 756

<210> 405

<211> 753

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 405

gacatcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gaacattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcatcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctcgaac gctcactttc 300

ggcggaggga ccaagctgga gatcaaacgt ggaggcggtg gctccggagg cgggggtagt 360

ggaggcggag ggagcggagg gggaggaagc gccgaggtgc agctgttgca gtctggggga 420

ggcttggtac agcctggagg gtccatgaga ctctcctgtg cggcctctgg attccccttc 480

agtggttatt atggattgaa ctgggtccgc cgggctccag gaaaggggct ggagtgggtt 540

gcatacattg ctggtcgcga cagttccata tactacgcgg actctgtgag gggccgcttc 600

acagtctcca gagacagcga cagggattca gtgtttctgc agatgaacag actgagagtc 660

gacgacacgg ctgtttacta ctgtgcgaca gatgggagtg gccacttggg ctttgactac 720

tggggccagg gaaccctggt caccgtctcc tca 753

<210> 406

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 406

gatgttgtga tgactcagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagagacct 120

gggaaagccc ctaagttcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggctc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgatcac cttcggccaa 300

gggacacgac tggagattaa acgtggaggc ggtggctccg gagggggagg aagcgcccag 360

gtcaacttaa gggagtctgg gggaggcttg gtacagcctg gagggtccct gagactctcc 420

tgtgcagcct ctcgattcac cttcagtagt tatgaaatga actgggtccg ccaggctcca 480

gggaaggggc tggagtgggt ttcatacatt agtagtagtg gtactaccat gtactacgca 540

gactctgtga agggccgatt caccatctcc agagacaacg ccaagaactc actgtatctg 600

caaatgaaca gcctgagagc cgaggacacg gctgtttatt actgtgcgag agacggcatg 660

cggttagtgg atcggggtgc ttttgatatc tggggccaag ggacaatggt caccgtctct 720

tca 723

<210> 407

<211> 786

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 407

gaaattgtgc tgactcagtc tccatcctcc ctctctgcat ctgttggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcatttcc accaatttaa attggtatca acagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcgtccagtt tccaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacaaca cccctccgta cacttttggc 300

caggggacca aggtggagat caaacgtgga ggcggtggct ccggcggtgg tgggtccggt 360

ggcggcggat ccggaggcgg gggtagtgga ggcggaggga gcggaggggg aggaagcgcc 420

caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 480

tcctgtgcag cctctcgatt caccttcagt agttatgaaa tgaactgggt ccgccaggct 540

ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtactac catgtactac 600

gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 660

ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagagacggc 720

atgcggttag tggatcgggg tgcttttgat atctggggcc aagggacaat ggtcaccgtc 780

tcttca 786

<210> 408

<211> 741

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 408

gacatcgtga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctta cacttttggc 300

caggggacca agctggagat caaacgtgga ggcggtggct ccggaggggg aggaagcgcc 360

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 420

tcctgcaagg cttctggagg caccttcagc agatatgaaa tcaactgggt gcggcaggcc 480

cctggacaag ggcttgagtg gatgggaagg atcatcccta accttggtat agcaaactat 540

gcacaggagc tccagggcag agtcacgatt atcgcggaca aatccacgag tacagcctac 600

atggaattga gcagcctgag atctgacgac acggccgtct attactgtgc gagggggtct 660

aagtggctgg cacccccccc ctcctactac tactacggta tggacgtctg gggccaaggg 720

accacggtca ccgtctcctc a 741

<210> 409

<211> 768

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 409

gaaacgacac tcacgcagtc tccgtccatc ctgtctgcat ctgtaggaga cagagtcacc 60

ttcacttgcc gggccagtca gacaattagt aactggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctataag gcgtctattt tagaaactgg ggtcccatca 180

aggttcagcg gcagtggctc tgggacggaa ttcactctca ccatcagcag cgtgcagcct 240

gatgattttg caacttatta ctgccaacaa tatagtgatt acgttacttt tggccagggg 300

accaaagtgg atatcaaacg tggaggcggt ggctccggcg gtggtgggtc cggtggcggc 360

ggatccggag gcgggggtag tggaggcgga gggagcggag ggggaggaag cgcccaggtg 420

cagctgcagg agtctggggg aggcttggta cagcctggca ggtccctgag actctcctgt 480

gcagcctctg gattcacctt tgatgaccat gccatgcact gggtccggca agctccaggg 540

aagggcttgg agtgggtctc aggaatttat cggaggggtg gtgcggtagg ctatgcggac 600

tctgtgaagg gccgattcac catctccaga gacgacgcca agaattccct ctatctggaa 660

atgaacagtc tgagacctga ggacacggcc ttgtattact gtggaagcga tcgctccccg 720

gggggtatgg acgtctgggg ccaagggaca atggtcaccg tctcttca 768

<210> 410

<211> 801

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 410

gacatcgtga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgcc cctgtacact 300

tttggccagg ggaccaagct ggagatcaaa cgtggaggcg gtggctccgg cggtggtggg 360

tccggtggcg gcggatccgg aggcgggggt agtggaggcg gagggagcgg agggggagga 420

agcgcccagg tgcagctggt gcagtctggg gctgaggtga agaagcctgg ggcctcagtg 480

acggtttcct gcaaggcatc tggatacccc ttcctcagtc actatgtcca ctgggtgcga 540

caggcccctg gacaagggct tgagtggatg ggaataatcg acgctcgtgg tggtggcaca 600

agttaccccc tggaattcca gggcagagtc accatgacca gggacacgtc cacgaacaca 660

gtgttcctga aactgaccaa cctgagatct actgacacgg ccgtctatta ttgtgcgaga 720

gatcccgtcg gatatggtgg taaccccggt ggcccctatc ttgacctttg gggccaggga 780

accctggtca ccgtctcctc a 801

<210> 411

<211> 738

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 411

gaaattgtgt tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aggagccgcc 60

ctctcctgca gggccagtca gagtgttagc agtaatgtgg cctggtacca gcagaaacct 120

ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tgtcccagcc 180

aggttccgtg gcggtggggc tgggacagac ttcactctca ccatcagcag cctgcagtct 240

gaagatgttg gtacttatta ctgtcagcag tatactcggg gcagggaaag ttattttggc 300

caggggacca aagtggatat caaacgtgga ggcggtggct ccggaggcgg agggagcgga 360

gggggaggaa gcgcccaggt gcagctgcag gagtcggggg gcggcgtggt ccagcctggg 420

gggtccctga gactctcctg tgcagcctct ggattcacct tcagtaaata tggcatgcac 480

tgggtccgcc aggctccagg caagggactg gaatgggtgg cagttgtctc atataatgga 540

ggtaatgatt tctatgcaga ctccgtgaag ggccgattca ccatttccag agacaattca 600

aagaacacgc tggatttgca aatggacagt ctgaaagctg aggacacggc tttatatttc 660

tgtgcgaaag atcagggagg ctattacttt tacggcatgg acgtctgggg ccaggggacc 720

acggtcaccg tctcctca 738

<210> 412

<211> 720

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 412

gacatcgtga tgacccagtc tccaggcatc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagcgttaac agaaggtact tggcctggta tcagcagaag 120

cctggccagg ctcccagcct cctcatctat ggtgcatcca atagggccac tggcgtccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag caatatggtg gctcaccggg gtacactttt 300

ggccagggga ccaagctgga gatcaaacgt ggaggcagct cccggtcaag cgcccaggtg 360

cagctgcagg agtctggggg aggcttggta cagcctggca ggtccctgag actctcctgt 420

gcagcctctg gattcacctt tgatgattat gccatgcact gggtccggca agctccaggg 480

aagggcctgg agtgggtctc aggtattagt tggaatagtg gtagcatagg ctatgcggac 540

tctgtgaagg gccgattcac catctccaga gacaacgcca agaactccct gtatctgcaa 600

atgaacagtc tgagagctga ggacacggcc ttgtattact gtgcaaaggg ccagagctcc 660

ttcctccccc gaggcggtat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 720

<210> 413

<211> 768

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 413

gaaattgtgt tgacgcagtc tccatcttcc ctgtctgcat ctgtggggga cagagtcacc 60

atttcctgcc gggcaggtca gggaattgga tcctatttaa attggtatca gcagagacca 120

gggaaggccc ctaacctcct gatctctggt ggaaccgatt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtgaatc tgggacggac ttcactctca ccatcagtag tctgcaacct 240

gaggattttg caacctactt ctgtcaacag agctacagtt cccctccgac gttcggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gtggcggcgg atccggaggc 360

gggggtagtg gaggcggagg gagcggaggg ggaggaagcg ccgaggtaca gctgcagcag 420

tctgggggag gcttggtaaa gcctgggggg tcccttagac tctcctgtgc agcctctgga 480

ttcactttca gtaacgcctg gatgagctgg gtccgccagg ctccagggaa ggggctggag 540

tgggttggcc gtattaaaag caaaactgat ggtgggacaa cagactacgc tgcacccgtg 600

aaaggcagat tcaccatctc aagagatgat tcaaaaaaca cgctgtatct gcaaatgaac 660

agcctgaaaa ccgaggacac agccgtgtat tactgtacca caggtcccga cgtattacga 720

ttggcaggtg gctactgggg ccagggaacc ctggtcaccg tctcctca 768

<210> 414

<211> 762

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 414

gaaattgtgt tgacgcagtc tccatcttcc gtgtctgctt ctgtgggaga cagagtcact 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctatgct acatccagtt tagagagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcaa cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tccccctcac tttcggcgga 300

gggaccaagg tggagatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgt tgcagtctgg gggaggcttg 420

gtagagcctg gcaggtccct gagactctcc tgtgcaggct ctggattcac ctttggtgat 480

tatgccatgc actgggtccg gcaagctcca gggaagggcc tggagtgggt ctcaggcatt 540

agttggaata gtgatagcat aggccaagcg gactctgtga agggccgatt caccatctcc 600

cgagacaacg ccaagaactc cctgtatctg cagatgaaca gtctgagagc tgaggacgcg 660

gccctgtatt actgtgcaaa agataccacg ggttattact atggtttggg gaggcgcgct 720

atggacgtct ggggccaagg gacaatggtc accgtctcct ca 762

<210> 415

<211> 729

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 415

gacatcgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcctg 60

atcacttgcc gggccagtca gagtattggt agttggttgg cctggtatca gcagaaacca 120

gggagagccc ctaaactcct gatctatgag gcgtctggtt tacaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacacaa ttcactctcg ccatcagcag cctgcaggct 240

gatgattttg caacttatta ctgccaacat cgggaaattt tcggcggagg gaccaagctg 300

gagatcaaac gtggaggcgg tggctccgga ggcggaggga gcggaggggg aggaagcgcc 360

gaggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 420

tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 480

ccagggaagg gcctggagtg ggtctcaggt attagttgga atagtggtag cataggctat 540

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 600

ctgcaaatga acagtctgag agctgaggac acggccttgt attactgtgc aaaagatatg 660

agtgagtggc tggtacgagg agatgctttt gatatctggg gccaagggac aatggtcacc 720

gtctcttca 729

<210> 416

<211> 798

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 416

gacatcgtga tgacccagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

ggaaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gcggtggtgg gtccggtggc 360

ggcggatccg gaggcggggg tagtggaggc ggagggagcg gagggggagg aagcgcccag 420

gtgcagctgc aggagtcggg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 480

tgtgcagcct ctggattcac cttcagtagc tatgctatgc actgggtccg ccaggctcca 540

ggcaaggggc tggagtgggt ggcagttata tcatatgatg gaagcaataa atactacgca 600

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 660

caaatgaaca gcctgagagc tgaggacacg gctgtgtatt actgtgcgag tctagggggg 720

tattgtactg gtggtgtatg cgatgcccca ttaccccttg actactgggg ccagggaacc 780

ctggtcaccg tctcctca 798

<210> 417

<211> 741

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 417

gccgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggggg gtccctgaga 60

ctctcctgtt cagcctctgg attcaccttc agtaactatg ctatgcactg ggtccgccag 120

gctccaggga agggactgga gtatgtttca gctattacta gtaatggggg tagcacatac 180

tacgcagact ccgtgagggg cagattcacc atctccagag acaattccaa gaacacacta 240

tatcttcaat tgagcagtct gagagctgag gacacgggtc tgtattactg tgtgaaagac 300

gaagggggcc ctacggtgac tacgggtccc tttgactact ggggccaggg caccctggtc 360

accgtctcct caggaggcgg tggctccgga ggcggaggga gcggaggggg aggaagcgaa 420

acgacactca cgcagtctcc atcttccctg tctgcatctg taggagacag agtcaccatc 480

acttgccagg cgagtcagga cattaggcac tatttaagtt ggtttcagca gaaaccaggg 540

aaagccccta aggccctgat ctacgaagcg tccaatttgg aaacaggcgt cccatcaaga 600

ttcagtggaa gtggatctgg gacagatttt actttcacca tcagcaacct gcagccacaa 660

gatgttgcaa catattactg tcaacaggct gataagctcc ccctcacttt cggcggaggg 720

accaagctgg aaatcaaacg t 741

<210> 418

<211> 774

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 418

gatgttgtga tgactcagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaagc tggagatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtgcagctgc aggagtctgg gggaggcctg 420

gtccagcctg gggggtccct gagactctcg tgtgcagcct ctggattcac cttcagtagc 480

tatagcatga actgggtccg ccaggctcca gggaaggggc tggagtgggt ctcatccatt 540

agtagtcgta gtaatgacat atactacgca gactcagtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 660

gctgtgtatt actgtgcgag accctattac tttgatagta gtggttatgt caattactac 720

tcctactacg gtttggacgt ctggggccaa gggaccacgg tcaccgtctc ctca 774

<210> 419

<211> 750

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 419

gaaattgtgc tgactcagtc tccatcctcc ctctctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc aggtatttaa attggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcagcag agatacagta ttccctacga tttcggccct 300

