CN111479921A - Methods and compositions for genetically modifying and expanding lymphocytes and modulating their activity - Google Patents

Abstract

The present invention provides methods and compositions for genetically modifying lymphocytes and related methods comprising genetically modifying T cells and/or NK cells, the methods using replication-defective recombinant retroviral particles comprising on their surface a pseudotyped component and optionally a membrane-bound T cell activation component (such as anti-CD 3), and encoding one or more engineered signaling polypeptides that may comprise a lymphoproliferative component and/or a Chimeric Antigen Receptor (CAR).

Description

Methods and compositions for genetically modifying and expanding lymphocytes and modulating their activity

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a partial continuation application of international application No. PCT/US2018/020818 applied on 3.3.2018; and claims the benefit of: us provisional application No. 62/560,176, filed 2017, 9, 18; us provisional application No. 62/564,253, filed on 27/9/2017; us provisional application No. 62/564,991, filed on 28.9.2017; and us provisional application No. 62/728,056, filed on 6/9/2018; the partial continuation application of the international application No. PCT/US2017/023112, which is applied on 3, 19 and 2017 in International application No. PCT/US 2018/020818; part of international application No. PCT/US2017/041277 applied on 7, 8 and 7 in 2017 is a continuation-in-part application; a partial continuation-in-us application No. 15/462,855 filed on 19/3/2017; and us application No. 15/644,778, filed on 8.7.2017; and claims the benefit of: us provisional application No. 62/467,039, filed on 3/2017; us provisional application No. 62/560,176, filed 2017, 9, 18; us provisional application No. 62/564,253, filed on 27/9/2017; and us provisional application No. 62/564,991, filed on 28/9/2017; international application No. PCT/US2017/023112 claims the benefit of the following applications: united states provisional application No. 62/390,093, filed on 3/19/2016; united states provisional application No. 62/360,041, filed on 8/7/2016; and us provisional application No. 62/467,039, filed on 3/2017; international application No. PCT/US2017/041277 claims the benefit of the following applications: international application No. PCT/US2017/023112, filed on 3/19/2017; us patent application No. 15/462,855 filed on 3/19/2017; united states provisional application No. 62/360,041, filed on 8/7/2016; and us provisional application No. 62/467,039, filed on 3/2017; U.S. application No. 15/462,855 claims the benefit of the following applications: united states provisional application No. 62/390,093, filed on 3/19/2016; united states provisional application No. 62/360,041, filed on 8/7/2016; and us provisional application No. 62/467,039, filed on 3/2017; and U.S. application No. 15/644,778 is a partially-filed continuation-in-part application of international application No. PCT/US2017/023112 filed on 3/19/2017; and us patent application No. 15/462,855, filed 3/19, 2017, partially in continuation; and claims the benefit of: united states provisional application No. 62/360,041, filed on 8/7/2016 and united states provisional application No. 62/467,039, filed on 3/2017. These applications are incorporated herein by reference in their entirety.

Sequence listing

The material in the electronic Sequence listing is submitted as a text (.txt) archive (with archive size 529KB) entitled "F1 _001_ TW _04_ Sequence _ L identifying _2018_09_ 17" created on 2018, 9, 17, and is incorporated by reference herein in its entirety.

Technical Field

The present invention relates to the field of immunology, or more specifically, to genetic modification of T lymphocytes or other immune cells, and methods for preparing replication-defective recombinant retroviral particles and controlling expression of genes therein.

Background

Lymphocytes isolated from an individual (e.g., a patient) can be activated in vitro and genetically modified to express synthetic proteins that are capable of relocating binding to other cells and the environment based on the incorporated genetic program. One example of such a synthetic protein is a Chimeric Antigen Receptor (CAR). One CAR currently in use is a fusion of an extracellular recognition domain (e.g., an antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains encoded by a replication-defective recombinant retrovirus.

Although recombinant retroviruses have shown efficacy in infecting non-dividing cells, resting CD4 and CD8 lymphocytes are not susceptible to gene transduction by these vectors. To overcome these difficulties, stimulators are often used to activate these cells in vitro before genetic modification of the CAR gene vector can occur. Following stimulation and transduction, the genetically modified cells are expanded in vitro and subsequently reintroduced into a patient with lymphodepletion. Following antigen engagement in vivo, the intracellular signaling portion of the CAR can begin an activation-related response in the immune cell and release a cytolytic molecule to induce tumor cell death.

Such current methods require extensive manipulation and in vitro production of proliferative T cells prior to reinfusion of the T cells into a patient, as well as lymphokinesOnce introduced into the body, such CAR therapies additionally fail to control the in vivo propagation rate, nor safely target targets that are also expressed outside the tumor⁵One cell/kg to 1 × 10⁸A dose of individual cells/kg expanded ex vivo for 12 to 28 days of cell infusion and targeted to a target (e.g., tumor target) is generally acceptable for this CAR therapy to be toxic to the target away from the tumor. These relatively long ex vivo expansion times create cell viability and sterility issues, as well as sample consistency issues in addition to the challenge of tunability. Thus, there is a significant need for safer, more effective, tunable T cell or NK cell therapies.

Since our understanding of the processes that drive transduction, proliferation and survival of lymphocytes is central to the various potential commercial uses involving immune processes, improved methods and compositions are needed for studying lymphocytes. For example, it would be helpful to identify methods and compositions that could be used to better characterize and understand how lymphocytes can be genetically modified and factors that affect their survival and proliferation. In addition, it would help identify compositions that drive lymphocyte proliferation and survival. These compositions are useful for studying the regulation of these processes. In addition to methods and compositions for studying lymphocytes, there is a need for improved viral packaging cell lines and methods for making and using the same. For example, these cell lines and methods would be suitable for analyzing different components of recombinant viruses (such as recombinant retroviral particles), as well as for methods of using packaging cell lines for the production of recombinant retroviral particles.

Disclosure of Invention

Provided herein are methods, compositions, and kits that help overcome the efficacy and safety concerns for transducing and/or genetically modifying lymphocytes, such as T cells and/or NK cells. Certain embodiments of these methods are suitable for performing adoptive cell therapy with these cells. Thus, in some aspects, provided herein are methods, compositions, and kits for genetically modifying and/or transducing lymphocytes (particularly T cells and/or NK cells) and/or for modulating transduced and/or genetically modified T cells and/or NK cells. These methods, compositions and kits provide improved efficacy and safety over the current art, particularly with respect to T cells and/or NK cells expressing Chimeric Antigen Receptors (CARs) and in illustrative embodiments microenvironment-restricted biological CARs. Transduced and/or genetically modified T cells and/or NK cells produced using the methods provided herein include, in illustrative embodiments delivered from a retroviral (e.g., lentiviral) genome via a retroviral (e.g., lentiviral) particle, functionality and functional combinations that provide improved characteristics for such cells and for methods of utilizing such cells, such as research methods, commercial production methods, and adoptive cell therapies. For example, these cells can be produced ex vivo in a shorter time, and the cells have improved growth characteristics that can be better regulated.

In some aspects, provided herein are regulatory components for modulating CARs, mrnas, inhibitory RNAs, and/or lymphoproliferative components in lymphocytes (such as B cells, T cells, and NK cells), e.g., chimeric lymphoproliferative components. In addition, in some aspects, provided herein are recombinant retroviruses that express and carry on their surface various functional components, as well as methods and packaging cell lines for producing the recombinant retroviruses. These recombinant retroviruses and methods and cells for producing them overcome the limitations of the prior art with respect to the number and size of genomes, with different functional components that provide benefits when delivered into T cells and/or NK cells.

In some aspects, methods are provided for transducing and/or genetically modifying lymphocytes (such as T cells and/or NK cells), and in illustrative embodiments, ex vivo methods are provided for transducing and/or genetically modifying resting T cells and/or NK cells. Some of these aspects can be performed more rapidly than previous methods, which can facilitate more efficient research, more efficient commercial production, and improved patient care methods. Further, provided herein are methods that in some embodiments utilize recombinant retroviruses provided herein in some aspects, in combination with pharmaceutical agents, to provide improved safety mechanisms to help modulate the activity of transduced and/or genetically modified lymphocytes (such as T cells and/or NK cells). These methods, compositions, and kits can be used as research tools in commercial generation and in adoptive cell therapy with transduced and/or genetically modified T cells and/or NK cells expressing a CAR.

Additional details regarding aspects and embodiments of the present invention are provided throughout the present patent application. Sections and section headings are for ease of reading and are not intended to limit the combinations of the invention, such as methods, compositions and kits or functional components thereof throughout the sections.

Drawings

FIG. 1 shows a schematic diagram of an illustrative composition comprising a packaging cell (100) and a replication-deficient recombinant retroviral particle (200) produced by the packaging cell (100) of one illustrative, non-limiting embodiment of the invention in FIG. 1, various vectors capable of encoding aspects of the invention, referred to as recombinant polynucleotides (110), are packaged into a recombinant retroviral particle (200) comprising in its genome a first engineered signaling polypeptide comprising one or more lymphoproliferative components and in some embodiments a second engineered signaling polypeptide which is a chimeric antigen receptor or CAR, the replication-deficient recombinant viral particle expressing on its membrane a pseudotyped component (in one non-limiting embodiment, a measles virus hemagglutinin (H) polypeptide and a virus fusion (F) polypeptide, or deletion variants thereof) (240), an activating component capable of binding and activating a resting T cell (in a non-limiting embodiment, a morbillivirus (H) polypeptide and a virus fusion (F) polypeptide, or a deletion variant of its domain) (240), an activating component capable of binding and activating a resting T cell (in a non-limiting embodiment, a polypeptide having a binding ability to bind 28 and a pol virus fusion (F) polypeptide, or a gag gene fusion protein, a gag 220, a gag-75, a fusion protein, a gag-220, a gag-75, a T5, a T cell binding and a HIV-5-75, a fusion protein, a T binding to a cell.

Figure 2 shows schematic diagrams of non-limiting illustrative compositions comprising replication-deficient recombinant retroviral particles (200) produced by packaging cells (100) and resting T cells (300) transfected by the replication-deficient recombinant retroviral particles (200) components on the surface of the replication-deficient recombinant retroviral particles (200) bind to receptors and/or ligands on the surface of resting T cells in non-limiting embodiments pseudotyping components may comprise binding polypeptides and fusogenic polypeptides that facilitate binding and fusion of the replication-deficient recombinant retroviral particles (200) to T cells (in non-limiting embodiments measles virus hemagglutinin (H) polypeptides and measles virus fusion (F) polypeptides, or cytoplasmic domain deletion variants thereof) in non-limiting embodiments, replication-deficient recombinant retroviral particles (200) comprise on their surface an activation component capable of activating resting T cells by binding to a T cell receptor complex and optionally a co-receptor (320) (in non-limiting embodiments, having a polypeptide capable of binding to CD 8663 and an activation component capable of binding to CD3) and optionally a regulatory component for binding to a cell surface of a regulatory DNA molecule encoding a non-proliferating cell receptor antagonist polypeptide (CD 75) that is capable of binding to CD 5 and binds to a cell-T cell receptor-effector molecule (CD) and further includes a regulatory polypeptide that binds to a non-regulatory component encoding a non-regulatory DNA encoding a non-proliferating cell-T cell binding to a cell-regulatory protein encoding a polypeptide that promotes the replication-binding to a cell receptor antagonist polypeptide that is capable of binding to a cell receptor (CD 9) and binds to a cell-binding to a cell receptor antagonist polypeptide that binds to a cell, a cell receptor antagonist polypeptide that is capable of a cell-binding to a cell receptor antagonist polypeptide that binds to a cell, a cell-binding to a cell receptor antagonist polypeptide that binds to a cell-binding to a cell, a cell-binding to a cell receptor antagonist polypeptide that is capable of a cell-binding to a cell receptor antagonist polypeptide that binds to a cell receptor antagonist polypeptide that is capable of controlling cell, a cell receptor antagonist polypeptide that binds to a cell, a cell-binding to a cell, a cell-binding to a cell-effector molecule that is capable of increasing cell receptor antagonist polypeptide that is capable of a cell-binding to a cell, a cell receptor antagonist polypeptide that is capable of a cell-binding to a cell receptor antagonist polypeptide that is capable of a cell.

Fig. 3A-3E show schematic diagrams of non-limiting, exemplary vector constructs for transfecting packaging cells to produce replication-defective recombinant retroviral particles described herein. Figure 3A shows a construct containing a polynucleotide sequence encoding an FRB domain fused to the NF κ B p65 activation subdomain (p65AD) and a ZFHD1 DNA binding domain fused to three FKBP repeats that are constitutively expressed. The construct in figure 3A also included HIV1 REV and Vpx as a fusion of SrcFlagVpx under the rapamycin-inducible ZFHD1/p65 AD promoter. Figure 3B shows a construct containing a polynucleotide encoding rtTA sequence under the control of ZFHD1/p65 AD promoter. Figure 3C shows a construct containing a polynucleotide encoding a puromycin resistance gene flanked by loxP sites and an extracellular MYC marker flanked by lox2272 sites. Both selectable markers are under the control of a BiTRE promoter flanked by FRT sites. Figure 3D shows a construct containing a polynucleotide encoding RFP flanked by loxP sites under the control of the TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of the RFP. Figure 3E shows a construct containing a polynucleotide encoding GFP flanked by loxP sites under the control of the TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of GEP. The constructs in figures 3C to 3E serve as landing sites for insertion of further polynucleotide sequences into the genome of the packaging cell line.