gggacacgac tggagattaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgt tgcagtctgg agcagaggtg 420

aaaaagcccg gggagtctct gaagatctcc tgtaaggttt ctggatacag cttttccagc 480

tactggatcg gctgggtgcg ccagatgccc gggaaaggcc tggaatggat ggggatcatc 540

tatcctggtg actccaatac cagatacagc ccgtccttcc aaggccaggt ctccttctcg 600

gccgacaagt ccatcagcac cgcctacctg cagtggagca gcctgaaggc ctcggacacc 660

gccatgtatt actgtgcgag gcttatctat ggtgactacg gtgggggtct tgactactgg 720

ggccagggaa ccctggtcac cgtctcctca 750

<210> 420

<211> 750

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 420

gaaattgtgc tgactcagtc tccatcctcc ctctctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc aggtatgtca attggtatca gcaaaaacca 120

gggaaagccc ttacgctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcagcag agatacagta ttccctacga tttcggccct 300

gggacacgac tggagattaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgt tgcagtctgg agcagaggtg 420

aaaaagcccg gggagtctct gaagatctct tgtaaggttt ctggatacag cttttccagc 480

tactggatcg gctgggtgcg ccagatgccc gggaaaggcc tggaatggat ggggatcatc 540

tatcctggtg actccaatac cagatacagc ccgtccttac acggccaggt ctccttctcg 600

gccgacaagt ccatcagcac cgcctacctg cagtggagca gcctgaaggc ctcggacacc 660

gccatgtatt actgtgcgag gcttatctat ggtgactacg gtgggggtct tgactactgg 720

ggccagggaa ccctggtcac cgtctcctca 750

<210> 421

<211> 744

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 421

gaaattgtgc tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccgaccgtg gacgttcggc 300

caagggacca agctggagat caaacgtgga ggcggtggct ccggaggcgg gggtagtgga 360

ggcggaggga gcggaggggg aggaagcgcc gaggtgcagc tgttgcagtc tggggctgag 420

gtgaagaagc ctgggtcctc ggtgaaggtc tcctgcaagg cttctggagg caccttcagc 480

agctatgcta tcagctgggt gcgacaggcc cctggacaag ggcttgagtg gatgggaggg 540

atcatcccta tctttggtac agcaaactac gcacagaagt tccagggcag agtcacgatt 600

accgcggacg aatccacgag cacagcctac atggagctga gcagcctgag atctgaggac 660

acggccgtgt attactgtgc gagggtgtat agcagtggct ggtttgacta ctggggccag 720

ggcaccctgg tcaccgtctc ctca 744

<210> 422

<211> 744

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 422

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccgtccgta cacttttggc 300

caggggacca aggtggagat caaacgtgga ggcggtggct ccggaggcgg gggtagtgga 360

ggcggaggga gcggaggggg aggaagcgcc gaggtacagc tgcagcagtc aggggctgag 420

gtgaagaagc ctgggtcctc ggtgaaggtc tcctgcaagg cttctggagg caccttcagc 480

agctatgcta tcagctgggt gcgacaggcc cctggacaag ggcttgagtg gatgggaggg 540

atcatcccta tctttggtac agcaaactac gcacagaagt tccagggcag agtcacgatt 600

accgcggacg aatccacgag cacagcctac atggagctga gcagcctgag atctgaggac 660

acggccgtgt attactgtgc gagggtgtat agcagtggct ggtttgacta ctggggccag 720

ggaaccctgg tcaccgtctc ctca 744

<210> 423

<211> 786

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 423

gacatcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gcggtggtgg gtccggtggc 360

ggcggatccg gaggcggggg tagtggaggc ggagggagcg gagggggagg aagcgcccag 420

gtgcagctgg tgcagtcagg tccaggactg gtgaagccct cgcagaccct ctcactcacc 480

tgtgccatct ccggggacag tgtctctagc aacagtgctg cttggaactg gatcaggcag 540

tccccatcga gaggccttga gtggctggga aggacatact acaggtccaa gtggtataat 600

gattatgcag tatctgtgaa aagtcgaata accatcaacc cagacacatc caagaaccag 660

ttctccctgc agctgaactc tgtgactccc gaggacacgg ctgtgtatta ctgtgcaaga 720

ttggggtata gcagcagctg gccgtttgac tactggggcc agggaaccct ggtcaccgtc 780

tcctca 786

<210> 424

<211> 735

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 424

gaaacgacac tcacgcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccgtacac ttttggccag 300

gggaccaagc tggagatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtgcagctgc aggagtcggg cccaggactg 420

gtgaagcctt cggagaccct gtccctcacc tgcactgtct ctggttactc catcaccagt 480

cgttcctact ggggctgggt ccggcagtcc ccagggaagg ggctggaatg gcttgggagt 540

ttgtcctgga ctggaagtac tcaatacaac ccgtccctca ggggtcgagt cacaatatca 600

ctggacaggt ccaacagtca attctccctg aggttgacct ctgtgtccgc cgccgacacg 660

gccacttact actgtgtgag agataataga gcccttgact cctggggcca gggaaccctg 720

gtcaccgtct cctca 735

<210> 425

<211> 774

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 425

gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccagg ggtgtacact 300

tttggccagg ggaccaaagt ggatatcaaa cgtggaggcg gtggctccgg cggtggtggg 360

tccggtggcg gcggatccgg aggcgggggt agtggaggcg gagggagcgg agggggagga 420

agcgcccagg tgcagctgca ggagtcgggc ccaggactgg tgaagccttc ggagaccctg 480

tccctcacct gcactgtctc tggttactcc atcaccagtc gttcctactg gggctgggtc 540

cggcagtccc cagggaaggg gctggaatgg cttgggagtt tgtcctggac tggaagtact 600

caatacaacc cgtccctcag gggtcgagtc acaatatcac tggacaggtc caacagtcaa 660

ttctccctga ggttgacctc tgtgtccgcc gccgacacgg ccacttacta ctgtgtgaga 720

gataatagag cccttgactc ctggggccag ggaaccctgg tcaccgtctc ctca 774

<210> 426

<211> 765

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 426

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaacca 120

gggaaagctc ctaagctcct gatctatgat gcctccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacatta cccctcacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtcaacttaa gggagtctgg gggaggcttg 420

gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac ctttagtagc 480

tattggatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ggccaacata 540

aagcaagatg gaagtgagaa atactatgtg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc actgtatctg caaatgaaca gcctgagagc cgaggacacg 660

gctgtgtatt actgtgcgag agcggtgggc gatagcagtg gctggtacca agactattac 720

ggtatggacg tctggggcca agggacaatg gtcaccgtct cttca 765

<210> 427

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 427

gatgttgtga tgactcagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgctcac tttcggcgga 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggagg gagcggaggg 360

ggaggaagcg ccgaggtgca gctggtggag tctgggggag gcttggtaca gcctgggggg 420

tccttgagac tctcctgtgc agcctctgga ttcacctttg cccacgatgc catgagctgg 480

gtccgccagg ctccagggaa ggggctggaa tgggtctcaa ctattactta tggtggtggt 540

ggcacatact acgcagactc cgtgaagggc cggttcacca tctccagaga caattccaag 600

aacacgctat ttctgcaaat gaacggcctg agagccgagg acacggccgt atattcctgt 660

gcgagaggcg gcgtgggggc gtttgactcc tggggccagg gaaccctggt caccgtctcc 720

tca 723

<210> 428

<211> 747

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 428

gatgttgtga tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagta tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccgtatgta cacttttggc 300

caggggacca aggtggagat caaacgtgga ggcggtggct ccggaggcgg gggtagtgga 360

ggcggaggga gcggaggggg aggaagcgcc gaggtgcagc tggtggagtc tgggggaggc 420

ttggtacagc ctggcaggtc cctgagactc tcctgtgcag cctctggatt cacctttgat 480

gattatgcca tgcactgggt ccggcaagct ccagggaagg gcctggagtg ggtctcaggt 540

attagttgga atagtggtag cataggctat gcggactctg tgaagggccg attcaccatc 600

tccagagaca acgccaagaa ctccctgtat ctgcaaatga acagtctgag agctgaggac 660

acggccttgt attactgtgc aaaagatttc acaggggata atgcttttga tatctggggc 720

caagggacaa tggtcaccgt ctcttca 747

<210> 429

<211> 741

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 429

gatgttgtga tgactcagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaaag tggatatcaa acgtggaggc ggtggctccg gaggcggagg gagcggaggg 360

ggaggaagcg ccgaggtaca gctgcagcag tcaggtccag gactggtgaa gccctcgcag 420

accctctcac tcacctgcgc catctccggg gacagtgtca ctgacaacag tgctgcttgg 480

aactggatca ggcagtcccc atcgagaggc ctggagtggc tgggaaggac atactacagg 540

ttcaagtggt ataatgaata tgcagaatct gtgaaaagtc gaataggcat caatccagac 600

atatccaaga accagttctc cctgcagctg agctctgtga ctcccgagga cacggctgtg 660

tattactgtg taagaggggg gacccggtcc tatagcgact ttgactactg gggccaggga 720

accctggtca ccgtctcctc a 741

<210> 430

<211> 747

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 430

gaaattgtgc tgactcagtc tccatcctcc ctgtccgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

cggtttagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ggcctcctac gttcggccaa 300

gggaccaaag tggatatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgcccag gtgcagctgg tgcagtctgg ggctgaggtg 420

aagaagcctg gggcctcagt gaaggtttcc tgcaaggcat ctggatacac cttcaccagc 480

tactatatgc actgggtgcg acaggcccct ggacaagggc ttgagtggat gggaataatc 540

aaccctagtg gtggtagcac aagctacgca cagaagttcc agggcagagt caccatgacc 600

agggacacgt ccacgagcac agtctacatg gagctgagca gcctgagatc tgaggacacg 660

gccgtgtatt actgtgcgag agccgcaccg gagttccgtg gtgcttttga tatctggggc 720

caagggacaa tggtcaccgt ctcttca 747

<210> 431

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 431

gacatccaga tgacccagtc tccttccacc ctgtcagcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt acctggttgg cctggtatca acaaaaacca 120

gggaaagccc ctaacctcct gatctataag gcgtctactt taaaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tatgatagtt ccccgtacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgg tggagtctgg gggaggcttg 420

gtacagcctg gcaggtccct gagactctcc tgtgcagcct ctggattcac ctttgatgat 480

tatgccatgc actgggtccg gcaagctcca gggaagggcc tggagtgggt ctcaggtatt 540

agttggaata gtggtagcat aggctatgcg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc cctgtatctg caaatgaaca gtctgagagc tgaggacacg 660

gccttgtatt actgtgcaaa agatatctcc cctgactgga gcaggggggg atggttcgac 720

ccctggggcc agggcaccct ggtcaccgtc tcctca 756

<210> 432

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 432

gacatccaga tgacccagtc tccttccacc ctgtcagcat ctgtaggaga cagagtcacc 60

atcacttgcc gcgccagtca gagtattagt acctggttgg cctggtatca acaaaaacca 120

gtgaaagccc ctaacctcct gatctataag gcgtctactt taaaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tatgatagtt ccccgtacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgg tggagtctgg gggaggcttg 420

gtacagcctg gcaggtccct gagactctcc tgtgcagcct ctggattcac ctttgatgat 480

tatgccatgc actgggtccg gcaagctcca gggaagggcc tggagtgggt ctcaggtatt 540

agttggaata gtggtagcat aggctatgcg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc cctgtatctg caaatgaaca gtctgagagc tgaggacacg 660

gccttgtatt actgtgcaaa agatatctcc cctgactgga gcaggggggg atggttcgac 720

ccctggggcc agggcaccct ggtcaccgtc tcctca 756

<210> 433

<211> 756

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 433

gacatccaga tgacccagtc tccttccacc ctgtcagcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt acctggttgg cctggtatca acaacaacca 120

gggaaagccc ctaacctcct gatctataag gcgtctactt taaaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tatgatagtt ccccgtacac ttttggccag 300

gggaccaagc tggaaatcaa acgtggaggc ggtggctccg gaggcggggg tagtggaggc 360

ggagggagcg gagggggagg aagcgccgag gtgcagctgg tggagtctgg gggaggcttg 420

gtacagcctg gcaggtccct gagactctcc tgtgcagcct ctggattcac ctttgatgat 480

tatgccatgc actgggtccg gcaagctcca gggaagggcc tggagtgggt ctcaggtatt 540

agttggaata gtggtagcat aggctatgcg gactctgtga agggccgatt caccatctcc 600

agagacaacg ccaagaactc cctgtatctg caaatgaaca gtctgagagc tgaggacacg 660

gccttgtatt actgtgcaaa agatatctcc cctgactgga gcaggggggg atggttcgac 720

ccctggggcc agggcaccct ggtcaccgtc tcctca 756

<210> 434

<211> 753

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 434

gacatcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gaacattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcatcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctcgaac gctcactttc 300

ggcggaggga ccaagctgga gatcaaacgt ggaggcggtg gctccggagg cgggggtagt 360

ggaggcggag ggagcggagg gggaggaagc gccgaggtgc agctgttgca gtctggggga 420

ggcttggtac agcctggagg gtccatgaga ctctcctgtg cggcctctgg attccccttc 480

agtggttatt atggattgaa ctgggtccgc cgggctccag gaaaggggct ggagtgggtt 540

gcatacattg ctggtcgcga cagttccata tactacgcgg actctgtgag gggccgcttc 600

acagtctcca gagacagcga cagggattca gtgtttctgc agatgaacag actgagagtc 660

gacgacacgg ctgtttacta ctgtgcgaca gatgggagtg gccacttggg ctttgactac 720

tggggccagg gaaccctggt caccgtctcc tca 753

<210> 435

<211> 753

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<220>

<221> misc_feature

<222> (15)..(15)