FIGS. 4A-4C show a schematic diagram of a non-limiting, exemplary vector construct for transfecting a packaging cell to produce a replication-deficient recombinant retroviral particle described herein FIG. 4A shows a construct containing a tricistronic polynucleotide encoding anti-CD 3 (homozygous UCHT1) scFvFc with a CD14 GPI anchor linkage site, CD80 extracellular domain (ECD) capable of binding CD28 to a CD16B GPI anchor linkage site, and I L-7 fused to Decay Accelerating Factor (DAF) with transposon sequences flanking the polynucleotide region for integration into the HEK293S genome, FIG. 4B shows a construct containing a polynucleotide having a BiTRE promoter and a polynucleotide region encoding a gag polypeptide and pol polypeptide in one direction, and a polynucleotide region encoding a measles protein and H Δ y protein in the other direction, and a polynucleotide construct containing a polynucleotide sequence encoding a PPIN 3 promoter (which is integrated under HECAR activity in HEK 293) and a polynucleotide region encoding a PPIN protein and H Δ y protein in the other direction, and a polynucleotide construct containing a DNA sequence encoding a PPINS-11-7 construct containing a PPINS-9 cDNA sequence encoding a PPINS 2, a PPINS-9-7 construct, a PPINS-9-2-9-a DNA sequence encoding a ribosome binding motif with a ribosome binding motif sequence encoding a ribosome binding motif, a.

FIG. 5 shows a schematic of a lentiviral expression vector encoding GFP, an anti-CD 19 chimeric antigen receptor and eTAG (referred to herein as F1-0-03).

Fig. 6A and 6B show histograms of the percentage (%) of CD3+ GFP + cells and histograms of the absolute cell counts per well of the CD3+ GFP + population at day 3, day 6, day 9, day 13 and day 17 after transduction of freshly isolated and unstimulated PBMC from donor 12M with VSV-G pseudotyped lentiviral particles encoding F1-0-03 and displaying GPI-anchored anti-CD 3 scfvcfc on their surface as indicated for 14 hours (fig. 6A) and CD3+ GFP + populations (fig. 6B). Each bar represents the mean +/-SD of two replicates.

FIGS. 7A and 7B show histograms of the percentage (%) of CD3+ GFP + cells in the total CD3+ population on days 3 and 6 after 14 hours of transduction of freshly isolated and unstimulated PBMC from donor 13F with lentiviral particles indicated as F1-0-03 (FIG. 7A) and histograms of the absolute cell counts per well of the CD3+ GFP + population (FIG. 7B). Please note that "A" shows the results of using VSV-G pseudotyped lentiviral particles (three replicates), "B" shows the results of using VSV-G pseudotyped lentiviral particles (two replicates) with OKT3Ab (1 μ G/m L) added to the transduction medium, "C" shows the results of using VSV-G pseudotyped lentiviral particles (three replicates) expressing GPI UCFvFc for GPI-anchored GPI 1scFvFc (three replicates), and "GPID" shows the results of using VSV-anchored GPI anchor and two replicates of the results of the VSV-G pseudotyped lentiviral particles (three replicates) for the surface of the GPI-anchored GPI anchor and GPI 7G bar representation of each of the results of the two replicates (7A. 7H + GCS).

Fig. 8A and 8B show histograms for the percentage of CD3+ GFP + cells in the total CD3+ population at day 3, day 6 and day 9 (fig. 8A) and histograms for the absolute cell counts per well of the CD3+ GFP + population (fig. 8B) after transduction of freshly isolated and unstimulated PBMCs from donor 12M with VSV-G pseudotyped lentiviral particles encoding F1-0-03 and manifesting GPI-anchored UCHT1 scfvcfc and GPI-anchored CD80 on their surface as indicated for the indicated exposure time (2h to 20 h). Transduction was performed in a tray or shaker flask as indicated. Each bar represents the mean +/-SD of two replicates of lentiviral particles pseudotyped with VSV-G ("VSV-G"); other experiments were not repeated.

Fig. 9A and 9B show histograms for the percentage (%) of CD3+ GFP + cells in the live CD3+ population at day 3 after transduction of freshly isolated and unstimulated PBMCs from donor 18 with lentiviral particles indicated encoding F1-0-03 (fig. 9A) and histograms for the absolute cell counts per microliter of the total live population (fig. 9B). Each bar represents the mean +/-SD of two replicates.

Fig. 10A and 10B show histograms of the percentage (%) of GFP + cells in the total viable cell population at day 6 after 4 hours of transduction of unstimulated PBMCS by different replication-defective lentiviral particles at 1MOI (fig. 10A) and the number of GFP + cells per microliter of culture (fig. 10B). The VSV-G pseudotyped lentiviral particles encoded F1-0-03[ VSV-G ] and were shown as indicated for UCHT1scFvFc-GPI [ VSV-G + U ] alone or with CD80-GPI [ VSV-G + U + CD80 ]. Samples were treated with the antiretroviral drugs dapivirine (dapivirine) and dolutegravir (dolutegravir) as indicated. "cells only" (untransduced) was included as a control.

FIGS. 11A and 11B show histograms of the percentage (%) of F L AG + cells in the total viable cell population at day 6 after 4 hours of transduction of unstimulated PBMC by different replication-defective lentiviral particles (FIG. 11A) and the number of F L AG + cells per microliter of culture (FIG. 11B). Lentiviral particles encode F1-3-219 and are used at 1 MOI.

FIGS. 12A and 12B show histograms of the percentage (%) of CD3+ F L AG + cells and the number of CD3+ F L AG + cells per microliter (FIG. 12B) in the total viable cell population at day 6 after 4 hours of transduction of unstimulated PBMC by different replication-defective lentiviral particles (FIG. 12A) and the number of CD3+ F L AG + cells in culture (FIG. 12B). Lentiviral particles encoded F1-3-451 and used at 1 MOI.

FIG. 13 is a histogram showing the number of CD3+ F L AG + cells per microliter of cultures on day 6 after unstimulated PBMCs were transduced by different replication-defective lentiviral particles at 1MOI for the indicated period F1-3-253 encodes an anti-CD 19CAR and F1-3-451 encodes C L E except for the same CAR the lentiviral particles were pseudotyped with VSV-G [ VSV-G ] and UCHT1ScFvFc-GPI [ VSV-G + U ] as indicated, the samples were treated with daproline (inhibitor of reverse transcription (RT Inb)) or dolutegravir (INT Inb)).

Figure 14A provides a schematic of the I L7R α variants tested for lymphoproliferative/survival activity when expressed in PBMCs figure 14B provides a bar graph showing the percentage of PBMC viability in the presence and absence of I L-2.

Figure 15 is a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding CAR and candidate C L E of pools 3A, 3B, 3.1A and 3.1B.

FIG. 16 is a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding a CAR and a candidate chimeric lymphoproliferative component of libraries 1A, 1.1A and 1.1B (C L E).

FIG. 17 is a schematic diagram of a non-limiting exemplary transgene expression cassette containing a polynucleotide sequence encoding candidate C L E of libraries 2B and 2.1B.

FIG. 18 is a schematic diagram of a non-limiting exemplary transgene expression cassette containing a polynucleotide sequence encoding candidate C L E of libraries 4B and 4.1B.

Figure 19 is a graph showing fold expansion of PBMCs transduced with lentiviral particles encoding individual C L E and cultured for 35 days in the absence of exogenous interleukins.

Figure 20 is a diagram showing fold expansion of PBMCs transduced with lentiviral particles encoding an anti-CD 19CAR construct and individual C L E and cultured for 35 days in the presence of donor-matched PBMCs but in the absence of exogenous interleukins.

Figure 21 is a graph showing the efficiency of transduction of resting PBMCs by indicated lentiviral particles within 4 hours in the absence of exogenous interleukins, the transduction efficiency was measured as CAR + PBMC% in culture after 6 days, as determined by FACS each lentiviral particle encoded CAR and C L e lentiviral particles F1-1-228U and F1-3-219U developed UCHT1 scfvffc-GPI on its surface.

FIGS. 22A and 22B are diagrams showing the temporal course of the total number of viable cells after 4 hours of transduction of resting PBMCs with the indicated lentiviral particles and 6 days of in vitro culture in the absence of exogenous interleukins each lentiviral particle encodes CAR and C L E, lentiviral particles F1-1-228U and F1-3-219U exhibit UCHT1scFvFc-GPI on its surface.

Fig. 23A, 23B and 23C are diagrams showing the time course of replication of lentiviral genome per microgram of genomic DNA from blood of tumor-bearing NSG mice administered with human PBMC transduced with the indicated lentiviral particles for 4 hours and injected intravenously without ex vivo expansion of PBMC each lentiviral particle encodes car.f 1-1-228, F1-1-228U, F1-3-219 and F1-3-219U also encodes C L e lentiviral particles F1-1-228U and F1-3-219U also display UCHT1 scfvcfc-GPI on their surface.

Figure 24 is a graph showing the number of CAR + cells per 200 μ Ι of blood of tumor-bearing NSG mice administered with human PBMC transduced with the indicated lentiviral particles for 4 hours and injected intravenously without ex vivo expansion of PBMCs.

Figure 25A is a graph showing the average volume of CHO-ROR2 tumors in NSG mice intravenously dosed with 4 hours of PBS or human PBMC transduced with the indicated lentiviral particles encoding anti-ROR 2 MRB CAR and C L E without ex vivo expansion of PBMC figure 25B is a graph showing the average volume of Raji tumors in NSG mice intravenously dosed with 4 hours of PBS or human PBMC transduced with the indicated lentiviral particles encoding anti-CD 19CAR and C L E without ex vivo expansion of PBMC figure 1-1-228U and F1-3-219U of lentiviral particles display UCHT1 scfvcfc-GPI on their surface.

FIG. 26A is a schematic representation of the lentiviral vector backbone F1-0-02, including the transgenic expression cassette driving expression of GFP and eTag, and the synthetic EF-1 α promoter and intron A upstream of GFP FIG. 26B shows insertion of miRNA into the EF1 α intron A of the F1-0-02 backbone, ` 1 ` denotes an EF1 α overlap ` 2 ` denotes a5 'arm ` 3 ` denotes the miRNA 15' stem ` 4 ` denotes a loop ` 5 ` denotes the miRNA 13 'stem ` 6 ` denotes the 3' arm `.

Figure 27 is a graph showing that mirnas targeting CD3 ζ, located in the promoter intron of EF-1 α, are capable of blocking expression of the CD3 complex.

FIG. 28 is a histogram showing the Δ Δ Ct values for samples transduced with miR-TCR α containing replication-defective lentiviral particles.

Stator

As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that specifically transplants antigens onto cells, such as T cells, NK cells, macrophages, and stem cells, the CARs of the present invention include at least one antigen-specific targeting region (ASTR), transmembrane domain (TM), and Intracellular Activation Domain (IAD), and may include a stem and one or more co-stimulatory domains (CSD). in another embodiment, the CARs are bispecific CARs specific for two different antigens or epitopes.

As used herein, the term "microenvironment" means any part or region of a tissue or body that has constant or temporal, physical or chemical differentiation from other tissue regions or body regions. For example, as used herein, "tumor microenvironment" refers to the environment in which a tumor resides, which is a non-cellular region within the tumor, and a region located just outside of the tumor tissue does not belong to the intracellular compartment of the cancer cells themselves. A tumor microenvironment may refer to any and all conditions of the tumor environment, including conditions that create a structural and/or functional environment for a malignant process to survive and/or expand and/or spread. For example, a tumor microenvironment may include alterations 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 an oxidizing agent, reducing agent or cofactor, and other conditions as will be appreciated by the skilled artisan.

As used interchangeably herein, the term "polynucleotide" and the term "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 multiple 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, the term "antibody" includes both polyclonal and monoclonal antibodies, including intact antibodies and antibody fragments that retain specific binding to an antigen. An antibody fragment may be (but is not limited to): fragment antigen binding fragment (Fab) fragment, Fab 'fragment, F (ab')₂Fragments, Fv fragments, Fab '-SH fragments, (Fab')₂Fv 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., sdAb, sdFv, nanobodies). The term includes genetically engineered and/or other modified forms of immunoglobulins such as: intrabodies, peptide antibodies, chimeric antibodies, single chain antibodies, fully human antibodies, humanized antibodies, fusion proteins including antigen-specific targeting regions of antibody and non-antibody proteins, heteroconjugate antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies), tandem bis-scfvs, and tandem tri-scfvs. Unless otherwise stated, the term "antibody" is understood to include functional antibody fragments thereof. The term also includes whole or full-length antibodies, which include antibodies of any class or subclass, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD.