<223> n is a, c, g or t

<400> 435

gacatcgtga tgacncagtc tccatcctcc ctgtgtgcat ctgtaggaga cagagtcacc 60

atcacttgcc ggtcaagtca gaacattagc agatatttaa cttggtatca gcagaaacca 120

gggatagccc gtaaactcct gatgtatgtt gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcatcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctcgaac gctcactttc 300

ggcggaggga ccaagctgga gatcaaacgt ggaggcggtg gctccggagg cgggggtagt 360

ggaggcggag ggagcggagg gggaggaagc gccgaggtgc agctgttgca gtctggggga 420

ggcttggtac agcctggagg gtccatgaga ctctcctgtg cggcctctgg attccccttc 480

agtggttatt atggattgaa ctgggtccgc cgggctccag gaaaggggct ggagtgggtt 540

gcatacattg ctggtcgcga cagttccata tactacgcgg actctgtgag gggccgcttc 600

acagtctcca gagacagcga cagggattca gtgtttctgc agatgaacag actgagagtc 660

gacgacacgg ctgtttacta ctgtgcgaca gatgggagtg gccacttggg ctttgactac 720

tggggccagg gaaccctggt caccgtctcc tca 753

<210> 436

<211> 768

<212> DNA

<213> artificial sequence

<220>

<223> scFv

<400> 436

gaaaggacac tcacgcagtc tccgttcatc ctgtctgcat ctgtaggaga cagagtcacc 60

ttcacttgcc gggccagtca gacaattagt aactggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctataag gcgtctattt tagaaactgg ggtcccatca 180

aggttcagcg gcagtggctc tgggacggaa ttcactctca ccatcagcag cgtgcagcct 240

gatgattttg caacttatta ctgccaacaa tatagtgatt acgttacttt tggccagggg 300

accaaagtgg atatcaaacg tggaggcggt ggctccggcg gtggtgggtc cggtggcggc 360

ggatccggag gcgggggtag tggaggcgga gggagcggag ggggaggaag cgcccaggtg 420

cagctgcagg agtctggggg aggcttggta cagcctggca ggtccctgag actctcctgt 480

gcagcctctg gattcacctt tgatgaccat gccatgcact gggtccggca agctccaggg 540

aagggcttgg agtgggtctc aggaatttat cggaggggtg gtgcggtagg ctatgcggac 600

tctgtgaagg gccgattcac catctccaga gacgacgcca agaattccct ttatctggaa 660

atgaacagta tgagacctga ggacacggcc ttgtattact gtggaagcga tcgctccccg 720

gggggtatgg acgtctgggg ccaagggaca atggtcaccg tctcctca 768

<210> 437

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 437

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

145 150 155 160

Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Ser Ile Ser Ser Arg Ser Asn Asp Ile Tyr Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Pro Tyr Tyr Phe Asp Ser Ser Gly Tyr Val Asn Tyr Tyr

225 230 235 240

Ser Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

245 250 255

Ser Ser

<210> 438

<211> 250

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 438

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Ile Pro Tyr

85 90 95

Asp Phe Gly Pro Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Ser Ser

145 150 155 160

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asn Thr Arg Tyr Ser Pro Ser

180 185 190

Phe Gln Gly Gln Val Ser Phe Ser Ala Asp Lys Ser Ile Ser Thr Ala

195 200 205

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

210 215 220

Cys Ala Arg Leu Ile Tyr Gly Asp Tyr Gly Gly Gly Leu Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 439

<211> 248

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 439

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Arg Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser

145 150 155 160

Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

165 170 175

Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln

180 185 190

Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr

195 200 205

Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Ala Arg Val Tyr Ser Ser Gly Trp Phe Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 440

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 440

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Asn Gln Gly Ile Ser Ser Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Asn Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu

130 135 140

Val Lys Lys Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly

145 150 155 160

Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met Pro Gly

165 170 175

Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Glu Ser Asp Ile

180 185 190

Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys

195 200 205

Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp

210 215 220

Thr Ala Met Tyr Tyr Cys Ala Arg Gln Arg Tyr Trp Tyr Phe Asp Leu

225 230 235 240

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 441

<211> 243

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 441

Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Ser Thr Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Gly Gly Ser Ser Arg Ser Ser Ala Gln Val Gln Leu Val Gln

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Ala Val Ser Gly Asp Ser Ile Arg Gly His Tyr Trp Ser Trp Ile Arg

145 150 155 160

Gln Thr Ala Gly Lys Gly Leu Glu Trp Ile Gly Tyr Thr Ser Asp Thr

165 170 175

Gly Asp Thr Lys Tyr Asn Pro Ser Leu Gly Ser Arg Val Thr Met Ser

180 185 190

Leu Asp Thr Ser Lys Asn Gln Leu Ser Leu Asn Leu Arg Ser Val Thr

195 200 205

Ala Thr Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Val Asp Pro Glu Asp

210 215 220

Pro Ser Gly Ala Leu Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210> 442

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 442

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

145 150 155 160

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Ser Lys Gly Lys Gly Pro Phe Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 443

<211> 260

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 443

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

130 135 140

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

145 150 155 160

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Arg

165 170 175

Ser Tyr Trp Gly Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp

180 185 190

Leu Gly Ser Leu Ser Trp Thr Gly Ser Thr Gln Tyr Asn Pro Ser Leu

195 200 205

Arg Gly Arg Val Thr Ile Ser Leu Asp Arg Ser Asn Ser Gln Phe Ser

210 215 220

Leu Arg Leu Thr Ser Val Ser Ala Ala Asp Thr Ala Thr Tyr Tyr Cys

225 230 235 240

Val Arg Asp Asn Arg Ala Leu Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 444

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 444

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Gly Val Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val

130 135 140

Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu

145 150 155 160

Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Arg Ser Tyr

165 170 175

Trp Gly Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly

180 185 190

Ser Leu Ser Trp Thr Gly Ser Thr Gln Tyr Asn Pro Ser Leu Arg Gly

195 200 205

Arg Val Thr Ile Ser Leu Asp Arg Ser Asn Ser Gln Phe Ser Leu Arg

210 215 220

Leu Thr Ser Val Ser Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Val Arg

225 230 235 240

Asp Asn Arg Ala Leu Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser

<210> 445

<211> 255

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 445

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Thr Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Asn Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

145 150 155 160

Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Ala Val Gly Asp Ser Ser Gly Trp Tyr Gln Asp Tyr Tyr

225 230 235 240

Gly Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250 255

<210> 446

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 446

Asp Val Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp Ala Met Ser Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Thr

165 170 175

Tyr Gly Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn

195 200 205

Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys Ala Arg Gly Gly

210 215 220

Val Gly Ala Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser

<210> 447

<211> 266

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 447

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Gln Ala Glu Asp Val Gly Val Tyr Phe Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

130 135 140

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

145 150 155 160

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

165 170 175

Pro Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

180 185 190

Ala Val Ile Ser His Asp Glu Ser Asn Lys Tyr Tyr Ala Asp Ser Val

195 200 205

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

Ala Arg Glu Arg Gly Gly Gly Tyr Ser Tyr Gly Ile Gly Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265

<210> 448

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 448

Glu Ile Val Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Tyr His Thr Tyr Trp Thr

85 90 95

Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

115 120 125

Glu Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

130 135 140

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

145 150 155 160

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

165 170 175

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

180 185 190

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

195 200 205

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Asp Arg Ser Glu Arg Ala Phe Asp Ile Trp Gly Gln Gly

225 230 235 240

Thr Met Val Thr Val Ser Ser

245

<210> 449

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 449

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ile

85 90 95

Thr Phe Gly Arg Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu

115 120 125

Leu Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ala Ser Val Lys Val

130 135 140

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Asn Tyr Ile His Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Met Gly Trp Ile Asn

165 170 175

Pro Asn Thr Asp Gly Thr Arg Tyr Ser Pro Asn Phe Gln Gly Arg Leu

180 185 190

Thr Phe Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr Leu Glu Leu Asn

195 200 205

Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Thr

210 215 220

Thr Gly Asn Ala Phe His Ile Trp Gly Gln Gly Thr Met Val Thr Val

225 230 235 240

Ser Ser

<210> 450

<211> 245

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 450

Ala Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser

20 25 30

Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gln Pro Gly Ala Arg Phe Asp Pro Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu

130 135 140

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Ala Cys Arg Ala Ser Gln

145 150 155 160

Ser Ile Ser Gly Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

165 170 175

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

180 185 190

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

195 200 205

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

210 215 220

Tyr Ser Ser Pro Gln Leu Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 451

<211> 253

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 451

Asp Val Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Gly Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Val Glu Ser Gly Gly

130 135 140

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

145 150 155 160

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

165 170 175

Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser

180 185 190

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

195 200 205

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Pro Glu Met Ala Thr Ile

225 230 235 240

His Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 452

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 452

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Ile Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Phe Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

130 135 140

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

145 150 155 160

Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

180 185 190

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

195 200 205

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Ser Asp Gly Tyr Ser Tyr Pro Arg His Phe Asp

225 230 235 240

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 453

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 453

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Arg Ile Gly Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Val

130 135 140

Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser

145 150 155 160

Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val

165 170 175

Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro

180 185 190

Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr

195 200 205

Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser

210 215 220

Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Pro Gly

225 230 235 240

Trp Gly Gly Pro Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser

<210> 454

<211> 238

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 454

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Ala

115 120 125

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

130 135 140

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro

145 150 155 160

Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp

165 170 175

Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

180 185 190

Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

195 200 205

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Pro Gly Trp Gly Gly Pro

210 215 220

Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

225 230 235

<210> 455

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 455

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Gly Ile Gly Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Val

130 135 140

Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser

145 150 155 160

Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val

165 170 175

Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro

180 185 190

Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr

195 200 205

Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser

210 215 220

Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Pro Gly

225 230 235 240

Trp Gly Gly Pro Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser

<210> 456

<211> 259

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 456

Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Gln

130 135 140

Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Val Thr

145 150 155 160

Cys Thr Phe Ser Gly Gly Ser Ile Ser Ser Ser Gly Tyr Tyr Trp Asn

165 170 175

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

180 185 190

Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Arg Arg Leu

195 200 205

Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser

210 215 220

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu

225 230 235 240

Arg Leu Tyr Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

245 250 255

Val Ser Ser

<210> 457

<211> 249

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 457

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Arg Met

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

130 135 140

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

145 150 155 160

Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

165 170 175

Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp

180 185 190

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

195 200 205

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr

210 215 220

Tyr Cys Ala Lys Asp Phe Thr Gly Asp Asn Ala Phe Asp Ile Trp Gly

225 230 235 240

Gln Gly Thr Met Val Thr Val Ser Ser

245

<210> 458

<211> 250

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 458

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Tyr Ser Phe Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Ala Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Leu Ala Met Val Gln Gly Ala Pro Ala Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 459

<211> 245

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 459

Ala Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp

20 25 30

Ser Tyr Met Thr Trp Ile Arg Gln Thr Pro Gly Gln Ser Leu Glu Trp

35 40 45

Val Ser Tyr Ile Ser Asn Ser Gly Asp Ala Ile Tyr Tyr Ala Gly Ser

50 55 60

Val Arg Gly Arg Phe Ile Val Ser Arg Asp Asn Ser Lys Asn Leu Leu

65 70 75 80

Phe Leu Gln Met Asn Asn Leu Arg Ala Asp Asp Ser Ala Thr Tyr Tyr

85 90 95

Cys Thr Thr Gly Lys Arg Ser Tyr Arg Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Tyr Ser Thr Pro Gln Thr Phe Gly Gln Gly Thr Arg

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 460

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 460

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp

145 150 155 160

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Ala Lys Asp Ile Ser Pro Asp Trp Ser Arg Gly Gly Trp Phe Asp

225 230 235 240

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 461

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 461

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp

145 150 155 160

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Ala Lys Asp Ile Ser Pro Asp Trp Ser Arg Gly Gly Trp Phe Asp

225 230 235 240

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 462

<211> 240

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 462

Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

1 5 10 15

Glu Ser Leu Lys Ile Ser Cys Arg Asp Ser Gly His Thr Phe Gly Asp

20 25 30

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser

50 55 60

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

65 70 75 80

Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Ser Ala Ile Tyr Tyr

85 90 95

Cys Ala Ala Arg Gly Ser Gly Trp Tyr Ser Asp Ala Phe Asp Ala Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Ser Cys Gln Gln Ser Tyr

210 215 220

Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

225 230 235 240

<210> 463

<211> 245

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 463

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ser Ala Ser Gly Phe Thr Phe Asn Asp Tyr Ala Met His Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ser Ala Ile Asn

165 170 175

Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Ile Gln Met Ser

195 200 205

Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Lys Val Gly

210 215 220

Gly Ser Ser Ser Trp Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser

245

<210> 464

<211> 248

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 464

Asp Val Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln

115 120 125

Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp Met Ser

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile

165 170 175

Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro Val Lys

180 185 190

Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu

195 200 205

Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr

210 215 220

Thr Glu Pro Gly Ile Ala Val Ala Gly Leu Gly Gly Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 465

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 465

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn

20 25 30

Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala

50 55 60

Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn

65 70 75 80

Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Thr Thr Val Thr Gly Val Leu Pro Lys Gly Gly Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr

130 135 140

Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Gly

145 150 155 160

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Gly Asn Leu

165 170 175

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Phe

180 185 190

Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Leu Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

210 215 220

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Met Tyr

225 230 235 240

Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

245 250

<210> 466

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 466

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ile Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Arg

85 90 95

Thr Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Met Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe

145 150 155 160

Ser Gly Tyr Tyr Gly Leu Asn Trp Val Arg Arg Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ala Tyr Ile Ala Gly Arg Asp Ser Ser Ile Tyr Tyr

180 185 190

Ala Asp Ser Val Arg Gly Arg Phe Thr Val Ser Arg Asp Ser Asp Arg

195 200 205

Asp Ser Val Phe Leu Gln Met Asn Arg Leu Arg Val Asp Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Thr Asp Gly Ser Gly His Leu Gly Phe Asp Tyr

225 230 235 240

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 467

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 467

Asp Val Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Ala Gln Val Asn Leu Arg Glu Ser Gly Gly

115 120 125

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

130 135 140

Arg Phe Thr Phe Ser Ser Tyr Glu Met Asn Trp Val Arg Gln Ala Pro

145 150 155 160

Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Thr Thr

165 170 175

Met Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

180 185 190

Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

195 200 205

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly Met Arg Leu Val Asp

210 215 220

Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

225 230 235 240

Ser

<210> 468

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 468

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Asn

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Phe Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Thr Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln Leu

130 135 140

Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

145 150 155 160

Ser Cys Ala Ala Ser Arg Phe Thr Phe Ser Ser Tyr Glu Met Asn Trp

165 170 175

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser

180 185 190

Ser Ser Gly Thr Thr Met Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

195 200 205

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

210 215 220

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly

225 230 235 240

Met Arg Leu Val Asp Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr

245 250 255

Met Val Thr Val Ser Ser

260

<210> 469

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 469

Asp Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln Leu Val Gln Ser Gly

115 120 125

Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala

130 135 140

Ser Gly Gly Thr Phe Ser Arg Tyr Glu Ile Asn Trp Val Arg Gln Ala

145 150 155 160

Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Ile Pro Asn Leu Gly

165 170 175

Ile Ala Asn Tyr Ala Gln Glu Leu Gln Gly Arg Val Thr Ile Ile Ala

180 185 190

Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser

195 200 205

Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser Lys Trp Leu Ala

210 215 220

Pro Pro Pro Ser Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly

225 230 235 240

Thr Thr Val Thr Val Ser Ser

245

<210> 470

<211> 256

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 470

Glu Thr Thr Leu Thr Gln Ser Pro Ser Ile Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ile Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Val Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Tyr Val Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln Leu Gln Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Phe Thr Phe Asp Asp His Ala Met His Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Tyr Arg Arg

180 185 190

Gly Gly Ala Val Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

195 200 205

Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr Leu Glu Met Asn Ser Leu

210 215 220

Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Gly Ser Asp Arg Ser Pro

225 230 235 240

Gly Gly Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250 255

<210> 471

<211> 267

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 471

Asp Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Pro Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val

130 135 140

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

145 150 155 160

Thr Val Ser Cys Lys Ala Ser Gly Tyr Pro Phe Leu Ser His Tyr Val

165 170 175

His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile

180 185 190

Ile Asp Ala Arg Gly Gly Gly Thr Ser Tyr Pro Leu Glu Phe Gln Gly

195 200 205

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Asn Thr Val Phe Leu Lys

210 215 220

Leu Thr Asn Leu Arg Ser Thr Asp Thr Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Asp Pro Val Gly Tyr Gly Gly Asn Pro Gly Gly Pro Tyr Leu Asp Leu

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265

<210> 472

<211> 246

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 472

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Gly Ala Ala Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Arg Gly

50 55 60

Gly Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Val Gly Thr Tyr Tyr Cys Gln Gln Tyr Thr Arg Gly Arg Glu

85 90 95

Ser Tyr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln

115 120 125

Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr Gly Met His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Val

165 170 175

Ser Tyr Asn Gly Gly Asn Asp Phe Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asp Leu Gln Met

195 200 205

Asp Ser Leu Lys Ala Glu Asp Thr Ala Leu Tyr Phe Cys Ala Lys Asp

210 215 220

Gln Gly Gly Tyr Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

225 230 235 240

Thr Val Thr Val Ser Ser

245

<210> 473

<211> 240

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 473

Asp Ile Val Met Thr Gln Ser Pro Gly Ile Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn Arg Arg

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ser Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Gly Ser Pro

85 90 95

Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly

100 105 110

Ser Ser Arg Ser Ser Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly

115 120 125

Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile

165 170 175

Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

180 185 190

Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

195 200 205

Thr Ala Leu Tyr Tyr Cys Ala Lys Gly Gln Ser Ser Phe Leu Pro Arg

210 215 220

Gly Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235 240

<210> 474

<211> 256

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 474

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Gly Gln Gly Ile Gly Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Ser Gly Gly Thr Asp Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Tyr Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Gln Gln Ser Gly Gly Gly

130 135 140

Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

145 150 155 160

Phe Thr Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly

165 170 175

Lys Gly Leu Glu Trp Val Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly

180 185 190

Thr Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg

195 200 205

Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

210 215 220

Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr Gly Pro Asp Val Leu Arg

225 230 235 240

Leu Ala Gly Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250 255

<210> 475

<211> 254

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 475

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Glu Pro Gly

130 135 140

Arg Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Gly Asp

145 150 155 160

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Gly Ile Ser Trp Asn Ser Asp Ser Ile Gly Gln Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Leu Tyr Tyr

210 215 220

Cys Ala Lys Asp Thr Thr Gly Tyr Tyr Tyr Gly Leu Gly Arg Arg Ala

225 230 235 240

Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250

<210> 476

<211> 243

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 476

Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Leu Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Glu Ala Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Ala Ile Ser Ser Leu Gln Ala

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Arg Glu Ile Phe Gly Gly

85 90 95

Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu Val Gln Ser Gly

115 120 125

Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala

130 135 140

Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala

145 150 155 160

Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly

165 170 175

Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

180 185 190

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Met Ser Glu Trp Leu

210 215 220

Val Arg Gly Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

225 230 235 240

Val Ser Ser

<210> 477

<211> 266

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 477

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln Leu Gln

130 135 140

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val

165 170 175

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser Tyr

180 185 190

Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

195 200 205

Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

210 215 220

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Leu Gly Gly

225 230 235 240

Tyr Cys Thr Gly Gly Val Cys Asp Ala Pro Leu Pro Leu Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265

<210> 478

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 478

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Asn

20 25 30

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

Val Ser Ala Ile Thr Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Gly Leu Tyr Tyr

85 90 95

Cys Val Lys Asp Glu Gly Gly Pro Thr Val Thr Thr Gly Pro Phe Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr Thr Leu Thr

130 135 140

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

145 150 155 160

Thr Cys Gln Ala Ser Gln Asp Ile Arg His Tyr Leu Ser Trp Phe Gln

165 170 175

Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Glu Ala Ser Asn

180 185 190

Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Phe Thr Ile Ser Asn Leu Gln Pro Gln Asp Val Ala Thr

210 215 220

Tyr Tyr Cys Gln Gln Ala Asp Lys Leu Pro Leu Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg

245

<210> 479

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 479

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

145 150 155 160

Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Ser Ile Ser Ser Arg Ser Asn Asp Ile Tyr Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Pro Tyr Tyr Phe Asp Ser Ser Gly Tyr Val Asn Tyr Tyr

225 230 235 240

Ser Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

245 250 255

Ser Ser

<210> 480

<211> 250

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 480

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Ile Pro Tyr

85 90 95

Asp Phe Gly Pro Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Ser Ser

145 150 155 160

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asn Thr Arg Tyr Ser Pro Ser

180 185 190

Phe Gln Gly Gln Val Ser Phe Ser Ala Asp Lys Ser Ile Ser Thr Ala

195 200 205

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

210 215 220

Cys Ala Arg Leu Ile Tyr Gly Asp Tyr Gly Gly Gly Leu Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 481

<211> 250

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 481

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Leu Thr Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Ile Pro Tyr

85 90 95

Asp Phe Gly Pro Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Ser Ser

145 150 155 160

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asn Thr Arg Tyr Ser Pro Ser

180 185 190

Leu His Gly Gln Val Ser Phe Ser Ala Asp Lys Ser Ile Ser Thr Ala

195 200 205

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

210 215 220

Cys Ala Arg Leu Ile Tyr Gly Asp Tyr Gly Gly Gly Leu Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 482

<211> 248

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 482

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Arg Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser

145 150 155 160

Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

165 170 175

Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln

180 185 190

Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr

195 200 205

Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Ala Arg Val Tyr Ser Ser Gly Trp Phe Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 483

<211> 248

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 483

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Arg Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Ala Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser

145 150 155 160

Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

165 170 175

Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln

180 185 190

Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr

195 200 205

Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Ala Arg Val Tyr Ser Ser Gly Trp Phe Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 484

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 484

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln Leu Val

130 135 140

Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

145 150 155 160

Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn

165 170 175

Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr

180 185 190

Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser

195 200 205

Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln

210 215 220

Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Leu Gly Tyr Ser Ser Ser Trp Pro Phe Asp Tyr Trp Gly Gln Gly Thr

245 250 255

Leu Val Thr Val Ser Ser

260

<210> 485

<211> 245

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 485

Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

130 135 140

Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser

145 150 155 160

Arg Ser Tyr Trp Gly Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu

165 170 175

Trp Leu Gly Ser Leu Ser Trp Thr Gly Ser Thr Gln Tyr Asn Pro Ser

180 185 190

Leu Arg Gly Arg Val Thr Ile Ser Leu Asp Arg Ser Asn Ser Gln Phe

195 200 205

Ser Leu Arg Leu Thr Ser Val Ser Ala Ala Asp Thr Ala Thr Tyr Tyr

210 215 220

Cys Val Arg Asp Asn Arg Ala Leu Asp Ser Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser

245

<210> 486

<211> 258

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 486

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Gly Val Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val

130 135 140

Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu

145 150 155 160

Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Arg Ser Tyr

165 170 175

Trp Gly Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly

180 185 190

Ser Leu Ser Trp Thr Gly Ser Thr Gln Tyr Asn Pro Ser Leu Arg Gly

195 200 205

Arg Val Thr Ile Ser Leu Asp Arg Ser Asn Ser Gln Phe Ser Leu Arg

210 215 220

Leu Thr Ser Val Ser Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Val Arg

225 230 235 240

Asp Asn Arg Ala Leu Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser

<210> 487

<211> 255

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 487

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Thr Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Asn Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

145 150 155 160

Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Ala Val Gly Asp Ser Ser Gly Trp Tyr Gln Asp Tyr Tyr

225 230 235 240

Gly Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250 255

<210> 488

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 488

Asp Val Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala His Asp Ala Met Ser Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Thr

165 170 175

Tyr Gly Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn

195 200 205

Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys Ala Arg Gly Gly

210 215 220

Val Gly Ala Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser

<210> 489

<211> 249

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 489

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Met Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Arg Met

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

130 135 140

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

145 150 155 160

Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

165 170 175

Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp

180 185 190

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

195 200 205

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr

210 215 220

Tyr Cys Ala Lys Asp Phe Thr Gly Asp Asn Ala Phe Asp Ile Trp Gly

225 230 235 240

Gln Gly Thr Met Val Thr Val Ser Ser

245

<210> 490

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 490

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Val Gln Leu

115 120 125

Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu

130 135 140

Thr Cys Ala Ile Ser Gly Asp Ser Val Thr Asp Asn Ser Ala Ala Trp

145 150 155 160

Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg

165 170 175

Thr Tyr Tyr Arg Phe Lys Trp Tyr Asn Glu Tyr Ala Glu Ser Val Lys

180 185 190

Ser Arg Ile Gly Ile Asn Pro Asp Ile Ser Lys Asn Gln Phe Ser Leu

195 200 205

Gln Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val

210 215 220

Arg Gly Gly Thr Arg Ser Tyr Ser Asp Phe Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 491

<211> 249

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 491

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

145 150 155 160

Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Ala Ala Pro Glu Phe Arg Gly Ala Phe Asp Ile Trp Gly

225 230 235 240

Gln Gly Thr Met Val Thr Val Ser Ser

245

<210> 492

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 492

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp

145 150 155 160

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Ala Lys Asp Ile Ser Pro Asp Trp Ser Arg Gly Gly Trp Phe Asp

225 230 235 240

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 493

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 493

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Val Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp

145 150 155 160

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Ala Lys Asp Ile Ser Pro Asp Trp Ser Arg Gly Gly Trp Phe Asp

225 230 235 240

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 494

<211> 252

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 494

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Gln Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp

145 150 155 160

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Ala Lys Asp Ile Ser Pro Asp Trp Ser Arg Gly Gly Trp Phe Asp

225 230 235 240

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 495

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 495

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ile Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Arg

85 90 95

Thr Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Met Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe

145 150 155 160

Ser Gly Tyr Tyr Gly Leu Asn Trp Val Arg Arg Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ala Tyr Ile Ala Gly Arg Asp Ser Ser Ile Tyr Tyr

180 185 190

Ala Asp Ser Val Arg Gly Arg Phe Thr Val Ser Arg Asp Ser Asp Arg

195 200 205

Asp Ser Val Phe Leu Gln Met Asn Arg Leu Arg Val Asp Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Thr Asp Gly Ser Gly His Leu Gly Phe Asp Tyr

225 230 235 240

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 496

<211> 251

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 496

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Cys Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asn Ile Ser Arg Tyr

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Ile Ala Arg Lys Leu Leu Met

35 40 45

Tyr Val Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ile Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Arg

85 90 95

Thr Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Ala Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Met Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe

145 150 155 160

Ser Gly Tyr Tyr Gly Leu Asn Trp Val Arg Arg Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ala Tyr Ile Ala Gly Arg Asp Ser Ser Ile Tyr Tyr

180 185 190

Ala Asp Ser Val Arg Gly Arg Phe Thr Val Ser Arg Asp Ser Asp Arg

195 200 205

Asp Ser Val Phe Leu Gln Met Asn Arg Leu Arg Val Asp Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Thr Asp Gly Ser Gly His Leu Gly Phe Asp Tyr

225 230 235 240

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 497

<211> 256

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 497

Glu Arg Thr Leu Thr Gln Ser Pro Phe Ile Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ile Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Val Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Tyr Val Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Val Gln Leu Gln Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Phe Thr Phe Asp Asp His Ala Met His Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Tyr Arg Arg