As used herein, the term "antibody fragment" includes a portion of an intact antibody, e.g., an antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; a bifunctional antibody; linear antibodies (Zapata et al, 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 (called "Fab" fragments, each with a single antigen-binding site) and a residual "Fe" fragment (an indication of the ability to reflect facile crystallization). Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking the antigen₂And (3) fragment.

As used interchangeably herein, the terms "single chain Fv", "scFv" or "sFv" antibody fragment include the V of an antibody_HDomain and V_LDomains, wherein the domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide is further comprised in V_HDomain and V_LBetween the domains, such that the sFv can form a polypeptide linker or spacer of desired structure for antigen binding. For an overview of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269 to 315 (1994).

As used herein, "naturally occurring" V_HDomain and V_LA domain refers to a V that has been isolated from a host without further molecular evolution to alter its affinity when produced as an scFv under specific conditions_HDomain and V_LDomains such as those disclosed in U.S. patent 8709755B2 and application WO/2016/033331A 1.

As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two reagents, and is expressed as the dissociation constant (Kd). The affinity can 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 greater than the affinity of the antibody for an unrelated amino acid sequence, or higher. The affinity of an antibody for a target protein can be, for example, about 100 nemo (nM) to about 0.1nM, about 100nM to about 1 picomole (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 upon dilution. With respect to antibodies and/or antigen binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein.

As used herein, the term "binding" refers to a binding due to, for example, covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic and/or hydrogen bonding interactions, includingSuch as salt and water bridge interactions) to associate directly between two molecules. Non-specific binding shall mean an affinity of less than about 10^-7Binding of M, e.g. with an affinity of less than 10^-6M、10^-5M、10^-4M, and the like.

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

As used herein, the term "module" includes polypeptides (including fusions to 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 "stalk" or "stalk domain" refers to a flexible polypeptide attachment region that provides structural flexibility and is spaced apart from flanking polypeptide regions, and may be composed of a natural or synthetic polypeptide. The stalk may be derived from the hinge or hinge region of an immunoglobulin (e.g., IgGl), which is generally defined as extending from Glu216 to Pro230 of human IgGl (Burton (1985) molecular. Immunol.,22: 161-206). The hinge region of other IgG isotypes can be aligned to the IgG1 sequence by allowing the first cysteine to form an inter-heavy chain disulfide bond (S-S) at the same position as the last cysteine. The handle may be naturally occurring or non-naturally occurring, including but not limited to an altered hinge region, as disclosed in U.S. Pat. No. 5,677,425. The handle may include an intact hinge region derived from any class or subclass of antibodies. 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 in that the vector or composition is not part of its natural environment.

As used herein, a "polypeptide" is a single chain of amino acid residues joined by peptide bonds. 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 can be "purified" to remove impurity components of the polypeptide's natural environment, 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 can be (1) purified to greater than 90%, greater than 95%, or greater than 98% by weight of the antibody, as determined using the lorentzian method (L owry method), e.g., greater than 99% by weight, (2) purified by means of a rotating cup sequencer to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) purified to homogeneity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using coomassie blue or silver dyes.

As used herein, the term "immune cell" 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 cells derived from bone marrow (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

As used herein, "T cell" includes expression of CD3Including helper T cells (CD 4)⁺Cells), cytotoxic T cells (CD 8)⁺Cells), regulatory T cells (tregs), and gamma-T cells.

As used herein, "cytotoxic cell" includes CD8⁺T cells, Natural Killer (NK) cells, NK-T cells, gamma T cells (a CD 4)⁺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 (pluralityor multipotent stem cells). "Stem cells" include, for example, embryonic stem cells (ES), Mesenchymal Stem Cells (MSC), induced differentiated pluripotent stem cells (iPS), and committed progenitor cells (hematopoietic stem cells (HSC), bone marrow-derived cells, etc.).

As used herein, the term "treating" and the like refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms 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 development of a disease in an individual who is predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

As used interchangeably herein, the terms "individual", "subject", "host" and "patient" refer to a mammal, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (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 the amount of an agent or a combined amount of two agents that, when administered to a mammal or other subject for the treatment of a disease, is sufficient to effect such treatment of the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the individual to be treated.

As used herein, the term "evolution" refers to the use of one or more mutation methods to produce a different polynucleotide encoding an otherwise polypeptide that is itself an improved biomolecule and/or facilitates 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 oxidizing, reducing, or co-factors, and other conditions that would be considered to be within normal ranges for an individual at the site of administration or at the tissue or organ at the site of action.

As used herein, "genetically modified cell" includes cells that contain an exogenous nucleic acid, whether or not the exogenous nucleic acid is integrated into the genome of the cell.

As used herein, a "polypeptide" may include a portion of a proteinaceous molecule or a complete proteinaceous molecule as well as any post-translational or other modifications.

A pseudotyped component as used herein may include: "binding polypeptides" which include one or more polypeptides (typically glycoproteins) that recognize and bind to a target host cell; and one or more "fusogenic polypeptides" that mediate the fusion of the retrovirus with the target host cell membrane, thereby allowing the retroviral genome to enter the target host cell. As used herein, a "binding polypeptide" may also be referred to as a "T cell and/or NK cell binding polypeptide" or "target engaging component", and a "fusogenic polypeptide" may also be referred to as a "fusogenic component".

Resting "lymphocytes (such as resting T cells) are lymphocytes in the G0 stage of the cell cycle that do not express an activation marker (such as Ki-67). Resting lymphocytes may include both naive T cells that have not been exposed to a specific antigen and memory T cells that have been altered by prior exposure to an antigen. "resting" lymphocytes may also be referred to as "quiescent" lymphocytes.

As used herein, "lymphodepletion" relates to a method of reducing the number of lymphocytes in a subject (e.g., with administration of a lymphodepleting agent). Fractionated radiation therapy of parts of the body or the whole body can also lead to lymphatic depletion. The lymphodepleting agent may be a chemical compound or composition capable of reducing the number of functional lymphocytes in a mammal upon administration thereof to the mammal. One example of such an agent is one or more chemotherapeutic agents. Such agents and dosages are known and can be selected by the treating physician, depending on the individual to be treated. Examples of lymphodepleting agents may include, but are not limited to, fludarabine (fludarabine), cyclophosphamide, cladribine (cladribine), dinierein (denileukin diftotox), or combinations thereof.

RNA interference (RNAi) is a biological process in which an RNA molecule inhibits gene expression or translation by neutralizing through a target RNA molecule. The RNA target may be mRNA, or it may be any other RNA that is sensitive to functional inhibition of RNAi. As used herein, an "inhibitory RNA molecule" refers to an RNA molecule whose presence produces RNAi within a cell and results in reduced expression of a transcript targeted by the inhibitory RNA molecule. An inhibitory RNA molecule as used herein has a5 'stem and a 3' stem capable of forming an RNA duplex. The inhibitory RNA molecule can be, for example, a miRNA (endogenous or artificial) or shRNA, a precursor of a miRNA (i.e., Pri-miRNA or pre-miRNA) or shRNA, or a dsRNA that is directly transcribed or introduced into a cell or an individual as an isolated nucleic acid.

As used herein, "double-stranded RNA" or "dsRNA" or "RNA duplex" refers to an RNA molecule consisting of two strands. Double-stranded molecules include those consisting of two RNA strands that hybridize to form a duplex RNA structure, or single-stranded RNA 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 contains sequences 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 in length, or in some embodiments between 18, 19, 20, 21, 22, 23, 24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 on the high end, with a given range typically having a lower end than the high end. Such structures typically include a5 'stem, loops, and a 3' stem connected by loops (which are not part of a double helix) adjacent to each stem. 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 at the low end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 at the high end, with a given range typically having 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 the components of microRNA processing. The microRNA processing component is the minimal nucleic acid sequence that facilitates the production of mature microRNAs from precursor microRNAs. These modules are typically located within a 40 nucleotide sequence flanking a microrna stem-loop structure. In some cases, the microrna processing components 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 multiple inhibitory RNA molecules refers to a linking member that adds two inhibitory RNA molecules.

As used herein, unless it is specifically indicated as replication competent retrovirus, the term "recombinant retrovirus" refers to a replication incompetent or "replication defective" retrovirus the terms "recombinant retrovirus" and "recombinant retroviral particle" are used interchangeably herein such retrovirus/retroviral particles can be any type of retroviral particle, including, for example, gamma retrovirus and, in illustrative embodiments, 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 a plasmid (which includes a packaging component such as Gag, Pol and Rev) and an enveloped or pseudotyped plasmid (which encodes a pseudotyped component) and a transferred, genomic or retroviral (e.g., lentiviral) expression vector (which typically is a plasmid encoding a gene or other related coding sequence thereon), thus, a retroviral (e.g., lentiviral) expression vector includes sequences that facilitate expression and packaging after transfection into the cell (e.g., such as psi and heterologous target packaging component coding sequence flanking 355 '84 and the term "TR 3' 84" is used interchangeably herein.

The "framework" of a miRNA is composed of "5 'microrna flanking sequences" and/or "3' microrna flanking sequences" surrounding the miRNA and (in some cases) loop sequences separating the stems of the stem-loop structures in the miRNA. In some examples, a "framework" is derived from a naturally occurring miRNA, such as miR-155. The terms "5 'microRNA flanking sequence" and "5' arm" are used interchangeably herein. The terms "3 'microrna flanking sequence" and "3' arm" are used interchangeably herein.

As used herein, the term "miRNA precursor" refers to an RNA molecule of any length that can be enzymatically processed into a miRNA, such as a primary RNA transcript, pri-miRNA or 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 component. The skilled person will understand that a construct refers to an isolated polynucleotide or an isolated polypeptide, depending on the context.

It is understood that the invention and the aspects and embodiments provided herein are not limited to the specific examples disclosed, and thus, variations are, of course, possible. It is also to be understood that the technology used herein is for the purpose of disclosing specific examples and embodiments, and is not intended to be limiting, since the scope of the invention 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 invention. 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 excluded limit in the stated range. Where stated ranges include one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. When overlapping low and high values are given for the range, one 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 to be construed as 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 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 cited.

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 is further noted that the claims may be directed to excluding any optional elements. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

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 subcombination. All combinations that are embodiments of the invention are specifically embraced by the invention and are disclosed herein just as if each and every combination were individually and clearly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically encompassed by the invention and are disclosed herein as if each and every such subcombination is individually and clearly disclosed.

Detailed Description

The present invention overcomes the prior art challenges by providing improved methods and compositions for genetically modifying lymphocytes, such as NK cells, and in illustrative embodiments, T cells. For example, some of these methods do not include pre-activating lymphocytes, and some of these methods are performed in less time than previous methods. In addition, compositions having a number of uses, including their use in these improved methods, are provided. Some of these compositions are genetically modified lymphocytes with improved proliferation and survival quality, including when cultured in vitro, for example in the absence of growth factors. These genetically modified lymphocytes will have uses such as: as a research tool to better understand the factors that influence T cell proliferation and survival; and commercial production, e.g., production of certain factors (such as growth factors and immunomodulators) that can be collected and tested or used in commercial products.

Some embodiments provided herein are methods for performing adoptive cell therapy that include transducing T cells and/or NK cells ex vivo requiring a fraction of time (e.g., 24 hours, 12 hours, or 8 hours or less), and in some embodiments without requiring prior ex vivo stimulation. These methods are preferably suitable for closed systems that process blood from an individual ex vivo, and may be performed for an individual present in the same room as some embodiments and/or for an individual in some embodiments at all times during the performance of the method within their line of sight of their blood or their isolated blood cells. More specifically, aspects and embodiments of the disclosure herein overcome problems associated with current adoptive cell therapies by providing methods for transducing resting T cells and/or resting NK cells, which generally utilize pseudotyping components that facilitate binding and fusion of replication-defective recombinant retroviral particles to resting T cells and/or resting NK cells, to facilitate genetic modification of resting T cells and/or resting NK cells with the replication-defective recombinant retroviral particles. Furthermore, the methods provided herein overcome the problems in the art by, in illustrative embodiments, utilizing a chimeric antigen receptor and one or more lymphoproliferative components, the expression of which is under the control of a control component, such that exposure of an individual to a compound that binds to the control component, or termination of such exposure promotes the expansion of genetically modified T cells and/or NK cells in vivo.

In view of these and other improvements disclosed in detail herein, in one aspect, provided herein is a method for genetically modifying resting T cells and/or resting NK cells of an individual (such as a patient having a disease or disorder), wherein blood from the individual is collected; resting T cells and/or NK cells are genetically modified by contacting them with replication-defective recombinant retroviral particles; and the genetically modified cells are reintroduced into the individual, typically within a shorter period of time (e.g., within 24 hours and in some non-limiting embodiments within 12 hours) than in prior art methods and/or without further ex vivo expansion of the population of genetically modified T cells and/or NK cells, e.g., such that the genetically modified resting T cells and/or NK cells do not undergo more than 4 ex vivo cell divisions. Thus, the methods provided herein can be performed in a much shorter time than current CAR therapies, thereby providing a method in which an individual can remain in the clinic for the entire time of the ex vivo step. This facilitates performing the ex vivo step in a closed system, which reduces the chance of contamination and mixing of the patient sample and can be performed more easily by a clinical laboratory.

Accordingly, fig. 1 and 2 provide schematic block diagrams of illustrative compositions for use in the methods provided herein. FIG. 1 provides a block diagram of packaging cells (100) and replication-defective recombinant retroviral particles (200) produced by such packaging cells. The packaging cell (100) comprises a recombinant polynucleotide (110) incorporated into its genome comprising a recombinant transcription component that expresses a retroviral protein and a variety of different membrane-bound polypeptides under the control of an inducible promoter regulated by a ligand-bound and ligand-activated transactivator. These transactivators, inducible promoters and ligands are used to induce sequential expression and accumulation of cell membrane-bound polypeptides to be incorporated into the membrane of the replication-defective recombinant retroviral particle and the retroviral components necessary for packaging and assembly of the replication-defective recombinant retroviral particle.

The illustrative packaging cell (100) illustrated in fig. 1 is produced as a result of sequential induced expression of various polynucleotides as discussed in detail below, and can be used in illustrative methods to produce replication-defective recombinant retroviral particles for use in methods of transfecting resting T cells and/or NK cells (300 in fig. 2) provided herein. In non-limiting embodiments, the packaging cell (100) includes in its genome nucleic acid encoding a packagable retroviral RNA genome that includes at least some of the components of the retroviral genome necessary for packaging and assembly of replication-defective recombinant retroviral particles (as non-limiting illustrative examples, a retroviral psi component, a retroviral gag polypeptide, and a retroviral pol polypeptide).

For example, a packaging cell and, in particular, a replication-defective recombinant retroviral particle formed may comprise a retroviral Vpx polypeptide (250), which in non-limiting embodiments may be expressed as a membrane-associated fusion protein, such as a Src-Flag-Vpx polypeptide, a pseudotyping component (240) which may comprise a binding polypeptide and a fusogenic polypeptide, in non-limiting embodiments a measles virus prothrombin (H) polypeptide and a measles virus fusion (F) polypeptide, or cytoplasmic domain deleted variants thereof, optionally one or more activating components (210, 220), in non-limiting embodiments comprising a membrane-binding polypeptide capable of binding to CD3 and a membrane-binding polypeptide capable of binding to CD28, and/or optionally a membrane-binding interleukin (230), in non-limiting embodiments comprising a variety of other membrane-binding polypeptides of the type L-7 fused to DAF or fragments thereof.

Replication-defective recombinant retroviral particles are produced as a result of sequential expression of the transcription modules by the packaging cell. The RNA retroviral genome within and typically integrated into the genome of a packaging cell that becomes the genome of a replication-defective recombinant retroviral particle includes retroviral components (as non-limiting illustrative examples, retroviral Gag and Pol polynucleotides) necessary for retroviral production, infection and integration into the genome of a host cell, which is typically a resting T cell and/or NK cell. In addition, the retroviral genome further comprises a polynucleotide encoding one or, typically, two engineered signaling polypeptides provided herein. One of the engineered signaling polypeptides typically encodes at least one lymphoproliferative component, the lymphoproliferative component (in a non-limiting example, a constitutive interleukin 7 receptor mutation) and the other engineered signaling polypeptide typically encodes a chimeric antigen receptor.

Subsequently, the replication-defective recombinant retroviral particle (200) is used to transduce resting T cells and/or resting NK cells (300) in the methods provided herein. As shown in fig. 2, after contacting resting T cells and/or NK cells (300) with the replication deficient recombinant retroviral particle (200), the membrane polypeptides discussed above on the surface of the replication deficient recombinant retroviral particle bind to receptors and/or ligands on the surface of resting T cells and/or NK cells (300). For example, pseudotyped components that may include binding polypeptides and fusogenic polypeptides that bind to molecules on the surface of resting T cells and/or resting NK cells as indicated above facilitate the binding and fusion of replication-defective recombinant retroviral particles (200) to T cell and/or NK cell membranes. The activation component (210, 220) activates resting T cells and/or NK cells (300) by engaging the T cell receptor complex, a process that occurs during the time course of contact or subsequent incubation. In addition, membrane-bound interleukins (230) may be present on the surface of the replication-defective recombinant retroviral particles and bind to the interleukin receptors (310) on the surface of resting T cells and/or NK cells (300), thereby further facilitating binding and activation. Thus, without being limited by theory, in the illustrative embodiments provided herein, due to one or more of these replication-defective recombinant retroviral particle (200) components, there is no need to utilize ex vivo stimulation or activation of components that are not already in or on the replication-defective recombinant retroviral particle (200). This in turn helps to reduce the ex vivo time required to complete the methods of the illustrative methods provided herein.

Upon binding to the T cell and/or NK cell (200), the replication-defective recombinant retroviral particle is then fused to the T cell and/or NK cell (300), and the polypeptides and nucleic acids in the replication-defective recombinant retroviral particle enter the T cell and/or NK cell (300). As indicated above, one of these polypeptides in the replication-defective recombinant retroviral particle is a Vpx polypeptide (250). The Vpx polypeptide (250) binds to and induces degradation of SAMHD1 restriction factor (350) that degrades free dntps in the cytoplasm. Thus, the concentration of free dntps in the cytoplasm increases as Vpx degrades SAMHD1 and increases reverse transcription activity, thereby facilitating reverse transcription and integration of the retroviral genome into the T cell and/or NK cell genome.

Following integration of the retroviral genome into a T cell and/or NK cell (200), the T cell and/or NK cell genome comprises a nucleic acid encoding a signalling polypeptide encoding a lymphoproliferative component (370) and optionally a signalling polypeptide encoding a CAR (360). Lymphoproliferative component the expression of the lymphoproliferative component and optionally the CAR is under the control of a control component. Exposure to a compound that binds to a control component, which can occur in vitro or in vivo by administering the compound to an individual transduced with its T cells and/or NK cells (300), promotes proliferation of T cells and/or NK cells (300) in vitro or in vivo by expressing the lymphoproliferative component and optionally due to expression of the CAR and binding of the CAR to its target cells. Thus, T cells and/or NK cells transduced herein with replication-defective recombinant retroviral particles have one or more signals that drive proliferation and/or inhibit cell death (in illustrative embodiments, this in turn avoids the need for prior methods to lymphodeplete the host prior to returning the transduced T cells and/or NK cells to the individual). In illustrative embodiments, this in turn further reduces the number of days required for treatment prior to reintroduction of the transduced T cells and/or NK cells into the individual. Thus, in illustrative embodiments, the time required from blood collection to reintroduction of blood to the subject is no more than 36 hours, 24 hours, 12 hours, or in some cases even no more than 8 hours, which radically alters the CAR-T method from prior art methods. In addition, the control component also provides one of the security mechanisms provided herein. For example, discontinuing administration of the compound can down-regulate or even terminate expression of lymphoproliferative component, and optionally a CAR, thereby ending proliferation and/or survival signals to the transduced T cells and/or NK cells and their progeny.

Method for transducing and/or genetically modifying lymphocytes

In certain aspects, provided herein is a method of transducing and/or genetically modifying Peripheral Blood Mononuclear Cells (PBMCs) or lymphocytes, typically T cells and/or NK cells, and in certain illustrative embodiments typically resting T cells and/or resting NK cells, comprising contacting the lymphocytes with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles typically comprise pseudotyped components on their surfaces, wherein the contacting (and incubating under the contacting conditions) facilitates transduction of resting T cells and/or NK cells by the replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. The pseudotyped module is generally capable of binding resting T cells and/or NK cells and may facilitate membrane fusion of itself or binding to other proteins of replication-defective recombinant retroviral particles.

Various components or steps of such method aspects for transducing and/or genetically modifying PBMCs, lymphocytes, T cells, and/or NK cells are provided herein (e.g., in this section and in the exemplary embodiments section), and as further discussed herein, such methods include embodiments provided throughout this specification, for example, embodiments such as any of the aspects for transducing and/or genetically modifying PBMCs or lymphocytes (e.g., NK cells, or, in illustrative embodiments, T cells) provided for examples in this section and in the exemplary embodiments section can include any of the embodiments of replication-defective recombinant retroviral particles provided herein, including those comprising: one or more lymphoproliferative components, CARs, pseudotyped components, riboswitches, activating components, membrane-bound interleukins, mirnas, Kozak-type sequences, WPRE components, triple stop codons, and/or other components disclosed herein, and can be combined with the methods herein for producing retroviral particles using packaging cells. In certain illustrative embodiments, the retroviral particle is a lentiviral particle. Such methods for genetically modifying and/or transducing PBMCs or lymphocytes, such as T cells and/or NK cells, may be performed in vitro or ex vivo. The skilled artisan will recognize that the details provided herein for transducing and/or genetically modifying PBMCs or lymphocytes (such as T cells and/or NK cells) are applicable to any aspect that includes such steps.

In certain illustrative embodiments, the cells are genetically modified and/or transduced without prior in vivo, in vitro, or ex vivo activation or stimulation. In certain illustrative embodiments, the cells are activated during the contacting and are not activated at all or are activated for more than 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours prior to the contacting. In certain illustrative embodiments, activation by components not present on the surface of a retroviral particle does not require genetically modifying and/or transducing the cell. Thus, no such activating or stimulating means are required, other than on the retroviral particle, before, during or after contact. Thus, as discussed in more detail herein, these illustrative embodiments that do not require pre-activation or stimulation provide the ability to quickly perform in vitro experiments aimed at better understanding T cells and the biological mechanisms therein. Furthermore, such methods provide for more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells and development of such commercial production methods. Finally, such methods provide for more rapid ex vivo processing of PBMCs for adoptive cell therapies, e.g., by providing point-of-care methods to substantially simplify the delivery of such therapies.

The contacting step of the methods for transduction and/or for genetically modified methods provided herein generally comprises an initial step in which retroviral particles (typically a population of retroviral particles) are contacted with cells (typically a population of cells) while in a liquid buffer and/or culture medium comprising a suspension to form a transduction reaction mixture, followed by an optional incubation period in this reaction mixture comprising the retroviral particles and the cells in suspension. The contacting may be performed, for example, in a chamber of a closed system suitable for processing PMBC, as discussed in more detail herein. The transduction reaction mixture may include one or more buffers or ions, and in illustrative embodiments includes culture media, such as those known in the art for ex vivo processing involving lymphocytes, as provided in further detail herein.

The transduction reaction mixture may be incubated between 23 ℃ and 39 ℃, and in some illustrative embodiments at 37 ℃. In certain embodiments, the transduction reaction may be performed at 37 ℃ to 39 ℃ for faster fusion/transduction. When contacted in the transduction reaction mixture, the cells and retroviral particles can be immediately treated to remove from the cells the retroviral particles that remain free in suspension and unassociated with the cells. Optionally, the cells and retroviral particles, whether free in suspension or in suspension 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. Prior to other steps, washing, such as in culture media used in transduction reactions, may be performed, regardless of whether such cells are to be studied in vitro, ex vivo, or introduced into an individual.

In some embodiments, the replication-defective recombinant retroviral particle may further comprise an activation module, which may be any activation module provided herein. In illustrative embodiments, the activating component may be anti-CD 3, such as anti-CD 3scFv or anti-CD 3 scfvffc.

In some embodiments, the contacting step in the methods provided herein of transducing and/or genetically modifying PBMCs or lymphocytes (typically T cells and/or NK cells) may be performed (or may occur) for between 1 and 24 hours, such as between 1 and 12 hours, or between 1 and 6 hours. In some illustrative embodiments, the contacting can be performed for less than 24 hours, e.g., 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 one initial contacting step in which the retroviral particles are contacted with the cells in suspension in the transduction reaction mixture. Such suspensions may include precipitation of cells and retroviral particles, or such precipitation of the bottom of a container or chamber by application of a force such as a centrifugal force. Following such initial contact, additional optional incubations can be performed in the reaction mixture (containing the cells and retroviral particles in suspension in the reaction mixture) without removing retroviral particles that remain free in solution and unassociated with the cells. In illustrative embodiments, the contacting can occur (or occur) for 30 seconds or 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 45 minutes, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours at the lower end of the range and between 10 minutes, 15 minutes, 30 minutes, or 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, and 72 hours at the upper end of the range. In certain illustrative embodiments, the contacting step can be performed for a time between 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes at the low end of the range and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours at the high end of the range. In some embodiments, the contacting step is performed for between 30 seconds, 1 minute, and 5 minutes at the high end of the range and 10 minutes at the high end of the range. In another illustrative embodiment, the contacting is only between the initial contacting step (without any additional incubation in the reaction mixture comprising free retroviral particles in suspension and cells in suspension) and without any additional incubation in the reaction mixture, or between 5 minutes, 10 minutes, 15 minutes, 30 minutes or 1 hour incubation of the reaction mixture.