180 185 190

Gly Gly Ala Val Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

195 200 205

Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr Leu Glu Met Asn Ser Met

210 215 220

Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Gly Ser Asp Arg Ser Pro

225 230 235 240

Gly Gly Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250 255

<210> 498

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 498

Gly Phe Thr Phe Ser Ser Tyr Ser

1 5

<210> 499

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 499

Gly Tyr Ser Phe Ser Ser Tyr Trp

1 5

<210> 500

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 500

Gly Gly Thr Phe Ser Ser Tyr Ala

1 5

<210> 501

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 501

Gly Tyr Ser Phe Thr Ser Tyr Trp

1 5

<210> 502

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 502

Gly Asp Ser Ile Arg Gly His Tyr

1 5

<210> 503

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 503

Gly Tyr Thr Phe Thr Ser Tyr Tyr

1 5

<210> 504

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 504

Gly Tyr Ser Ile Thr Ser Arg Ser Tyr

1 5

<210> 505

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 505

Gly Phe Thr Phe Ser Ser Tyr Trp

1 5

<210> 506

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 506

Gly Phe Thr Phe Ala His Asp Ala

1 5

<210> 507

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 507

Gly Phe Thr Phe Ser Ser Tyr Pro

1 5

<210> 508

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 508

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr

1 5 10

<210> 509

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 509

Gly Tyr Thr Phe Thr Gly Asn Tyr

1 5

<210> 510

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 510

Gly Phe Thr Val Ser Ser Asn Tyr

1 5

<210> 511

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 511

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 512

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 512

Gly Gly Ser Ile Ser Ser Ser Gly Tyr Tyr

1 5 10

<210> 513

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 513

Gly Phe Thr Phe Asp Asp Tyr Ala

1 5

<210> 514

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 514

Gly Phe Thr Phe Ser Asp Ser Tyr

1 5

<210> 515

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 515

Gly His Thr Phe Gly Asp Tyr Trp

1 5

<210> 516

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 516

Gly Phe Thr Phe Asn Asp Tyr Ala

1 5

<210> 517

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 517

Gly Phe Thr Phe Ser Asn Ala Trp

1 5

<210> 518

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 518

Phe Pro Phe Ser Gly Tyr Tyr Gly

1 5

<210> 519

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 519

Arg Phe Thr Phe Ser Ser Tyr Glu

1 5

<210> 520

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 520

Gly Gly Thr Phe Ser Arg Tyr Glu

1 5

<210> 521

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 521

Gly Phe Thr Phe Asp Asp His Ala

1 5

<210> 522

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 522

Gly Tyr Pro Phe Leu Ser His Tyr

1 5

<210> 523

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 523

Gly Phe Thr Phe Ser Lys Tyr Gly

1 5

<210> 524

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 524

Gly Phe Thr Phe Gly Asp Tyr Ala

1 5

<210> 525

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 525

Gly Phe Thr Phe Ser Asn Tyr Ala

1 5

<210> 526

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 526

Val Ser Ser Asn Ser Ala Ala Trp Asn

1 5

<210> 527

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 527

Val Thr Asp Asn Ser Ala Ala Trp Asn

1 5

<210> 528

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 528

Ile Ser Ser Arg Ser Asn Asp Ile

1 5

<210> 529

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 529

Ile Tyr Pro Gly Asp Ser Asn Thr

1 5

<210> 530

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 530

Ile Ile Pro Ile Phe Gly Thr Ala

1 5

<210> 531

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 531

Ile Tyr Pro Gly Glu Ser Asp Ile

1 5

<210> 532

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 532

Thr Ser Asp Thr Gly Asp Thr

1 5

<210> 533

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 533

Ile Asn Pro Ser Gly Gly Ser Thr

1 5

<210> 534

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 534

Leu Ser Trp Thr Gly Ser Thr

1 5

<210> 535

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 535

Ile Lys Gln Asp Gly Ser Glu Lys

1 5

<210> 536

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 536

Ile Thr Tyr Gly Gly Gly Gly Thr

1 5

<210> 537

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 537

Ile Ser His Asp Glu Ser Asn Lys

1 5

<210> 538

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 538

Ile Tyr Tyr Ser Gly Ser Thr

1 5

<210> 539

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 539

Ile Asn Pro Asn Thr Asp Gly Thr

1 5

<210> 540

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 540

Ile Tyr Ser Gly Gly Ser Thr

1 5

<210> 541

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 541

Ile Ser Gly Ser Gly Gly Ser Thr

1 5

<210> 542

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 542

Ile Ser Tyr Asp Gly Ser Asn Lys

1 5

<210> 543

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 543

Ile Tyr Pro Gly Asp Ser Asp Thr

1 5

<210> 544

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 544

Ile Tyr His Ser Gly Ser Thr

1 5

<210> 545

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 545

Ile Ser Trp Asn Ser Gly Ser Ile

1 5

<210> 546

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 546

Ile Ser Asn Ser Gly Asp Ala Ile

1 5

<210> 547

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 547

Ile Tyr Pro Asp Asp Ser Asp Thr

1 5

<210> 548

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 548

Ile Asn Ser Asn Gly Gly Ser Thr

1 5

<210> 549

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 549

Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr

1 5 10

<210> 550

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 550

Ile Ala Gly Arg Asp Ser Ser Ile

1 5

<210> 551

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 551

Ile Ser Ser Ser Gly Thr Thr Met

1 5

<210> 552

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 552

Ile Ile Pro Asn Leu Gly Ile Ala

1 5

<210> 553

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 553

Ile Tyr Arg Arg Gly Gly Ala Val

1 5

<210> 554

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 554

Ile Asp Ala Arg Gly Gly Gly Thr

1 5

<210> 555

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 555

Val Ser Tyr Asn Gly Gly Asn Asp

1 5

<210> 556

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 556

Ile Ser Trp Asn Ser Asp Ser Ile

1 5

<210> 557

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 557

Ile Thr Ser Asn Gly Gly Ser Thr

1 5

<210> 558

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 558

Tyr Arg Ser Lys Trp Tyr Asn

1 5

<210> 559

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 559

Tyr Arg Phe Lys Trp Tyr Asn

1 5

<210> 560

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 560

Ala Arg Pro Tyr Tyr Phe Asp Ser Ser Gly Tyr Val Asn Tyr Tyr Ser

1 5 10 15

Tyr Tyr Gly Leu Asp Val

20

<210> 561

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 561

Ala Arg Leu Ile Tyr Gly Asp Tyr Gly Gly Gly Leu Asp Tyr

1 5 10

<210> 562

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 562

Ala Arg Val Tyr Ser Ser Gly Trp Phe Asp Tyr

1 5 10

<210> 563

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 563

Ala Arg Gln Arg Tyr Trp Tyr Phe Asp Leu

1 5 10

<210> 564

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 564

Ala Arg Val Asp Pro Glu Asp Pro Ser Gly Ala Leu Leu Asp Leu

1 5 10 15

<210> 565

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 565

Ala Arg Ser Lys Gly Lys Gly Pro Phe Asp Tyr

1 5 10

<210> 566

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 566

Val Arg Asp Asn Arg Ala Leu Asp Ser

1 5

<210> 567

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 567

Ala Arg Ala Val Gly Asp Ser Ser Gly Trp Tyr Gln Asp Tyr Tyr Gly

1 5 10 15

Met Asp Val

<210> 568

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 568

Ala Arg Gly Gly Val Gly Ala Phe Asp Ser

1 5 10

<210> 569

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 569

Ala Arg Glu Arg Gly Gly Gly Tyr Ser Tyr Gly Ile Gly Asp Tyr

1 5 10 15

<210> 570

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 570

Ala Arg Asp Arg Ser Glu Arg Ala Phe Asp Ile

1 5 10

<210> 571

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 571

Ala Arg Asp Thr Thr Gly Asn Ala Phe His Ile

1 5 10

<210> 572

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 572

Ala Arg Asp Gln Pro Gly Ala Arg Phe Asp Pro

1 5 10

<210> 573

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 573

Ala Lys Asp Pro Glu Met Ala Thr Ile His Tyr

1 5 10

<210> 574

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 574

Ala Arg Ser Asp Gly Tyr Ser Tyr Pro Arg His Phe Asp Tyr

1 5 10

<210> 575

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 575

Ala Arg Gln Pro Gly Trp Gly Gly Pro Phe Asp Pro

1 5 10

<210> 576

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 576

Ala Arg Ser Leu Arg Leu Tyr Gly Phe Phe Asp Tyr

1 5 10

<210> 577

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 577

Ala Lys Asp Phe Thr Gly Asp Asn Ala Phe Asp Ile

1 5 10

<210> 578

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 578

Ala Arg Leu Ala Met Val Gln Gly Ala Pro Ala Asp Tyr

1 5 10

<210> 579

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 579

Thr Thr Gly Lys Arg Ser Tyr Arg Val

1 5

<210> 580

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 580

Ala Lys Asp Ile Ser Pro Asp Trp Ser Arg Gly Gly Trp Phe Asp Pro

1 5 10 15

<210> 581

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 581

Ala Ala Arg Gly Ser Gly Trp Tyr Ser Asp Ala Phe Asp Ala

1 5 10

<210> 582

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 582

Val Lys Val Gly Gly Ser Ser Ser Trp Tyr Gly Phe Asp Tyr

1 5 10

<210> 583

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 583

Thr Thr Glu Pro Gly Ile Ala Val Ala Gly Leu Gly Gly Tyr

1 5 10

<210> 584

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 584

Thr Thr Val Thr Gly Val Leu Pro Lys Gly Gly Tyr

1 5 10

<210> 585

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 585

Ala Thr Asp Gly Ser Gly His Leu Gly Phe Asp Tyr

1 5 10

<210> 586

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 586

Ala Arg Asp Gly Met Arg Leu Val Asp Arg Gly Ala Phe Asp Ile

1 5 10 15

<210> 587

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 587

Ala Arg Gly Ser Lys Trp Leu Ala Pro Pro Pro Ser Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