The methods of genetic modification and/or transduction of cells provided herein generally comprise inserting into a cell a polynucleotide comprising one or more transcription units encoding a CAR or lymphoproliferative component, or, in illustrative embodiments, both a CAR and lymphoproliferative component according to any of the CAR and lymphoproliferative component embodiments provided herein, or, in illustrative embodiments, a lymphoproliferative component that promotes survival and/or proliferation under suitable conditions including, but not limited to, those provided in the examples provided herein, that cause survival and/or proliferation of a genetically modified and/or transduced cell, as described herein, thus, compared to a control cell that is genetically modified and/or transduced prior to its being genetically modified and/or transduced, or, in some embodiments, the same control recombinant retroviral particle modified and/or transduced as used to produce a genetically modified and/or transduced cell that is capable of increasing survival or expansion, or, in comparison to a control cell that is modified and/or transduced cell that is cultured with the same control recombinant retroviral particle that is used to produce a genetically modified and/or transduced cell that is capable of increasing survival or expanding, or that is capable of proliferating cells in vitro, or in vivo, in comparison to a control cell culture that comprises one or, in vitro, one or more of a cell-proliferating cell-expressing, or cell-proliferating cell, or cell-expressing, by addition of one or under in vitro transcription of a cell-expressing one or cell-expressing one or cell-expressing a cell-proliferating cell-expressing a cell-expressing a gene-expressing cell-expressing gene-expressing a gene-expressing cell-expressing a cell-expressing gene-expressing a gene-expressing cell-expressing a gene-expressing property-expressing cell-expressing a cell-expressing cell-expressing a gene-expressing cell-expressing.

Thus, in illustrative embodiments, the methods for genetically modifying and/or transducing herein provide a rapid means of providing new functionality by inserting into cells nucleic acids that, when expressed, provide the function of a CAR and/or lymphoproliferative component as discussed herein, for example, as provided in examples herein, such cells are capable of better survival and/or expansion in vitro and in vivo than control cells that are identical to the genetically modified cells but do not comprise nucleic acids encoding a lymphoproliferative component and optionally a CAR, for example, the examples herein demonstrate that genetically modified PBMCs expressing a CAR or expressing both a lymphoproliferative component and a lymphoproliferative component survive and/or proliferate more during the first 7 days in culture after contact compared to the same control cells that have not been genetically modified, wherein the culture survives or increases survival and/or proliferation of both the CAR and the lymphoproliferative component in the absence of any added interleukins (such as, but not limited to, I L-2, I L-15 or I L-7) or the cells that have increased mitotic characteristics after contact with these cells, such as do not have increased mitotic gene expression characteristics, as compared to cells that when contacted with control cells that when cells that are not genetically modified cells, the cells have increased mitotic gene expression of the cells, such as compared to when contacted on day 7, the same day, the cells have increased expression of the same day of contacting.

In certain embodiments, the genetically modified and/or transduced cell exhibits, is capable of, is suitable for, possesses, or is modified for amplification in a medium in the absence of an exogenously added interleukin, in the absence of an optionally bound antigen encoded by the genome of the retroviral particle, and/or in the absence of a lymphocyte mitotic agent, compared to a control cell that is the same as the genetically modified and/or transduced cell prior to its being genetically modified and/or transduced, the genetically modified and/or transduced cell exhibits, is capable of, is suitable for, possesses the following properties, or is modified for improved amplification in a medium in the absence of any exogenously added T cell stimulatory agent (e.g., I L-2, I L-7, I L-15, anti-CD 3, or anti-CD 28), or is modified for amplification in a medium.

For example, the medium that may be included in the contacting step or that may be used during cell culture and/or during various washing steps may include basal media, such as commercially available media for ex vivo T cell and/or NK cell culture, when the cells and retroviral particles are initially contacted with the retroviral particles (including the retroviral particles and cells in a suspension-containing medium) or thereafter contacted therewith during an optional incubation period. Non-limiting examples of such media include: X-VIVO^TM15 chemically defined serum-free hematopoietic cell culture medium (L onza) (catalog No. 2018 BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q or 04-418Q), ImmunoCult^TMXF T cell expansion Medium (STEMCE LL Technologies) (2018 Cat. No. 10981),

T cell expansion XSFM (Irvine scientific) (2018 Cat. No. 91141),

Medium CTS^TM(Therapeutic Grade) (Thermo Fisher Scientific (herein referred to as "Thermo Fisher") or CTS^TMOptimizer^TMMedia (Thermo Fisher) (2018 catalog No. a10221-01 (basal media (bottle)) and a10484-02 (supplement), a10221-03 (basal media (bag)), a1048501 (basal media and supplement kit (bottle)), and a1048503 (basal media and supplement kit (bag))^TMOpTmizer^TMT cell expansion SFM, flask type) or A1048503 (CTS)^TMOpTmizer^TMT cell expansion SFM, bag format) additives such as human serum albumin, human AB + serum, and/or serum derived from the individual may be added to the transduction reaction mixture, supporting interleukins such as I L2, I L7, or I L15 or those found in human serum may be added to the transduction reaction mixture.

In exemplary embodiments of such methods, the genetically modified T cells or NK cells can be transplanted and/or enriched in mice in vivo for at least 7, 14, or 28 days. The skilled person will recognise that such mice may be treated or otherwise genetically modified such that any immunological differences between the genetically modified T cells and/or NK cells do not result in an immune response in the mouse against any component of the lymphocytes transduced by the replication defective recombinant retroviral particles.

The packaging cells (and in illustrative embodiments, packaging cell lines, and in certain illustrative embodiments, packaging cells provided in certain aspects herein) are used to produce replication-defective recombinant retroviral particles. In some embodiments, the packaging cell line can be a suspension cell line. In an illustrative example, a packaging cell line can be grown in serum-free media. In some embodiments, the lymphocytes can be from an individual. In an illustrative embodiment, the lymphocytes can be from the blood of an individual.

Thus, in one aspect, provided herein is a method for transducing (and/or genetically modifying) lymphocytes (typically resting T cells and/or resting NK cells from isolated blood), the method comprising:

A. collecting blood from a subject;

B. isolating Peripheral Blood Mononuclear Cells (PBMCs) comprising resting T cells and/or resting NK cells; and

C. contacting ex vivo resting T cells and/or resting NK cells of an individual with a non-replicating recombinant retroviral particle, wherein the non-replicating recombinant retroviral particle comprises on its surface a pseudotyping component capable of binding to the resting T cells and/or resting NK cells and facilitating membrane fusion of the non-replicating recombinant retroviral particle with it, wherein the contacting facilitates transduction of at least 5% of the resting T cells and/or resting NK cells with the non-replicating recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells, thereby transducing the resting T cells and/or NK cells.

In some embodiments of any of the methods herein that include a step of transducing T cells and/or NK cells, the cells can be contacted with the retroviral particle without prior activation. In some embodiments of any of the methods herein comprising the step of transducing T cells and/or NK cells, prior to transduction, the T cells and/or NK cells are incubated on a substrate adhered to the monocyte for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in yet another embodiment. In one illustrative embodiment, prior to transduction, T cells and/or NK cells are incubated overnight on an adhesive matrix to remove monocytes. In another embodiment, the method may comprise incubating the T cells and/or NK cells on the monocyte-bound adhesion matrix for no more than 30 minutes, 1 hour, or2 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 matrix. In another embodiment, the T cells and/or NK cells are not incubated with or exposed to bovine serum (such as cell culture bovine serum, e.g., fetal bovine serum) prior to or during transduction.

Thus, in another aspect, provided herein is a method for genetically modifying or transducing a lymphocyte (in an illustrative embodiment, a T cell and/or NK cell, or a population of T cells and/or NK cells) of an individual, the method comprising contacting ex vivo a T cell and/or NK cell of a general individual with a replication deficient recombinant retroviral particle comprising 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, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a polypeptide comprising an Antigen Specific Targeting Region (ASTR), A Chimeric Antigen Receptor (CAR) of a transmembrane domain and an intracellular activation domain, wherein the contacting facilitates transduction of resting T cells and/or NK cells or at least some of resting T cells and/or NK cells with replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells.

In another aspect, provided herein is a method for genetically modifying or transducing a lymphocyte (e.g., T cell and/or NK cell) or a population thereof of an individual, the method comprising contacting ex vivo a lymphocyte (e.g., T cell and/or NK cell) or a population thereof of an individual with a replication-defective recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in the lymphocyte (e.g., T cell and/or NK cell), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a polypeptide comprising an antigen-specific targeting region (ASTR), a promoter, and a promoter, A Chimeric Antigen Receptor (CAR) of a transmembrane domain and an intracellular activation domain, wherein the contacting facilitates genetic modification and/or transduction of a lymphocyte (e.g., T cell and/or NK cell) or at least some of a lymphocyte (e.g., T cell and/or NK cell) with a replication-defective recombinant retroviral particle, thereby producing a genetically modified and/or transduced lymphocyte (e.g., T cell and/or NK cell).

In some embodiments of the methods provided immediately above, the genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or populations thereof are introduced into an individual. In some embodiments, the genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or populations thereof undergo 4 or fewer cell divisions ex vivo prior to introduction or reintroduction into an individual. In some embodiments, the lymphocyte is a resting T cell and/or resting NK cell that is contacted with the replication-defective recombinant retroviral particle for between 1 hour and 12 hours. In some embodiments, the time between the time blood is collected from the individual and the time the genetically modified T cells and/or NK cells are reintroduced into the individual is no more than 8 hours. In some embodiments, all steps after collection of blood and before reintroduction of blood are performed in a closed system that is monitored by a person throughout the treatment.

In any of the method aspects provided immediately above that include a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, the polynucleotide may further include a third nucleic acid sequence encoding at least one lymphoproliferative component that is not an inhibitory RNA molecule.

In any of the method aspects provided immediately above that include a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, which in some embodiments can include a5 'strand and a 3' strand that are partially or fully complementary to each other, wherein the 5 'strand and the 3' strand are capable of forming an 18 to 25 nucleotide RNA duplex. In some embodiments, the 5 'strand length can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and the 3' strand length can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the 5 'and 3' strand lengths may be the same or different. In some embodiments, the RNA duplex may include one or more mismatches. In alternative embodiments, the RNA duplex has no mismatches.

In any of the method aspects provided immediately above that include a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules, which may be mirnas or shrnas, directed against one or more RNA targets. In some embodiments, the inhibitory molecule may be a precursor of a miRNA, such as a Pri-miRNA or Pre-miRNA, or a precursor of a shRNA. In some embodiments, the inhibitory molecule may be an artificially-derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule can be dsRNA processed into siRNA (transcribed or artificially introduced) or siRNA itself. In some embodiments, the inhibitory RNA molecule can be a miRNA or shRNA having a sequence not found in nature, or having at least one functional fragment not found in nature, or having a combination of functional segments not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules is miR-155.

In any of the method aspects provided immediately above that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, in some embodiments, the inhibitory RNA molecules can comprise in a5 'to 3' orientation: a5 ' arm, a5 ' stem, a loop, a3 ' stem that is partially or fully complementary to the 5 ' stem, and a3 ' arm. In some embodiments, at least one of the two or more inhibitory RNA molecules has this arrangement. In other embodiments, all of the two or more inhibitory RNA molecules have this arrangement. In some embodiments, the 5' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop length can be 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the 5 'arm, the 3' arm, or both are derived from a naturally occurring miRNA. In some embodiments, the 5 'arm, the 3' arm, or both are derived from a naturally occurring miRNA selected from the group consisting of: miR-155, miR-30, miR-17-92, miR-122 and miR-21. In illustrative embodiments, the 5 'arm, the 3' arm, or both are derived from miR-155. In some embodiments, the 5 'arm, the 3' arm, or both are derived from mus musculus miR-155 or homo sapiens miR-155. In some embodiments, the 5' -arm has a sequence set forth in SEQ ID No. 256 or is a functional variant thereof, such as a sequence that is the same length as SEQ ID No. 256, or is 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID No. 256, or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 256. In some embodiments, the 3' arm has a sequence set forth in SEQ ID No. 260 or a functional variant thereof, such as a sequence that is the same length as SEQ ID No. 260, or is 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID No. 260, or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 260. In some embodiments, the 3' arm comprises nucleotides 221 to 283 of the mus musculus BIC.

In any of the method aspects provided immediately above that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets, in some embodiments, the two or more inhibitory RNA molecules can be contiguously located in the first nucleic acid sequence. In some embodiments, inhibitory RNA molecules can be linked to each other directly or indirectly using a non-functional linker sequence. In some embodiments, the connector sequence may be between 5 and 120 nucleotides in length, or between 10 and 40 nucleotides in length.

In any of the method aspects provided immediately above that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets, in some embodiments, the first nucleic acid sequence encodes two to four inhibitory RNA molecules. In illustrative embodiments, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 5, or between 3 and 6 inhibitory RNA molecules are included in the first nucleic acid sequence. In an illustrative embodiment, four inhibitory RNA molecules are included in the first nucleic acid sequence.

In any of the method aspects provided immediately above that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, the one or more (e.g., two or more) inhibitory RNA molecules may be in an intron.

In any of the method aspects provided immediately above that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets, in some embodiments, the two or more inhibitory RNA molecules may be directed against different targets in an alternative embodiment, the two or more inhibitory RNA molecules are directed against the same target in some embodiments, the RNA targets are mRNA transcribed from genes expressed by free T cells, such as, but not limited to, PD-1 (to prevent inactivation), CT L a4 (to prevent inactivation), TCRa (to prevent autoimmunity), TCRb (to prevent autoimmunity), CD3Z (to prevent autoimmunity), SOCS1 (to prevent inactivation), SMAD2 (to prevent inactivation), miR-155 targets (to promote activation of ifny (to reduce TCR transcription of CRS), TCR elongation of TCR signaling by TCR L (to prevent i 732 (to prevent death 2), tacs A (to prevent death from RNA molecules from RNA receptors) expressed by T cells, in some embodiments, RNA target RNA molecules encoding endogenous T receptors, mRNA for example, mRNA transcription of a T receptor mRNA for which inhibits cholesterol level of endogenous T receptor mRNA binding.

In any of the method aspects provided immediately above that include a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, in some embodiments, the CAR is a microenvironment-restricted organism (MRB) -CAR. In other embodiments, the ASTR of the CAR binds to a tumor-associated antigen. In other embodiments, the ASTR of the CAR is a microenvironment-restricted biological (MRB) -ASTR. In any of the aspects and embodiments disclosed herein that comprise MRB-ASTR or MRB-CAR, MRB-ASTR binds preferentially or only to its cognate antigen under certain aberrant conditions, such as those present in the tumor microenvironment. MRB-ASTR that binds preferentially or exclusively under aberrant conditions in the tumor microenvironment may provide reduced targeted extratumoral effects (on-target off-tumor effects) because of reduced binding to antigen under normal physiological conditions, in some cases to levels below those detected by immunoassay. Normal physiological conditions may include those that are considered to be within the normal range for the individual of temperature, pH, osmolality, oxidative stress and electrolyte concentration at the site of administration or at the tissue or organ at the site of action. An abnormal condition is a condition that deviates from a normal acceptable range. In some embodiments, the MRB-astrr is reversibly or irreversibly inactivated under normal conditions. As used herein, an MRB-ASTR may be an antibody, an antigen, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, or an affibody. In embodiments where the MRB-ASTR is an antibody, the MRB-ASTR can be a full-length antibody, a single chain antibody, a Fab fragment, a Fab 'fragment, a (Fab') 2 fragment, a Fv fragment, a bivalent single chain antibody, or a diabody, where the ASTR comprises heavy and light chains from the antibody. In some embodiments, the MRB-astrr is a single-stranded variable fragment.

In any of the method aspects provided immediately above that include a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and in some cases, a third nucleic acid sequence of the one or more nucleic acid sequences encodes at least one lymphoproliferative component that is not an inhibitory RNA molecule, and in some embodiments, any or all of the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are 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 some embodiments, methods are provided for activating and/or genetically modifying and generally transducing resting T cells or NK cells (in illustrative embodiments, resting T cells) by contacting the cells with retroviral particles disclosed herein and a soluble anti-CD 3 antibody at 25ng/ml to 200ng/ml, 50ng/ml to 150ng/ml, 75ng/ml to 125ng/ml, or 100 ng/ml. In illustrative embodiments, these methods are performed without prior activation, and may be performed, for example, for 8 hours or less, 4 hours or less, or between 2 hours and 8 hours, between 2 hours and 4 hours, or between 2 hours and 3 hours.

In certain aspects, provided herein are methods for performing adoptive cell therapy for an individual, which may include, as an illustrative example, the following:

A. collecting blood from a subject;

B. isolating Peripheral Blood Mononuclear Cells (PBMCs) comprising resting T cells and/or resting NK cells;

C. contacting ex vivo a resting T cell and/or a resting NK cell of an individual with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises on its surface a pseudotyping module capable of binding to the resting T cell and/or NK cell and facilitating fusion of the replication deficient recombinant retroviral particle to its membrane, wherein the contacting facilitates transduction of the resting T cell and/or NK cell with the replication deficient recombinant retroviral particle, thereby producing a genetically modified T cell and/or NK cell; and

D. the genetically modified cells are reintroduced into the individual within 36 hours, 24 hours, 12 hours, or even 8 hours of blood collection from the individual, thereby performing adoptive cell therapy in the individual.

In some aspects provided herein, methods having similar steps are referred to as methods for genetically modifying and expanding lymphocytes of an individual. The skilled artisan will appreciate that the discussion herein also applies to methods of genetically modifying and expanding lymphocytes of a subject as they apply to methods and compositions for performing adoptive cell therapy.

Typically, the adoptive cell therapy of the present invention is performed using autologous transfer, wherein cells are isolated and/or otherwise prepared from the individual receiving the cell therapy or from a sample derived from such individual. Thus, in some aspects, the cells are cells derived from an individual in need of treatment (e.g., a patient) and following isolation and processing from the same individual. In some embodiments of the methods and compositions disclosed herein, an individual having a disease or disorder enters a medical facility where the individual's blood is drawn using known methods, such as venipuncture. In certain embodiments, the volume of blood drawn from an individual is between 10ml, 15ml, 20ml, 25ml, 30ml, 35ml, 40ml, 50ml, 75ml, or 100ml at the lower end of the range and 200ml, 250ml, 300ml, 350ml, 400ml, 500ml, 750ml, 1000ml, 2000ml, or 2500ml at the upper end of the range. In some embodiments, between 10ml and 400ml of blood is drawn from the individual. In some embodiments, between 20ml and 250ml of blood is drawn from the individual. In some embodiments, the blood is fresh at the time of treatment. In any of the embodiments disclosed herein, the fresh blood may be blood drawn from an individual less than 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes prior. In some embodiments, blood is treated in the methods provided herein without storage.

Contact between T cells and/or NK cells and replication-defective recombinant retroviral particles generally facilitates transduction of T cells and/or NK cells with the replication-defective recombinant retroviral particles. Transduced T cells and/or NK cells throughout the present invention include progeny of the transduced cells ex vivo that retain at least some of the nucleic acid or polynucleotide incorporated into the cells during ex vivo transduction. In the methods herein that refer to "reintroducing" transduced cells, it is understood that such cells are not typically in a transduced state when they are collected from the blood of an individual. The subject in any of the aspects disclosed herein can be, for example, an animal, a mammal, and in illustrative embodiments a human.

Without being limited by theory, in a non-limiting illustrative method, ex vivo delivery of a polynucleotide encoding a lymphoproliferative component, such as an I L7 constitutively active mutant, which can integrate 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 lymphodepleting the host, thus, in illustrative embodiments, the individual is exposed to the lymphotrophic agent for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, or 28 days or for 1 month, 2 months, 3 months, or 6 months of performing the contact, during the contact and/or for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, or 28 days or for 1 month, 2 months, 3 months, or 6 months after reintroducing the modified T cell and/or NK cell back into the individual and is not exposed to the lymphotrophic agent in a non-limiting illustrative method, wherein the exposure to the lymphotrophic agent and/or the non-limiting method of performing the non-defective T cell replication step ex vivo.

Thus, amplifying genetically modified T cells and/or NK cells in vivo in an individual is a feature of some embodiments of the invention. In illustrative embodiments, these methods are ex vivo non-propagating or substantially non-propagating.

In the non-limiting illustrative examples herein, this entire method/process of reintroducing blood back to the individual after drawing blood from the individual to ex vivo transduction of T cells and/or NK cells may occur within the following time periods: 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 other embodiments, this entire method/process of reintroducing blood back into the individual after drawing/collecting blood from the individual to ex vivo transduction (in the non-limiting illustrative examples herein) of T cells and/or NK cells occurs within the following time period: between 1 hour and 12 hours, or between 2 hours and 8 hours, or between 1 hour and 3 hours, or between 2 hours and 4 hours, or between 2 hours and 6 hours, or between 4 hours and 12 hours, or between 4 hours and 24 hours, or between 8 hours and 36 hours, or between 8 hours and 48 hours, or between 12 hours and 24 hours, or between 12 hours and 36 hours, or between 12 hours and 48 hours; or during the following time period: between 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, and 240 minutes at the low end of the range and 120 minutes, 180 minutes, and 240 minutes, 300 minutes, 360 minutes, 420 minutes, and 480 minutes at the high end of the range. In other embodiments, this entire method/process of reintroducing blood back to the individual after drawing/collecting blood from the individual to ex vivo transduction of T cells and/or NK cells occurs within the following time periods: between 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 12 hours at the lower end of the range and 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours at the upper end of the range. In some embodiments, the genetically modified T cell and/or NK cell line is isolated from the replication-defective recombinant retroviral particle after the period of contact.

In some embodiments of any of the methods herein (which include a step of blood collection and a step of transducing lymphocytes (in illustrative embodiments, T cells and/or NK cells, including resting T cells and NK cells), the method of collecting from blood to transduced T cells and/or NK cells does not include a step of removing monocytes by incubation on an adhesion matrix for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in yet another embodiment. In one illustrative embodiment, the method of collecting from blood to transduced T cells and/or NK cells does not include overnight incubation on an adhesive matrix to remove mononuclear spheres. In another embodiment, the method of collecting from blood to transduced T cells and/or NK cells comprises the step of removing mononuclear spheres by incubation on an adhesive matrix for no more than 30 minutes, 1 hour, or2 hours. In another embodiment, the method of collecting blood from an individual to transduced lymphocytes (in an illustrative embodiment, T cells and/or NK cells, including resting T cells and/or NK cells) does not include the step of removing monocytes by incubation on an adhesive matrix. In another embodiment, a method of blood collection from an individual to transduced lymphocytes (in an illustrative embodiment, T cells and/or NK cells, including resting T cells and/or NK cells) includes a method during which the T cells and/or NK cells are not incubated with or exposed to bovine serum, such as cell culture bovine serum, e.g., fetal bovine serum.

In some embodiments of any of the methods herein (which include the step of blood collection and the step of transducing lymphocytes (in illustrative embodiments, T cells and/or NK cells, including resting T cells and NK cells), the method of blood collection from a subject to reintroduction of T cells and/or NK cells into a subject does not include the step of removing monocytes by incubation on an adhesive matrix for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in yet another embodiment. In one illustrative embodiment, the method of blood collection from an individual to reintroduction of T cells and/or NK cells into an individual does not include incubating overnight on an adhesive matrix to remove monocytes. In another embodiment, the method of blood collection from an individual to reintroduction of T cells and/or NK cells comprises the step of removing mononuclear spheres by incubation on an adhesive matrix for no more than 30 minutes, 1 hour or2 hours. In another embodiment, the method of collecting blood from an individual to reintroducing T cells and/or NK cells into an individual does not include the step of removing the mononuclear spheres by incubating overnight on an adhesive matrix. In another embodiment, a method of blood collection from an individual to reintroduction of T cells and/or NK cells into the individual is performed during which the T cells and/or NK cells are not incubated with or exposed to bovine serum, such as cell culture bovine serum, e.g., fetal bovine serum.

Because the methods for adoptive cell therapy provided herein, and related methods for ex vivo modification of resting T cells and/or NK cells prior to expansion of said T cells and/or NK cells in vivo, can be performed in significantly shorter times than previous methods, enabling substantial improvements in patient care and safety as well as product manufacturability. Thus, such methods are expected to be advantageous in view of regulatory agencies responsible for approving such methods for therapeutic purposes in vivo. For example, in a non-limiting example, the individual may remain in the same building (e.g., infusion clinic) or room as the facility that processed their blood or sample for the entire time of sample processing prior to reintroduction of the modified T cells and/or NK cells into the patient. In non-limiting illustrative embodiments, throughout the method/process of reintroducing blood into a subject following blood draw/collection from the subject to ex vivo transduced T cells and/or NK cells, the subject remains within the location line and/or within 100, 50, 25 or 12 feet or arm distance of the blood or cells being treated. In other non-limiting illustrative embodiments, the individual remains awake and/or at least one person may continue to monitor the individual's blood or cells being treated throughout the method/process and/or continuously of reintroducing blood into the individual after blood is drawn/collected from the individual to ex vivo transduced T cells and/or NK cells. Because of the improvements provided herein, the entire method/process for adoptive cell therapy and/or transducing resting T cells and/or NK cells for reintroduction of blood to an individual after blood withdrawal/collection from the individual to ex vivo transduced T cells and/or NK cells can be performed in conjunction with continuous monitoring of humans. In other non-limiting illustrative embodiments, blood cells are not cultured in a room where no person is present at any point of the overall method/process of reintroducing blood to an individual after blood is drawn/collected from the individual to transduce T cells and/or NK cells ex vivo. In other non-limiting illustrative embodiments, the entire method/process of reintroducing blood to an individual after blood draw/collection from the individual to ex vivo transduced T cells and/or NK cells is performed next to the individual and/or in the same room as the individual and/or next to the bed or chair of the individual. Thus, sample consistency confusion can be avoided, as well as long and expensive incubations of more than 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 a blood sample and its components reintroduced into an individual are contacted with only single, single-use components.