20

<210> 588

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 588

Gly Ser Asp Arg Ser Pro Gly Gly Met Asp Val

1 5 10

<210> 589

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 589

Ala Arg Asp Pro Val Gly Tyr Gly Gly Asn Pro Gly Gly Pro Tyr Leu

1 5 10 15

Asp Leu

<210> 590

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 590

Ala Lys Asp Gln Gly Gly Tyr Tyr Phe Tyr Gly Met Asp Val

1 5 10

<210> 591

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 591

Ala Lys Gly Gln Ser Ser Phe Leu Pro Arg Gly Gly Met Asp Val

1 5 10 15

<210> 592

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 592

Thr Thr Gly Pro Asp Val Leu Arg Leu Ala Gly Gly Tyr

1 5 10

<210> 593

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 593

Ala Lys Asp Thr Thr Gly Tyr Tyr Tyr Gly Leu Gly Arg Arg Ala Met

1 5 10 15

Asp Val

<210> 594

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 594

Ala Lys Asp Met Ser Glu Trp Leu Val Arg Gly Asp Ala Phe Asp Ile

1 5 10 15

<210> 595

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 595

Ala Ser Leu Gly Gly Tyr Cys Thr Gly Gly Val Cys Asp Ala Pro Leu

1 5 10 15

Pro Leu Asp Tyr

20

<210> 596

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 596

Val Lys Asp Glu Gly Gly Pro Thr Val Thr Thr Gly Pro Phe Asp Tyr

1 5 10 15

<210> 597

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 597

Ala Arg Leu Gly Tyr Ser Ser Ser Trp Pro Phe Asp Tyr

1 5 10

<210> 598

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 598

Val Arg Gly Gly Thr Arg Ser Tyr Ser Asp Phe Asp Tyr

1 5 10

<210> 599

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 599

Ala Arg Ala Ala Pro Glu Phe Arg Gly Ala Phe Asp Ile

1 5 10

<210> 600

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 600

Gln Ser Ile Ser Ser Tyr

1 5

<210> 601

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 601

Gln Ser Ile Ser Arg Tyr

1 5

<210> 602

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 602

Gln Gly Ile Ser Ser Phe

1 5

<210> 603

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 603

Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr

1 5 10

<210> 604

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 604

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 605

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 605

Gln Gly Ile Ser Ser Ala

1 5

<210> 606

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 606

Gln Gly Ile Ser Ser Trp

1 5

<210> 607

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 607

Gln Ser Ile Ser Ser Trp

1 5

<210> 608

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 608

Gln Thr Ile Arg Asn Tyr

1 5

<210> 609

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 609

Gln Ser Ile Ser Gly Tyr

1 5

<210> 610

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 610

Gln Ser Val Ser Gly Ser Tyr

1 5

<210> 611

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 611

Pro Arg Ile Gly Asn Asp

1 5

<210> 612

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 612

Pro Gly Ile Gly Asn Asp

1 5

<210> 613

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 613

Gln Ser Ile Ser Thr Trp

1 5

<210> 614

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 614

Gln Ser Ile Ser Thr Tyr

1 5

<210> 615

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 615

Gln Ser Val Ser Ser Gly Asn

1 5

<210> 616

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 616

Gln Asn Ile Ser Ser Tyr

1 5

<210> 617

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 617

Gln Ser Ile Ser Thr Asn

1 5

<210> 618

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 618

Gln Thr Ile Ser Asn Trp

1 5

<210> 619

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 619

Gln Ser Val Ser Ser Asn

1 5

<210> 620

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 620

Gln Ser Val Asn Arg Arg Tyr

1 5

<210> 621

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 621

Gln Gly Ile Gly Ser Tyr

1 5

<210> 622

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 622

Gln Ser Ile Gly Ser Trp

1 5

<210> 623

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 623

Gln Asp Ile Arg His Tyr

1 5

<210> 624

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 624

Gln Asn Ile Ser Arg Tyr

1 5

<210> 625

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 625

Ala Ala Ser

1

<210> 626

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 626

Leu Gly Ser

1

<210> 627

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 627

Ala Thr Ser

1

<210> 628

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 628

Asp Ala Ser

1

<210> 629

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 629

Glu Ala Ser

1

<210> 630

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 630

Gly Ala Ser

1

<210> 631

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 631

Gly Gly Thr

1

<210> 632

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 632

Lys Ala Ser

1

<210> 633

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 633

Val Ala Ser

1

<210> 634

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 634

Gln Gln Ser Tyr Ser Arg Pro Pro Thr

1 5

<210> 635

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 635

Gln Gln Arg Tyr Ser Ile Pro Tyr Asp

1 5

<210> 636

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 636

Gln Gln Ser Tyr Ser Thr Arg Pro Trp Thr

1 5 10

<210> 637

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 637

Gln Gln His Ser Ser Tyr Pro Leu Thr

1 5

<210> 638

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 638

Met Gln Ala Leu Gln Thr Ser Thr Tyr Ser

1 5 10

<210> 639

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 639

Met Gln Ala Leu Gln Thr Pro Tyr Thr

1 5

<210> 640

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 640

Gln Gln Tyr Gly Ser Ser Pro Gly Val Tyr Thr

1 5 10

<210> 641

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 641

Gln Gln Ser Tyr Ile Thr Pro His Thr

1 5

<210> 642

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 642

Gln Gln Ala Asn Ser Phe Pro Leu Thr

1 5

<210> 643

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 643

Met Gln Ala Leu Gln Thr Pro Pro Thr

1 5

<210> 644

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 644

Gln Gln Tyr His Thr Tyr Trp Thr

1 5

<210> 645

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 645

Gln Gln Ser Tyr Ser Thr Pro Ile Thr

1 5

<210> 646

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 646

Gln Gln Ser Tyr Ser Ser Pro Gln Leu Pro Met Tyr Thr

1 5 10

<210> 647

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 647

Gln His Tyr Gly Ser Ser Pro Leu Thr

1 5

<210> 648

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 648

Gln Gln Tyr Gly Ser Ser Pro Met Tyr Thr

1 5 10

<210> 649

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 649

Leu Gln His Asn Asn Tyr Pro Tyr Thr

1 5

<210> 650

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 650

Gln Gln Ala Asn Ser Phe Pro Tyr Thr

1 5

<210> 651

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 651

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

<210> 652

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 652

Gln Gln Ser Tyr Ser Thr Arg Met Tyr Thr

1 5 10

<210> 653

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 653

Gln Gln Ala Tyr Ser Phe Pro Pro Tyr Thr

1 5 10

<210> 654

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 654

Gln Gln Ser Tyr Ser Thr Pro Gln Thr

1 5

<210> 655

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 655

Gln Gln Tyr Asp Ser Ser Pro Tyr Thr

1 5

<210> 656

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 656

Gln Gln Ser Tyr Ser Tyr Pro Ile Thr

1 5

<210> 657

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 657

Gln Gln Tyr Gly Ser Ser Pro Arg Thr

1 5

<210> 658

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 658

Gln Gln Tyr Gly Ser Ser Pro Met Tyr Ser

1 5 10

<210> 659

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 659

Gln Gln Ser Tyr Ser Thr Pro Arg Thr Leu Thr

1 5 10

<210> 660

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 660

Gln Gln Ala Asn Ser Phe Pro Ile Thr

1 5

<210> 661

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 661

Gln Gln Ser Tyr Asn Thr Pro Pro Tyr Thr

1 5 10

<210> 662

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 662

Gln Gln Tyr Gly Ser Ser Pro Tyr Thr

1 5

<210> 663

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 663

Gln Gln Tyr Ser Asp Tyr Val Thr

1 5

<210> 664

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 664

Gln Gln Tyr Gly Ser Ser Pro Pro Leu Tyr Thr

1 5 10

<210> 665

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 665

Gln Gln Tyr Thr Arg Gly Arg Glu Ser Tyr

1 5 10

<210> 666

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 666

Gln Gln Tyr Gly Gly Ser Pro Gly Tyr Thr

1 5 10

<210> 667

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 667

Gln Gln Ser Tyr Ser Ser Pro Pro Thr

1 5

<210> 668

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 668

Gln His Arg Glu Ile

1 5

<210> 669

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 669

Gln Gln Ala Asp Lys Leu Pro Leu Thr

1 5

<210> 670

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 670

Gln Gln Ser Tyr Ser Thr Arg Pro Tyr Thr

1 5 10

<210> 671

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 671

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210> 672

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 672

Gln Gln Ser Tyr Ser Thr Pro Tyr Thr

1 5

<210> 673

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> connector 1

<400> 673

Gly Gly Ser Ser Arg Ser Ser

1 5

<210> 674

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> connector 2

<400> 674

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 675

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> connector 5

<400> 675

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 676

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> TM1

<400> 676

Ala Val Gly Gln Asp Thr Gln Glu Val Ile Val Val Pro His Ser Leu

1 5 10 15

Pro Phe Lys Val Val Val Ile Ser Ala Ile Leu Ala Leu Val Val Leu

20 25 30

Thr Ile Ile Ser Leu Ile Ile Leu Ile Met Leu Trp Gln Lys Lys Pro

35 40 45

Arg

<210> 677

<211> 63

<212> PRT

<213> artificial sequence

<220>

<223> TM2

<400> 677

Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys

1 5 10 15

Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val

20 25 30

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

35 40 45

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His

50 55 60

<210> 678

<211> 85

<212> PRT

<213> artificial sequence

<220>

<223> TM3

<400> 678

Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys

1 5 10 15

Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys

20 25 30

Gln Glu His Phe Pro Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu

35 40 45

Ile Ser Val Asn Gly Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe

50 55 60

Ala Pro Arg Cys Arg Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu

65 70 75 80

Ser Val Arg Pro Val

85

<210> 679

<211> 88

<212> PRT

<213> artificial sequence

<220>

<223> TM4

<400> 679

Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys

1 5 10 15

Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val

20 25 30

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

35 40 45

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Thr

50 55 60

Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg Arg Arg Asn Glu Arg Leu

65 70 75 80

Arg Arg Glu Ser Val Arg Pro Val

85

<210> 680

<211> 452

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(452)

<223> caspase_2 full Length

<400> 680

Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu

1 5 10 15

Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met

20 25 30

His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala

35 40 45

Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp

50 55 60

Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser

65 70 75 80

Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro

85 90 95

Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly

100 105 110

His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val

115 120 125

Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val

130 135 140

Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr

145 150 155 160

Val Glu His Ser Leu Asp Asn Lys Asp Gly Pro Val Cys Leu Gln Val

165 170 175

Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr

180 185 190

Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val

195 200 205

His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val

210 215 220

Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val

225 230 235 240

His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln

245 250 255

Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val

260 265 270

Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly

275 280 285

Lys Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn

290 295 300

Cys Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys

305 310 315 320

Arg Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn

325 330 335

His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys

340 345 350

Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr

355 360 365

Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser

370 375 380

Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp

385 390 395 400

Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp

405 410 415

Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met

420 425 430

Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly

435 440 445

His Pro Pro Thr

450

<210> 681

<211> 496

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(496)

<223> caspase_8 full Length

<400> 681

Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser

1 5 10 15

Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln

20 25 30

Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu

35 40 45

Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu

50 55 60

Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn

65 70 75 80

Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala

85 90 95

Gln Ile Ser Ala Tyr Arg Phe His Phe Cys Arg Met Ser Trp Ala Glu

100 105 110

Ala Asn Ser Gln Cys Gln Thr Gln Ser Val Pro Phe Trp Arg Arg Val

115 120 125

Asp His Leu Leu Ile Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val

130 135 140

Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile

145 150 155 160

Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile

165 170 175

Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu

180 185 190

Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn

195 200 205

Asp Tyr Glu Glu Phe Ser Lys Gly Glu Glu Leu Cys Gly Val Met Thr

210 215 220

Ile Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu Asp

225 230 235 240

Lys Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile

245 250 255

Asn Asn His Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His

260 265 270

Ser Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr

275 280 285

Thr Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pro His His Asp Cys

290 295 300

Thr Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu Met Asp

305 310 315 320

His Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp

325 330 335

Lys Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Ala Pro Ile Tyr Glu

340 345 350

Leu Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys

355 360 365

Pro Lys Val Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys

370 375 380

Gly Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met

385 390 395 400

Asp Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe

405 410 415

Leu Leu Gly Met Ala Thr Val Asn Asn Cys Val Ser Tyr Arg Asn Pro

420 425 430

Ala Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu

435 440 445

Arg Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn

450 455 460

Tyr Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gln Met

465 470 475 480

Pro Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp

485 490 495

<210> 682

<211> 416

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(416)

<223> caspase_9 full Length

<400> 682

Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu

1 5 10 15

Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Ser

20 25 30

Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser

35 40 45

Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr

50 55 60

Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr

65 70 75 80

Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala

85 90 95

Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg

100 105 110

Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro

115 120 125

Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser

130 135 140

Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys

145 150 155 160

Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly

165 170 175

Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg

180 185 190

Arg Phe Ser Ser Pro His Phe Met Val Glu Val Lys Gly Asp Leu Thr

195 200 205

Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His

210 215 220

Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln

225 230 235 240

Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys

245 250 255

Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys

260 265 270

Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly

275 280 285

Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu

290 295 300

Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln

305 310 315 320

Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro

325 330 335

Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val

340 345 350

Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp

355 360 365

Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu

370 375 380

Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met

385 390 395 400

Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser

405 410 415

<210> 683

<211> 478

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(478)

<223> caspase_10 full Length

<400> 683

Met Lys Ser Gln Gly Gln His Trp Tyr Ser Ser Ser Asp Lys Asn Cys

1 5 10 15

Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Ile Asp Ser Asn Leu Gly

20 25 30

Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly Leu Val Pro

35 40 45

Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Ser Asp Val Phe Glu His

50 55 60

Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe Phe Leu Ala

65 70 75 80

Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Leu Leu Gln His Leu Asn

85 90 95

Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pro Thr Arg Gln Arg Val

100 105 110

Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser Glu Gly Ile Asp Ser

115 120 125

Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Lys Asp Ser Leu Pro Lys

130 135 140

Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys Gln Gly

145 150 155 160

Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys Lys Thr

165 170 175

Val Val Pro Lys Leu Leu Arg Asn Ile Glu Lys Tyr Lys Arg Glu Lys

180 185 190

Ala Ile Gln Ile Val Thr Pro Pro Val Asp Lys Glu Ala Glu Ser Tyr

195 200 205

Gln Gly Glu Glu Glu Leu Val Ser Gln Thr Asp Val Lys Thr Phe Leu

210 215 220

Glu Ala Leu Pro Arg Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg

225 230 235 240

Gly Leu Cys Val Ile Val Asn Asn His Ser Phe Thr Ser Leu Lys Asp

245 250 255

Arg Gln Gly Thr His Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln

260 265 270

Trp Leu Gly Phe Thr Val His Ile His Asn Asn Val Thr Lys Val Glu

275 280 285

Met Glu Met Val Leu Gln Lys Gln Lys Cys Asn Pro Ala His Ala Asp

290 295 300

Gly Asp Cys Phe Val Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala

305 310 315 320

Val Tyr Ser Ser Asp Glu Ala Leu Ile Pro Ile Arg Glu Ile Met Ser

325 330 335

His Phe Thr Ala Leu Gln Cys Pro Arg Leu Ala Glu Lys Pro Lys Leu

340 345 350

Phe Phe Ile Gln Ala Cys Gln Gly Glu Glu Ile Gln Pro Ser Val Ser

355 360 365

Ile Glu Ala Asp Ala Leu Asn Pro Glu Gln Ala Pro Thr Ser Leu Gln

370 375 380

Asp Ser Ile Pro Ala Glu Ala Asp Phe Leu Leu Gly Leu Ala Thr Val

385 390 395 400

Pro Gly Tyr Val Ser Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile

405 410 415

Gln Ser Leu Cys Asn His Leu Lys Lys Leu Val Pro Arg Met Leu Lys

420 425 430

Phe Leu Glu Lys Thr Met Glu Ile Arg Gly Arg Lys Arg Thr Val Trp

435 440 445

Gly Ala Lys Gln Ile Ser Ala Thr Ser Leu Pro Thr Ala Ile Ser Ala

450 455 460

Gln Thr Pro Arg Pro Pro Met Arg Arg Trp Ser Ser Val Ser

465 470 475

<210> 684

<211> 362

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(362)

<223> CD95 protein

<400> 684

Met Leu Arg Val Cys Ala Gln Gly Trp His Ala Gln Gly Leu Pro His

1 5 10 15

Gly Thr Asn Ser Leu Leu Lys Gly Gly Thr Glu Thr Ser Leu Ser Thr

20 25 30

Pro Thr Arg Leu Val Val Leu Thr Ser Val Ala Arg Leu Ser Ser Lys

35 40 45

Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu

50 55 60

Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His

65 70 75 80

His Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys

85 90 95

Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys

100 105 110

Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys

115 120 125

Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile

130 135 140

Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe

145 150 155 160

Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys

165 170 175

Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys

180 185 190

Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp Leu Cys Leu Leu Leu

195 200 205

Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg Lys Glu Val Gln Lys

210 215 220

Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly Ser His Glu Ser Pro

225 230 235 240

Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu Ser Asp Val Asp Leu

245 250 255

Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met Thr Leu Ser Gln Val

260 265 270

Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile Asp Glu

275 280 285

Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val Gln Leu

290 295 300

Leu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr Asp Thr

305 310 315 320

Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala Glu Lys

325 330 335

Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser Asp Ser Glu Asn Ser

340 345 350

Asn Phe Arg Asn Glu Ile Gln Ser Leu Val

355 360

<210> 685

<211> 455

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(455)

<223> TNF-R1 protein

<400> 685

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1 5 10 15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

20 25 30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

35 40 45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

50 55 60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65 70 75 80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

85 90 95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

100 105 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

115 120 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

130 135 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145 150 155 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

165 170 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

180 185 190

Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser

195 200 205

Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu

210 215 220

Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gln Arg Trp Lys

225 230 235 240

Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys Glu

245 250 255

Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser

260 265 270

Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val

275 280 285

Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys

290 295 300

Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala Pro Pro Tyr Gln Gly

305 310 315 320

Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser Asp Pro Ile Pro Asn

325 330 335

Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp

340 345 350

Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val Glu Asn Val Pro Pro

355 360 365

Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu

370 375 380

Ile Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln

385 390 395 400

Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg Glu Ala

405 410 415

Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly

420 425 430

Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro

435 440 445

Pro Ala Pro Ser Leu Leu Arg

450 455

<210> 686

<211> 181

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(181)