Methods for performing the adoptive cell therapies provided herein generally include 1) methods for transducing lymphocytes, such as T cells and/or NK cells, which in illustrative embodiments are resting T cells and/or NK cells, and/or include 2) methods for genetically modifying lymphocytes, such as T cells and/or NK cells, which in illustrative embodiments are resting T cells and/or NK cells, both (1 and 2) themselves form distinct aspects of the invention.

As non-limiting examples, in some embodiments of the methods for transduction or genetic modification herein, PBMCs are isolated using a Sepax or Sepax 2 cell processing system (biosafee). in some embodiments, PBMCs are isolated using a CliniMACS progress cell processor (Miltenyi Biotec). in some embodiments, blood is collected from an individual using an automated blood cell separator, the blood is passed through a device that picks out peripheral cell types (such as PBMCs), and the remainder is returned to the individual density gradient centrifugation may be performed after blood cell separation.

During certain embodiments herein, e.g., in methods of modifying lymphocytes and methods of performing adoptive cell therapy, the T cells and/or NK cells contacted with the replication-defective recombinant retroviral particles of the invention are predominantly resting T cells^-). In some embodiments, T cells and/or NK cells contacted with the replication-defective recombinant retroviral particle are included between 90%, 91%, 92%, 93%, 94%, and 95% resting cells at the low end of the range and 96%, 97%, 98%, 99%, or 100% resting cells at the high end of the range. In some embodiments, the T cells and/or NK cells comprise primary cells.

In some embodiments of the methods and compositions disclosed herein, T cells and/or NK cells are contacted with replication-defective recombinant retroviral particles ex vivo to genetically modify the T cells and/or NK cells to elicit a targeted immune response in an individual upon reintroduction into the individual. During the contact period, the replication-defective recombinant retroviral particle recognizes and binds to T cells and/or NK cells, at which time the retrovirus begins to fuse with the host cell membrane. Then, via a transduction procedure, genetic material from the replication-defective recombinant retroviral particle enters the T cell and/or NK cell and is incorporated into the host cell DNA. Methods of lentivirus transduction are known. Exemplary Methods are described in, for example, Wang et al (2012) J.Immunother.35(9): 689-.

Many of the methods provided herein include transducing T cells and/or NK cells. Methods for the ex vivo transduction of T cells and/or NK cells with replication deficient recombinant retroviral particles, such as replication deficient recombinant lentiviral particles, are known in the art. In illustrative embodiments, the methods provided herein do not require ex vivo stimulation or activation. Thus, this conventional step in previous methods may be avoided in the present method, but ex vivo stimulatory molecules (such as anti-CD 3 and/or anti-CD 28 beads) may be present during transduction. However, with the illustrative methods provided herein, ex vivo stimulation is not required. In certain exemplary methods, replication-defective recombinant retroviral particles, such as lentiviruses, can be used in units between 3 and 10 superinfections (MOI), and in some embodiments, between 5 and 10 MOI.

The transduction reaction can be performed in a closed system (such as the Sepax system as discussed herein), where the transduction reaction can be performed in a disposable bag loaded on the system. Once blood cells (such as PBMCs) from a blood sample collected from an individual are separated, isolated and/or purified from granulocytes (including neutrophils), which are not normally present during the contacting step (i.e., transduction reaction), these blood cells can be contacted with the replication defective recombinant retroviral particles disclosed herein in a bag.

The replication-defective recombinant retroviral particles can be reintroduced into the bag containing the isolated PBMCs, thereby contacting the PBMCs. The time from the time blood is collected from the subject to the time blood cells (such as PBMCs) are added to the transduction reaction bag may be between 30 minutes and 4 hours, between 30 minutes and 2 hours, or in some examples about 1 hour.

In certain embodiments wherein the cells are infused into the individual, following transduction, prior to infusing the transduced T and/or NK cells back into the individual, the cells are washed to remove the transduction reaction mixture prior to infusion back into the individual. For example, a system (such as a Sepax instrument) may be used to wash the cells, e.g., with 10ml to 50ml of wash solution, prior to infusing the transduced cells back into the subject. In some embodiments, the neutrophils are removed prior to PBMC and/or T cells and/or NK cells being treated, contacted with replication defective recombinant retroviral particles, transduced or transfected.

In an illustrative embodiment for performing adoptive cell therapy, blood is collected from an individual into a blood bag, and the blood bag is connected to a cell processing system, such as a Sepax cell processing system. PBMCs isolated using a cell processing system are collected into bags, contacted with replication defective recombinant retroviral particles under conditions sufficient to transduce T cells and/or NK cells, and incubated. After incubation, bags containing a mixture of PBMCs and replication defective recombinant retroviral particles are connected to a cell processing system and the PBMCs are washed. The washed PBMCs were collected into bags and re-infused into subjects. In some embodiments, the entire method from collecting blood to reinfusing transduced T cells and/or NK cells is performed within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, or 24 hours. In the illustrative embodiment, the entire method is performed within 12 hours.

In some embodiments, the target cell of the replication-defective recombinant retroviral particle is PB L in some embodiments, the target cell is a T cell and/or an NK cell.

In some embodiments, the replication-defective recombinant retroviral particles provided herein have on their surface a pseudotyping component that is capable of binding T cells and/or NK cells and facilitates membrane fusion with the replication-defective recombinant retroviral particle. In other embodiments, the replication-defective recombinant retroviral particle has on its surface an activating module capable of binding resting T cells and/or NK cells. In yet other embodiments, the replication-defective recombinant retroviral particle has a membrane-bound interleukin on its surface. In some embodiments, the replication-defective recombinant retroviral particle comprises a polynucleotide having one or more transcription units encoding one or more engineered signaling polypeptides, one or more of which comprise one or more lymphoproliferative components. In other embodiments, when two signaling polypeptides are utilized, one comprises at least one lymphoproliferative component, a lymphoproliferative component, and the other is typically a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. As indicated herein, the activation module, which is typically associated with the surface of the replication-defective recombinant retroviral particle provided herein, is capable of contacting resting T cells and/or NK cells for a sufficient period of time and, as a result of the contacting and under appropriate conditions, activating resting T cells and/or NK cells. It is understood that this activation occurs over time during the contacting step of the methods herein. Furthermore, it is understood that in some embodiments wherein the pseudotyped component is found on the surface of replication deficient recombinant retroviral particles that bind T cells and/or NK cells, in the methods herein, activation may be induced by binding the pseudotyped component. An activation component is optionally present in those embodiments.

Additional descriptions of pseudotyped components, activated components, membrane-bound interleukins, engineered signaling polypeptides, lymphoproliferative components, and CARs are provided elsewhere herein.

In some embodiments of the methods and compositions disclosed herein, between 5% and 90% of the total lymphocytes collected from the blood are transduced. In certain embodiments, the percentage of transduced lymphocytes is between 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% at the low end of the range and 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% at the high end of the range. In some embodiments, the percentage of transduced lymphocytes is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

In some embodiments of the methods and compositions disclosed herein, the genetically modified T cells and/or NK cells are introduced back into, reintroduced into, or reinfused into the subject without additional 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 genetically modified T cells and/or NK cells prior to introduction of the genetically modified T cells and/or NK cells into the subject, which typically takes days or weeks, and requires the subject to return to blood infusion for days or weeks after initial blood draw-up, in some embodiments of the methods and compositions disclosed herein, before the T cells and/or NK cells are contacted with replication deficient recombinant viral particles, the T cells and/or NK cells are not exposed to anti-CD 3/anti-CD 28 solid vectors (such as anti-CD 3/anti-CD 28) for some hours in vivo recombinant CD 3/CD 9 cells, CD 9/NK cells, CD 9 cells are not exposed to CD 9, CD 9.

In any of the embodiments disclosed herein, the number of T cells and/or NK cells to be reinfused into an individual may be 1 × 10 at the low end of the range³、2.5×10³、5×10³、1×10⁴、2.5×10⁴、5×10⁴、1×10⁵、2.5×10⁵、5×10⁵、1×10⁶、2.5×10⁶、5×10⁶And 1 × 10⁷The cell/kg and the high end of the range are 5 × 10⁴、1×10⁵、2.5×10⁵、5×10⁵、1×10⁶、2.5×10⁶、5×10⁶、1×10⁷、2.5×10⁷、5×10⁷And 1 × 10⁸In illustrative embodiments, the number of T cells and/or NK cells to be reinfused into an individual may be 1 × 10 at the low end of the range⁴、2.5×10⁴、5×10⁴And 1 × 10⁵One cell/kg and the high end of the range is 2.5 × 10⁴、5×10⁴、1×10⁵、2.5×10⁵、5×10⁵And 1 × 10⁶Between one cell/kgIn some embodiments, the number of PBs L to be reinfused into an individual may be less than 5 × 10 at the low end of the range⁵、1×10⁶、2.5×10⁶、5×10⁶、1×10⁷、2.5×10⁷、5×10⁷And 1 × 10⁸Individual cell, with the high end of the range being 2.5 × 10⁶、5×10⁶、1×10⁷、2.5×10⁷、5×10⁷、1×10⁸、2.5×10⁸、5×10⁸And 1 × 10⁹In some embodiments, the number of T cells and/or NK cells available for reinfusion into a 70kg individual or patient is 7 × 10⁵Each cell is 2.5 × 10⁸In other embodiments, the number of T cells and/or NK cells available for transduction is about 7 × 10⁶Plus or minus 10%.

In the methods disclosed herein, the entire adoptive cell therapy procedure (from drawing blood to reinfusing genetically modified T cells and/or NK cells) can be advantageously performed in a shorter time than previous methods. In some embodiments, the entire adoptive cell therapy program can be performed in less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, or 24 hours. In illustrative embodiments, the entire adoptive cell therapy program can be performed in less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In some embodiments, the entire adoptive cell therapy program can be performed between 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or 15 hours at the low end of the range and 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, or 24 hours at the high end of the range.

In some embodiments herein, the closed system is used to treat PBMCs, for example in a method comprising genetically modifying PBMCs, NK cells, and in illustrative embodiments T cells, for exampleBy transducing PBMCs or a subset thereof. These methods can be used to genetically modify lymphocytes to be used in scientific research, commercial production, or therapeutic approaches. For example, these methods may include transferring Peripheral Blood Mononuclear Cells (PBMCs) (including NK cells, T cells, or both, and in some embodiments, resting T cells and/or resting NK cells) from a container into a transduction reaction mixture within a closed system, which thus does not require environmental exposure. The container may be, for example, a tube, bag, syringe, or other container. In some embodiments, the vessel is a vessel 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 overnight blood collection procedure. The methods for genetically modifying herein generally involve a contacting step in which the lymphocyte is contacted with a replication-defective recombinant retroviral particle. In some embodiments, the contacting may be performed in a container (e.g., within a blood bag). In other embodiments, the PBMCs, or a sub-portion thereof, may be transferred from the container to another container within the closed system (e.g., from the first container to the second container) for contact. The second vessel may be a cell processing compartment of a closed device, such as a G-Rex device. In some embodiments, after contacting, the genetically modified (e.g., transduced) cells can be transferred to a different container within a closed system (i.e., without exposure to the environment). Prior to or after such transfer, the cells are typically washed in a closed system to substantially removeAll or all ofA retroviral particle. In some embodiments, the process disclosed in this paragraph, wherein transferring the PBMCs or portion thereof after contacting into a container in which contacting (e.g., transduction) would occur via washing of the cells, is performed within 12 hours. In some embodiments, it is performed between 1 hour, 2 hours, 3 hours, or 4 hours at the lower end of the range and 4 hours, 8 hours, 10 hours, or 12 hours at the upper end of the range.

In some embodiments provided herein, the steps of drawing a blood sample from an individual, contacting T cells and/or NK cells with replication-defective recombinant retroviral particles, and/or introducing genetically modified T cells and/or NK cells into the individual, occur in a closed system. Closed systems are a culture method that is generally closed or completely closed to contamination. Thus, the system or process does not expose the cells to the environment. It is an advantage of the present invention that methods for performing CAR therapy in a closed system are provided herein. One of the highest risks for safety and regulation in cell processing procedures is the risk of contamination via frequent exposure to the environment, as found in traditional open cell culture systems. To mitigate this risk, some commercial approaches have been developed that focus on using disposable (single use) devices, especially in the absence of antibiotics. However, even if used under sterile conditions, opening the flask to sample or add additional growth medium always presents a contamination risk. To overcome this problem, a closed system process, a process designed and operable so that the product is not exposed to the outside environment, is provided herein. This is important because the external environment is typically not sterile. Material transfer is performed via sterile connection or welded tubing. Air for gas exchange is conducted through a 0.2 μm filter via a gas permeable membrane or similar other additives to prevent environmental exposure.