<223> death receptor-3

<400> 686

Met Glu Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu

1 5 10 15

Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg

20 25 30

Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys

35 40 45

Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu Pro

50 55 60

Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr Phe Leu Ala

65 70 75 80

Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys Gln Ala Cys Asp

85 90 95

Glu Gln Ala Ser Gln Val Ala Leu Glu Asn Cys Ser Ala Val Ala Asp

100 105 110

Thr Arg Cys Gly Cys Lys Pro Gly Trp Phe Val Glu Cys Gln Val Ser

115 120 125

Gln Cys Val Ser Ser Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys

130 135 140

Gly Ala Leu His Arg His Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr

145 150 155 160

Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys

165 170 175

Val Ser Cys Pro Thr

180

<210> 687

<211> 468

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(468)

<223> death receptor-4

<400> 687

Met Ala Pro Pro Pro Ala Arg Val His Leu Gly Ala Phe Leu Ala Val

1 5 10 15

Thr Pro Asn Pro Gly Ser Ala Ala Ser Gly Thr Glu Ala Ala Ala Ala

20 25 30

Thr Pro Ser Lys Val Trp Gly Ser Ser Ala Gly Arg Ile Glu Pro Arg

35 40 45

Gly Gly Gly Arg Gly Ala Leu Pro Thr Ser Met Gly Gln His Gly Pro

50 55 60

Ser Ala Arg Ala Arg Ala Gly Arg Ala Pro Gly Pro Arg Pro Ala Arg

65 70 75 80

Glu Ala Ser Pro Arg Leu Arg Val His Lys Thr Phe Lys Phe Val Val

85 90 95

Val Gly Val Leu Leu Gln Val Val Pro Ser Ser Ala Ala Thr Ile Lys

100 105 110

Leu His Asp Gln Ser Ile Gly Thr Gln Gln Trp Glu His Ser Pro Leu

115 120 125

Gly Glu Leu Cys Pro Pro Gly Ser His Arg Ser Glu His Pro Gly Ala

130 135 140

Cys Asn Arg Cys Thr Glu Gly Val Gly Tyr Thr Asn Ala Ser Asn Asn

145 150 155 160

Leu Phe Ala Cys Leu Pro Cys Thr Ala Cys Lys Ser Asp Glu Glu Glu

165 170 175

Arg Ser Pro Cys Thr Thr Thr Arg Asn Thr Ala Cys Gln Cys Lys Pro

180 185 190

Gly Thr Phe Arg Asn Asp Asn Ser Ala Glu Met Cys Arg Lys Cys Ser

195 200 205

Arg Gly Cys Pro Arg Gly Met Val Lys Val Lys Asp Cys Thr Pro Trp

210 215 220

Ser Asp Ile Glu Cys Val His Lys Glu Ser Gly Asn Gly His Asn Ile

225 230 235 240

Trp Val Ile Leu Val Val Thr Leu Val Val Pro Leu Leu Leu Val Ala

245 250 255

Val Leu Ile Val Cys Cys Cys Ile Gly Ser Gly Cys Gly Gly Asp Pro

260 265 270

Lys Cys Met Asp Arg Val Cys Phe Trp Arg Leu Gly Leu Leu Arg Gly

275 280 285

Pro Gly Ala Glu Asp Asn Ala His Asn Glu Ile Leu Ser Asn Ala Asp

290 295 300

Ser Leu Ser Thr Phe Val Ser Glu Gln Gln Met Glu Ser Gln Glu Pro

305 310 315 320

Ala Asp Leu Thr Gly Val Thr Val Gln Ser Pro Gly Glu Ala Gln Cys

325 330 335

Leu Leu Gly Pro Ala Glu Ala Glu Gly Ser Gln Arg Arg Arg Leu Leu

340 345 350

Val Pro Ala Asn Gly Ala Asp Pro Thr Glu Thr Leu Met Leu Phe Phe

355 360 365

Asp Lys Phe Ala Asn Ile Val Pro Phe Asp Ser Trp Asp Gln Leu Met

370 375 380

Arg Gln Leu Asp Leu Thr Lys Asn Glu Ile Asp Val Val Arg Ala Gly

385 390 395 400

Thr Ala Gly Pro Gly Asp Ala Leu Tyr Ala Met Leu Met Lys Trp Val

405 410 415

Asn Lys Thr Gly Arg Asn Ala Ser Ile His Thr Leu Leu Asp Ala Leu

420 425 430

Glu Arg Met Glu Glu Arg His Ala Arg Glu Lys Ile Gln Asp Leu Leu

435 440 445

Val Asp Ser Gly Lys Phe Ile Tyr Leu Glu Asp Gly Thr Gly Ser Ala

450 455 460

Val Ser Leu Glu

465

<210> 688

<211> 440

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(440)

<223> death receptor-5

<400> 688

Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys

1 5 10 15

Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro

20 25 30

Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu

35 40 45

Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln

50 55 60

Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu

65 70 75 80

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

85 90 95

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

100 105 110

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

115 120 125

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

130 135 140

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

145 150 155 160

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

165 170 175

Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Val Pro

180 185 190

Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro

195 200 205

Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val

210 215 220

Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val

225 230 235 240

Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu

245 250 255

Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu

260 265 270

Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu

275 280 285

Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser

290 295 300

Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser

305 310 315 320

Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu

325 330 335

Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp

340 345 350

Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu Ile

355 360 365

Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr

370 375 380

Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His

385 390 395 400

Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln

405 410 415

Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu

420 425 430

Gly Asn Ala Asp Ser Ala Met Ser

435 440

<210> 689

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> gamma secretase recognition sequence-Alcadein a

<400> 689

Val Val Pro Ser Thr Ala Thr Val Val Ile Val Val Cys Val Ser Phe

1 5 10 15

Leu Val Phe Met Ile Ile Leu Gly Val Phe Arg Ile Arg Ala Ala

20 25 30

<210> 690

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> gamma secretase recognition sequence-Alcadein

<400> 690

Met Ile Pro Ser Ala Ala Thr Leu Ile Ile Val Val Cys Val Gly Phe

1 5 10 15

Leu Val Leu Met Val Val Leu Gly Leu Val Arg Ile His Ser Leu His

20 25 30

<210> 691

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> gamma-secretase recognition sequence-Alcadein gamma

<400> 691

Ser Val Val Pro Ser Ile Ala Thr Val Val Ile Ile Ile Ser Val Cys

1 5 10 15

Met Leu Val Phe Val Val Ala Met Gly Val Tyr Arg Val Arg Ile Ala

20 25 30

<210> 692

<211> 33

<212> PRT

<213> artificial sequence

<220>

<223> Γ secretase recognition sequence-APLP 1

<400> 692

Gly Val Ser Arg Glu Ala Val Ser Gly Leu Leu Ile Met Gly Ala Gly

1 5 10 15

Gly Gly Ser Leu Ile Val Leu Ser Met Leu Leu Leu Arg Arg Lys Lys

20 25 30

Pro

<210> 693

<211> 34

<212> PRT

<213> artificial sequence

<220>

<223> Γ secretase recognition sequence-APLP 2

<400> 693

Phe Ser Leu Ser Ser Ser Ala Leu Ile Gly Leu Leu Val Ile Ala Val

1 5 10 15

Ala Ile Ala Thr Val Ile Val Ile Ser Leu Val Met Leu Arg Lys Arg

20 25 30

Gln Tyr

<210> 694

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> Γ secretase recognition sequence-Notch 1

<400> 694

Pro Ala Gln Leu His Phe Met Tyr Val Ala Ala Ala Ala Phe Val Leu

1 5 10 15

Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys Arg Arg

20 25 30

<210> 695

<211> 33

<212> PRT

<213> artificial sequence

<220>

<223> Γ secretase recognition sequence-Notch 2

<400> 695

Pro Glu Arg Thr Gln Leu Leu Tyr Leu Leu Ala Val Ala Val Val Ile

1 5 10 15

Ile Leu Phe Ile Ile Leu Leu Gly Val Ile Met Ala Lys Arg Lys Arg

20 25 30

Lys

<210> 696

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> Γ secretase recognition sequence-Notch 3

<400> 696

Pro Ser Val Pro Leu Leu Pro Leu Leu Val Ala Gly Ala Val Leu Leu

1 5 10 15

Leu Val Ile Leu Val Leu Gly Val Met Val Ala Arg Arg Lys Arg Glu

20 25 30

<210> 697

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> Γ secretase recognition sequence-Notch 4

<400> 697

Ala Asn Gln Leu Pro Trp Pro Val Leu Cys Ser Pro Val Ala Gly Val

1 5 10 15

Ile Leu Leu Ala Leu Gly Ala Leu Leu Val Leu Gln Leu Ile Arg Arg

20 25 30

<210> 698

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> E27 anti-IgE antibody-heavy chain

<400> 698

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Ser Ile Lys Tyr Ser Gly Glu Thr Lys Tyr Asn Pro Ser Val

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

Claims (68)

1. A polynucleotide, comprising: one or more transcriptional units, wherein each of the one or more transcriptional units is operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units comprise:

a) A nucleic acid encoding a first engineered signaling polypeptide, wherein the first engineered signaling polypeptide is a Chimeric Antigen Receptor (CAR), and

b) A nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain, a membrane association domain, and a handle connecting the anti-idiotype extracellular recognition domain to the membrane association domain, wherein the anti-idiotype extracellular recognition domain comprises an anti-idiotype antibody or antibody mimetic idiotype binding variable region that recognizes the idiotype of a target antibody or target antibody mimetic.

2. A modified mammalian cell comprising the polynucleotide of claim 1.

3. A method for delivering a modified T cell and/or NK cell to a subject, wherein the method comprises administering the modified T cell and/or NK cell to the subject, wherein the modified T cell and/or NK cell comprises the polynucleotide of claim 1.

4. Use of modified T cells and/or NK cells in the preparation of a kit, wherein the use of the kit comprises: administering the modified T cells and/or NK cells to a subject, wherein the modified T cells and/or NK cells comprise the polynucleotide of claim 1.

5. A polynucleotide vector comprising the polynucleotide of claim 1.

6. A polypeptide comprising an extracellular recognition domain, a membrane association domain, and a handle that connects the extracellular recognition domain to the membrane association domain, and further comprising an intracellular domain (ICD) of a first engineered signaling polypeptide, wherein the first engineered signaling polypeptide is a Chimeric Antigen Receptor (CAR), a recombinant T Cell Receptor (TCR), or a Lymphoproliferative Element (LE), and wherein the extracellular recognition domain is an anti-idiotype extracellular recognition domain that recognizes an idiotype of a target antibody.

7. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, the polynucleotide vector of claim 5 or the polypeptide of claim 6, wherein the anti-idiotype extracellular recognition domain is an antibody comprising an scFv.

8. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, the polynucleotide vector of claim 5 or the polypeptide of claim 6, wherein the idiotype-binding variable region comprises a framework region, and wherein the framework region is a human framework region.

9. The modified mammalian cell of claim 2, wherein the cell is one cell of a population of human cells within an infusion bag.

10. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, or the polynucleotide vector of claim 5, wherein the polynucleotide further encodes a second engineered signaling polypeptide, and wherein the second engineered signaling polypeptide is a lymphoproliferative element.

11. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 10, wherein the lymphoproliferative element comprises an intracellular domain that is a means for transmitting a signal that promotes proliferation or survival of T cells and/or NK cells.

12. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 11, wherein the lymphoproliferative element is constitutively active.

13. The polynucleotide, cell, method, use or polynucleotide vector of claim 10, wherein the polynucleotide comprises an Internal Ribosome Entry Site (IRES), ribosome jump sequence and/or cleavage signal between a nucleic acid encoding the first or second engineered signaling polypeptide and a nucleic acid encoding the anti-idiotype polypeptide.

14. The polynucleotide, cell, method, use or polynucleotide vector of claim 10, wherein the polynucleotide comprises an Internal Ribosome Entry Site (IRES), ribosome jump sequence and/or cleavage signal between the nucleic acid encoding the second engineered signaling polypeptide and the nucleic acid encoding the anti-idiotype polypeptide.

15. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, the polynucleotide vector of claim 5 or the polypeptide of claim 6, wherein the target antibody comprises an antigen binding site, and wherein the anti-idiotype polypeptide is capable of binding to the antigen binding site of the target antibody.

16. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 15, wherein the target antibody or target antibody mimetic is an approved biological antibody or antibody mimetic approved by the United States Food and Drug Administration (USFDA), european Medicines Administration (EMA), chinese National Medicines Administration (NMAN) (chinese FDA), or japanese medicines and food safety administration (PFSB).

17. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 16, wherein the approved biological antibody or antibody mimetic is the approved biological antibody, and wherein the approved biological antibody is cetuximab (cetuximab), moromib-CD 3 (muromonab-CD 3), efaciens-b (muromonab-CD 3), toximomab-i 131 (tositumomab-i 131), nebulomab (neucumab), ibritumomab (edeclomab), cetuximab (gummitumomab), daclizumab (daclizumab), atamab (olapariumab), aciumab (abamectin) at the same time as (basiliximab), palivizumab (palivizumab), infliximab (infliximab), trastuzumab (trauzumab), trauzumab (trauzumab) adalimumab (adalimumab), timumumab (ibritumomab tiuxetan), omalizumab (omalizumab), bevacizumab (bevacizumab), natalizumab (natlimumab), panitumumab (panitumab), ranibizumab (ranibizumab), eculizumab (eclipzumab), cetuximab (certolizumab pegol), uteumab (ustekumab), kanamab (canakinumab), golimumab (golimumab), oagulamumab (ofatumumab), tozumab (tocidab), denoumab (denosumab), belimumab (belimumab), ipilimumab (ipilimumab), ben-mab (brentuximab vedotin), toxib, pertuzumab (pertuzumab), enmetrastuzumab (ado-trastuzumab emtansine), risperiduzumab (raxibacumab), oxuzumab (obinutuzumab), cetuximab (siltuximab), ramucirumab (ramucirumab), vedolizumab (vedolizumab), nivolumab (nivolumumab), pembrolizumab (pembrolizumab), boaltumab (blinatumomab), alemtuzumab (alemtuzumab), everkuumab (evokuumab), idazouzumab (idakutuzumab), midtuzumab (desituzumab), dituximab), pertuzumab (sekukuumab), mepoisizumab (mevalizumab), alemtuzumab (iriumab), daclizumab (alemtuzumab), dacuzumab (dacuzumab) and daratuzumab (dacuzumab) Ai Luozhu mab (elotuzumab), exolizumab (ixekizumab), rebaudizumab (relizumab), bei Luotuo Shu Shan mab (bezlotoxumab), alemtuzumab (atezolizumab), otoximab (obilotoxin) bromomab, bromoticket mab (brodalumaab), degree-prize Li Youshan mab (dupilumab), ootuzumab (inotuzumab ozogamicin), gulkumab (guselkumab), sha Lilu mab (sarilumab), avermectin mab (aveumab), eimeriuzumab (emizumab), oxbezizumab (emizumab), deleuzumab (durvalumab), gemtuzumab (gemtuzumab ozogamicin), rebaudiuzumab (erenumab) (erenauab), erenauzumab (erenumab), gazeab (gazeab), gazeumab (gammagumab-gamuzumab), bromothalonil You Shan anti (burosum) (burosum-twza), ranolauzumab (landelumab) (landelumab-flyo), mo Geli set mab (mogamulizumab) (mogamulizumab-kpkc), tildrakizumab (tildrakizumab-asmn), repairaziumab (freezumab) (freemam-vfram), eculizumab (ravulizumab-cvz), simplica Li Shan anti (cemizumab) (cemiimab-rwlc), valsaluzumab (ibalazumab-uiek), epalizumab (emplastuzumab-zsg), panaxizumab (24), and oxazizumab (45-tetracalcalizumab) (54-25) are provided. Raschizumab (riscanlizumab) (riscanlizumab-rzaa), vistin-poisatozumab (polatuzumab vedotin) (polatuzumab vedotin-piiq), luo Moshan anti (polatuzumab vedotin) (romisozumab-aqqg), bruxizumab (broucizumab) (broucizumab-dbll), fuzilizumab (crizanlizumab-tmca), enrolment mab (enfortumab vedotin) (enfortumab vedotin-ejfv), desitrozumab ([ fam- ] trastuzumab deruxtecan) (fam trastuzumab deruxtecan-nxki), tetuzumab (tetuzumab) (tetuzumab-trbw), ai Punai bead mab (epotinizumab-jjjmmr), ai Satuo mab (isuzumab-isuqfcb), facituzumab govitecan (sacituzumab govitecan-hziy), inbizumab (sacituzumab govitecan) (inebilizumab-cdon), tavalizumab (tafasitamab) (tafasitamab-cxix), bei Lan Tatuzumab Mo Futing (belantamab mafodotin) (belantamab mafodotin-blmf), satelizumab (satraplizumab) (satraplizumab-mwge), ati Wei Shankang (atotivimab), macti Wei Shankang (maftivimab), odesivimab-ebgn, naxitamab-gqgk, margetuximab-cmkb, asuwimb-zykl, ivermectin Su Shan (evinacumab), multi-tarolimab (dostarlimab) (doslimab-gxyy), tetroximab (loncastuximab tesirine) (loncastuximab tesirine-lpyl), efamamab (amivanthaumab-vw), amantaginab (amamevalab-aduw), aviumab (avanamum-avaumab) (waumab-avanamum) Qu Luolu monoclonal antibody (tralokinumab), anistuzumab (anidrolimumab) (anidrolimumab-fnia), motoneuron monoclonal antibody (oportuzumab monatox), vitamin-tetroximab (tisotumab vedotin), bimetalizumab (bimekizumab), naso Li Shan antibody (narcoplimab), terstuzumab (tezepelumab), xindi Li Shan antibody (sincalimab), inolimumab (inolimumab), batirimumab (beltimlimab), rituximab (ublituximab), tertup Li Shan antibody (toripalimab), obromab (ombuamab), pezilimab (penpulimab), taliruximab (tanlizumab), farnesimab (faririma), su Timo monoclonal antibody (sulimab), telithromycin (teplizumab) and remifram Li Shan antibody (retifanlimab).

18. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 16, wherein the anti-idiotype polypeptide comprises means for binding to an idiotype of cetuximab.

19. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 17, wherein the approved biological antibody is cetuximab.

20. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 19, wherein the anti-idiotype polypeptide is capable of blocking binding of cetuximab to an epidermal growth factor receptor.

21. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, or the polynucleotide vector of claim 5, wherein the anti-idiotype polypeptide further comprises one or more intracellular domains (ICDs).

22. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 21, wherein the ICD is 1 to 35 amino acids in length.

23. The polynucleotide, cell, method, use, polynucleotide vector of claim 21 or polypeptide of claim 6, wherein the membrane associating domain is a transmembrane domain.

24. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 23, wherein the ICD comprises all or part of an intracellular signaling domain of one or more lymphoproliferative elements, and wherein the ICD is capable of activating a signaling pathway.

25. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 24, wherein the lymphoproliferative element is inducible, and wherein the inducible lymphoproliferative element is capable of activating proliferation or survival signaling after binding of the anti-idiotype polypeptide to the target antibody or target antibody mimetic.

26. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 25, wherein said ICD is capable of activating a Jak pathway, TRAF pathway, PI3K pathway and/or PLC pathway after dimerization.

27. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 25, wherein the idiotype is present on two target antibodies: a first target antibody that promotes cytotoxicity, and a second target antibody that promotes less cytotoxicity than the first target antibody.

28. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 24, wherein the one or more cytokine receptors are selected from the group consisting of CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL ST, IL7R, IL RA, IL9R, IL RA, IL10RB, IL11RA, IL12RB1, IL13R, IL RA1, IL13RA2, IL15R, IL RA, IL17RB, IL17RC 17RE, IL18R1, IL18RA, IL20RB, IL21R, IL RA1, IL23R, IL27RA, IL 7432 RA, IL7 8231, IL9R, IL RA, IL10RA, IL11RA, IL12RB1, IL13R, IL RA1, IL13RA, IL13R, IL RA 2RA, IL13RA, IL20RA, TNFRSF4 or TNFRSF18 receptor.

29. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 23, wherein the membrane associated domain is a transmembrane domain of the lymphoproliferative element, the CAR, or the recombinant TCR.

30. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, the polynucleotide vector of claim 5 or the polypeptide of claim 6, wherein the handle is 4 to 100 amino acids in length.

31. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 30, wherein the handle encodes a dimerizing moiety, wherein the dimerizing moiety is constitutively dimerized.

32. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 31, wherein the constitutively dimerized dimerization domain comprises a leucine zipper.

33. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, or the polynucleotide vector of claim 5, wherein the polynucleotide further comprises a nucleic acid encoding one or more inhibitory RNA molecules.

34. The polynucleotide, cell, method, use or polynucleotide vector of claim 33, wherein one of the one or more inhibitory RNA molecules targets ifny.

35. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, the polynucleotide vector of claim 5 or the polypeptide of claim 6, wherein the target antibody is structured such that binding of the anti-idiotype polypeptide to the target antibody induces cytotoxicity.

36. The method of claim 3 or the use of claim 4, further comprising instructing a user to deliver the target antibody to the subject if an adverse event of the subject is desired to be treated.

37. The modified mammalian cell of claim 2, wherein the modified mammalian cell is a CAR-T cell or a CAR-NK cell.

38. The modified mammalian cell population of claim 2, wherein the population comprises CAR-T cells, CAR-NK cells, or both CAR-T cells and CAR-NK cells.

39. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 23, wherein said ICD comprises an intracellular apoptosis domain capable of inducing an apoptosis signal upon binding of said anti-idiotype extracellular recognition domain to a target antibody or antibody mimetic comprising said idiotype.

40. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 39, wherein said anti-idiotype polypeptide further comprises a proteolytic cleavage site as part of and/or between said transmembrane domain and said intracellular apoptotic domain, wherein said proteolytic cleavage site has a cleavage site amino acid sequence such that when said anti-idiotype polypeptide is dimerized by binding a target antibody or antibody mimetic to said anti-idiotype extracellular recognition domain, said anti-idiotype polypeptide is cleaved at said proteolytic cleavage site.

41. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 40, wherein said proteolytic cleavage site is effective for cleavage by a gamma secretase complex or a Notch receptor following dimerization of said anti-idiotype polypeptide.

42. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 41, wherein the intracellular apoptosis domain comprises one or more of a caspase 2 polypeptide, a caspase 8 polypeptide, a caspase 9 polypeptide, and a caspase 10 polypeptide, wherein such an intracellular apoptosis domain is capable of activating an effector caspase upon cleavage from the anti-idiotype polypeptide.

43. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide according to claim 41, wherein the intracellular apoptosis domain comprises one or more caspase polypeptides, wherein the caspase polypeptide is amino acids 327 to 452 of SEQ ID NO:680 (caspase 2), amino acids 384 to 496 of SEQ ID NO:681 (caspase 8), amino acids 294 to 416 of SEQ ID NO:682 (caspase 9) and amino acids 365 to 478 of SEQ ID NO:683 (caspase 10).

44. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 39, wherein the ICD comprises one or more Caspase Activation and Recruitment Domains (CARDs), death Domains (DDs), death Effector Domains (DED), thermal protein domains (PYDs), and/or caspase proteolytic domains that are activated to transmit an apoptosis-inducing signal upon dimerization.

45. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 39, wherein said anti-idiotype polypeptide further comprises a dimerization moiety, wherein said dimerization moiety is constitutively dimerized, and/or wherein said target antibody is an IgA or IgM antibody.

46. The polynucleotide, cell, method, use, polynucleotide vector or polypeptide of claim 45, wherein said intracellular apoptosis domain comprises one or more Death Domains (DD) from FAS, TNF-R1, DR3, DR4 and/or DR 5.

47. The vector of claim 5, wherein the vector is a viral vector, and wherein the viral vector is replication defective.

48. The vector of claim 47, wherein the viral vector is a retroviral particle.

49. The vector of claim 48 wherein the surface of said retroviral particle comprises a membrane bound cytokine.

50. The vector of claim 49 wherein the membrane-bound cytokine is a chemokine.

51. The vector of claim 50, wherein the vector comprises a cleavage signal effective to cleave the membrane-bound cytokine from the membrane.

52. The vector of claim 48, wherein said retroviral particle is a lentiviral vector.

53. The vector of claim 47, further comprising an activating element on the surface of the retroviral particle, wherein the activating element is fused to a heterologous membrane attachment sequence, and wherein the activating element is a polypeptide capable of binding CD3 on the surface of resting T cells and activating the resting T cells, and wherein the activating element is not encoded by a polynucleotide in the retroviral particle.

54. The vector of claim 53, wherein the activating element comprises an anti-CD 3 antibody or antibody mimetic.

55. The polynucleotide of claim 1, the cell of claim 2, the method of claim 3, the use of claim 4, the polynucleotide vector of claim 5 or the polypeptide of claim 6, wherein the anti-idiotype polypeptide comprises the handle, membrane association domain and ICD of SEQ ID No. 676, SEQ ID No. 677, SEQ ID No. 678 or SEQ ID No. 679.

56. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 29, wherein the transmembrane domain is the transmembrane domain of the CAR and the ICD is the ICD of the CAR.

57. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 56, wherein said CAR is a bispecific CAR, wherein a first ASTR of said CAR comprises said anti-idiotype extracellular recognition domain.

58. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 57, wherein the second ASTR of the CAR is capable of binding to a tumor-associated antigen or a tumor-specific antigen.

59. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 58, wherein the first and second ASTRs of the CAR are scFv antibodies.

60. The polynucleotide, cell, method, use, polynucleotide vector, or polypeptide of claim 59, wherein the first and second ASTRs of the CAR comprise scFv-Fc, scFv-CH, and scFv-zipper antibodies.

61. A retroviral particle comprising

a) A polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide comprising an anti-idiotype extracellular recognition domain, a membrane association domain, and a handle connecting the anti-idiotype extracellular recognition domain to the membrane association domain, wherein the anti-idiotype extracellular recognition domain comprises an anti-idiotype antibody or antibody mimetic idiotype binding variable region that recognizes the idiotype of a target antibody or target antibody mimetic; and

b) An activating element on the surface of the retroviral particle, wherein the activating element is fused to a heterologous membrane attachment sequence, and wherein the activating element is a polypeptide capable of binding CD3 on the surface of a T cell.

62. A method for delivering a modified T cell and/or NK cell to a subject, the method comprising administering the modified cell to the subject, wherein the modified cell is modified with a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide and a nucleic acid encoding a CAR.

63. Use of modified T cells and/or NK cells in the preparation of a kit, wherein the use of the kit comprises: administering the modified T cells and/or NK cells to a subject, wherein the modified T cells and/or NK cells comprise a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide and a nucleic acid encoding a CAR.

64. A modified primary T cell or NK cell, wherein the modified primary T cell or NK cell expresses an anti-idiotype polypeptide.

65. The modified primary T cell or NK cell of claim 63, wherein the modified primary T cell or NK cell is a tumor infiltrating lymphocyte.

66. The modified primary T cell or NK cell of claim 63, wherein the modified primary T cell or NK cell is a CAR-T or NK-T cell.

67. The modified primary T cell or NK cell of claim 63, wherein the anti-idiotype polypeptide is expressed on the surface of the T cell or NK cell.

68. A polypeptide comprising an extracellular recognition domain, a membrane association domain, and a handle that connects the extracellular recognition domain to the membrane association domain, wherein the extracellular recognition domain is an anti-idiotype extracellular recognition domain that recognizes an idiotype of a target antibody, and optionally wherein the anti-idiotype polypeptide is a CAR, TCR, or lymphoproliferative element.

Images