In some embodiments, the ex vivo circulatory system comprises a system or device for separating PB L and/or a system or device for separating T cells and/or NK cells in combination with a system or device for exposing the cells to replication-defective recombinant retroviral particles.

Such closed system methods may be performed using commercially available devices. For example, the method may be performed in a device suitable for closed system T cell manufacturing. Such devices include G-Rex^TM、WAVEBioreactor^TM、OriGenPermaLife^TMBag and

and (4) a bag.

In some embodiments of the methods and compositions disclosed herein, the genetically modified T cells and/or NK cells in the individual are exposed to a compound that binds to an in vivo control component present therein, wherein the control component is part of the genetic material introduced by the replication-defective recombinant retroviral particle. In some embodiments, the control component can be a riboswitch, and the compound can bind to an aptamer domain of the riboswitch. In some embodiments, the control component may be a chaperone. Chaperones are compounds that directly affect the activity of the lymphoproliferative component or other component of the first or second engineered signaling polypeptides herein, typically by binding. In any of the embodiments disclosed herein, the compound can be a nucleoside analog. In some embodiments, the nucleoside analog may be a nucleoside-like antiviral Drug, wherein the antiviral Drug is a compound approved by the Food and Drug Administration for antiviral therapy or a compound in the U.S. antiviral clinical trial. In illustrative embodiments, the compound may be acyclovir or penciclovir. In some embodiments, the compound may be famciclovir (oral prodrug of penciclovir) or valaciclovir (oral prodrug of acyclovir). The binding of the compound to the control module influences the expression of the introduced genetic material and thus influences the spread of the genetically modified T cells and/or NK cells.

In some embodiments, the nucleoside-like antiviral drug or prodrug (e.g., acyclovir, valacyclovir, penciclovir, or famciclovir) is administered to the subject before, concurrently with, and/or after isolation of PB L from the blood of the subject and before T cells and/or NK cells are contacted with the replication-deficient recombinant retroviral particles, in some embodiments, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes at the lower end of the range and 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, or 24 hours at the upper end of the range, before, concurrently with, and/or after isolation of PB L from the blood of the subject, and/or after isolation of the nucleoside-deficient recombinant retroviral particles, the nucleoside-like antiviral drug or prodrug is administered to the subject.6 hours, or prodrug at least 5 hours, 6 hours, or prodrug is administered to the subject, the subject may be administered for at least 5 days, 6, or 6, or 3, 6, five, six, four, six, four, eight, four, eight, four, eight, five, four, five, four.

In some embodiments, the compound associated with the control component is administered to the subject once, twice, three times, or four times daily. In some embodiments, the daily dose of compound is provided for 1 week, 2 weeks, 4 weeks, 3 months, 6 months, 1 year, until the individual is disease-free (such as cancer-free) or indefinitely. In illustrative embodiments, the drug is a nucleoside-like antiviral drug conjugated to a nucleoside analog, such as a riboswitch, as disclosed in further detail in WO2017/165245a2, WO2018/009923a1, and WO2018/161064a 1.

Methods for delivering drugs, whether small molecules or biologics, and which can be used in the methods provided herein are known in the art. Any such method may be used to deliver a drug or candidate compound or antibody for use in the methods of the invention. For example, conventional routes of administration include non-invasive oral (via the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes. Many protein and peptide drugs (such as monoclonal antibodies) must be delivered by injection or nanoneedle arrays. For example, many immunizations are based on the delivery of protein drugs, and are typically performed by injection.

Engineered signaling polypeptides

In some embodiments, replication-deficient recombinant retroviral particles for contacting T cells and/or NK cells have a polynucleotide with one or more transcriptional units encoding one or more engineered signaling polypeptides in some embodiments, the engineered signaling polypeptides include any combination of an extracellular domain (e.g., an antigen-specific targeting region or ASTR), a stalk, and a transmembrane domain, in combination with one or more intracellular activation domains, optionally one or more regulatory domains such as a costimulatory domain, and optionally one or more T cell survival motifs in illustrative embodiments, at least one, two, or all of the engineered signaling polypeptides are Chimeric Antigen Receptors (CARs) or lymphoproliferative component (L E) such as chimeric lymphoproliferative component (C L E).

Extracellular domains

In some embodiments, the engineered signaling polypeptide comprises an extracellular domain that is a member of a specific binding pair. For example, in some embodiments, the extracellular domain can be an extracellular domain of an interleukin receptor or a mutant thereof or a hormone receptor or a mutant thereof. This mutant extracellular domain is reported in some embodiments to be constitutively active when expressed in at least some cell types. In illustrative embodiments, the extracellular domain and the transmembrane domain do not include a ligand binding region. It is believed that these domains do not bind ligands when present in engineered signaling polypeptides and expressed in B cells, T cells, and/or NK cells. Mutations in these receptor mutants can occur in the extracellular membrane proximal region. Without being limited by theory, mutations in at least some of the extracellular domains (and some extracellular-transmembrane domains) of the engineered signaling polypeptides provided herein are responsible for signaling of the engineered signaling polypeptides in the absence of ligands by bringing together activation chains that are not normally together. Further embodiments regarding ectodomains comprising mutations in the ectodomain can be found, for example, in the lymphoproliferative component section herein.

In certain illustrative embodiments, the extracellular domain comprises a dimerization motif. In an illustrative embodiment, the dimeric moiety comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. Further embodiments regarding extracellular domains comprising dimeric motifs can be found, for example, in the lymphoproliferative component 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; a ligand-receptor binding pair; and the like. Thus, members of a specific binding pair suitable for use in engineered signaling polypeptides of the invention include ASTRs, which are antibodies, antigens, ligands, receptor binding domains of ligands, receptors, ligand binding domains of receptors, and affinity antibodies.

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

In some embodiments, the ASTR is a single chain fv (scfv). In some embodiments, the heavy chain is N-terminal to the light chain in the engineered signaling polypeptide. In other embodiments, the light chain is N-terminal to the heavy chain in the engineered signaling polypeptide. In any of the disclosed embodiments, the heavy and light chains may be separated by a linker as discussed in more detail herein. In any of the disclosed embodiments, the heavy or light chain may be N-terminal to the engineered signaling polypeptide and typically C-terminal to another domain (such as 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 the engineered signaling polypeptides and in methods of using the engineered signaling polypeptides of the invention in some cases it is also suitable to use a T Cell (TCR) -based recognition domain, such as a single chain TCR (scTv, a single chain two domain TCR containing V α V β).

In some embodiments, the ASTR may be multispecific, e.g., a bispecific antibody. Multispecific antibodies have binding specificities directed against 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 may bind to two different epitopes of a ta-targeting antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the target antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

The cancer cell-associated antigen can be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a Hodgkin's (Hodgkin) lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin's B-cell lymphoma (B-NH L) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a neuroblastoma, a colon cancer cell, etc. the cancer cell-associated antigen can also be expressed by a non-cancer cell.

Non-limiting examples of ASTR-bindable antigens of engineered signaling polypeptides include, for example, CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, Prostate Specific Membrane Antigen (PSMA), CD44 surface adhesion molecules, mesothelin, carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-Al, I L-13R-a 2, GD2, Axl, Ror2, and the like.

In some cases, suitable members for engineering a specific binding pair in a signaling polypeptide are ASTRs that are ligands for a receptor. Ligands include, but are not limited to: hormones (e.g., erythropoietin, growth hormone, leptin, etc.); interleukins (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., regulatory proteins; vascular endothelial growth factor (VEGF; and the like); an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp); and the like.

When the 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 can be a VEGF receptor, including a soluble VEGF receptor.

As mentioned above, in some cases, the member of a specific binding pair included in an engineered signaling polypeptide is an ASTR, which is a receptor, e.g., a receptor for a ligand, a co-receptor, etc., which can be a ligand-binding fragment of a receptor suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptors), the killer lectin-like receptor subfamily K, member 1(NKG2D) polypeptides (receptors for MICA, MICB, and U L B6), interleukin receptors (e.g., I L-13 receptor, I L-2 receptor, etc.), CD27, Natural Cytotoxic Receptors (NCR) (e.g., NKP30(NCR3/CD337) polypeptides (receptors for H L A-B related transcript 3(BAT3) and B7-H6), etc.), and the like.

In certain embodiments of any of the aspects provided herein that include ASTRs, the ASTRs can be localized to an intermediate protein that links the ASTRs to a targeting molecule expressed on a target cell. The intermediate protein may be expressed endogenously or introduced exogenously and may be native, engineered or chemically modified. In certain embodiments, the ASTR may be an anti-label ASTR, such that at least one labeled intermediate (typically an anti-label conjugate) is included between the label recognized by the ASTR and the targeting molecule (typically a protein target expressed on a target cell). Thus, in these embodiments, the ASTR binds to the label and the label is conjugated to an antibody directed against an antigen on a target cell (such as a cancer cell). Non-limiting examples of labels include Fluorescein Isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, the polymethacrylic chlorophyll protein complex, green fluorescent protein, Phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein. Thus, ASTR comprises korean-bound labeled molecules.

Handle

In some embodiments, the engineered signaling polypeptide comprises a stalk located in a portion of the engineered signaling polypeptide that is outside of the cell and interposed between the ASTR and the transmembrane domain, in some cases, the stalk has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the wild-type CD8 stalk region (TTTPAPRPPTPAPTIASQP L S L RPEACRPAAGG AVHTRG L DFA (SEQ ID NO:79)), at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the wild-type CD28 stalk region (FCKIEVMYPPPY L DNEKSNGTIIHVKGKH L CPSP L FPGPSKP (SEQ ID NO:80)), or at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the wild-type immunoglobulin heavy chain stalk region.

The stalk region can be about 4 to about 50 amino acids in length, for example about 4 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

In some cases, the handle of the engineered signaling polypeptide includes at least one cysteine. For example, in some cases, the handle may include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 62). If present, cysteine in the handle of the first engineered signaling polypeptide may be capable of forming a disulfide bond with the handle in the second engineered signaling polypeptide.

The handles may comprise the amino acid sequence of an immunoglobulin hinge region known in the art; see, for example, Tan et al, (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al (1986) Nucl. 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 amino acids of any of the following amino acid sequences: DKTT (SEQ ID NO:63), CPPC (SEQ ID NO:62), CPEPKSCDTPPPCPR (SEQ ID NO: 64); see, for example, Glaser et al (2005) J.Biol. chem.280: 732; E25 KTP 3 GDTTHT (SEQ ID NO: KSCDKTHTCP; SEQ ID NO: 5842; see, for example, Glaser et al (2005) J.Biokl. J.2012: Chem.280: 11: L K.K.K.3 GDT.7 H.7; and optionally substituted with a human IgG hinge region (SEQ ID NO: 9670; see, SEQ ID NO: 35; such as a human hinge region of a human hinge region having amino acid sequence deletion of at least 50, preferably, or a human hinge region similar to a native amino acid sequence of at least 10, 15, 20 or all amino acid sequence of any of the amino acid sequence: 11; SEQ ID NO: 11; such as SEQ ID NO: 11; SEQ ID NO: 7: 11; and optionally, preferably a human hinge region (SEQ ID NO: 11; see, preferably a human hinge region of a human hinge region such as a human hinge region) (see, preferably a human hinge region).

TTTPAPRPPTPAPTIASQP L S L RPEACRPAAGGAVHTRG L DFACD (SEQ ID NO:73), or a variant thereof.

Transmembrane domain

The engineered signaling polypeptides of the invention may include a transmembrane domain for insertion into a eukaryotic cell membrane. A transmembrane domain may be inserted between the ASTR and the costimulatory domain. The transmembrane domain may be interposed between the stalk 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, stalk, transmembrane domain, and activation domain.

Non-limiting examples of TM domains suitable for use in any of the aspects or embodiments 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 TM domain or a stretch of at least 10, 15, 20 or all amino acids of any of the combined stalk and TM domains a) CD8 α TM (SEQ ID NO:46), b) CD8 β TM (SEQ ID NO:47), c) CD4 stalk (SEQ ID NO:48), d) CD3Z TM (SEQ ID NO:49), e) CD28 TM (SEQ ID NO:50), f) CD134 (40) TM (SEQ ID NO:51), g) CD7 TM (SEQ ID NO:52 h), and 8 (SEQ ID NO: 8676), and OX (28).

As non-limiting examples, a transmembrane domain of an aspect of the invention may have at least 80%, 90% or 95% or may have 100% sequence identity to the transmembrane domain of SEQ ID NO. 46, or may have 100% sequence identity to any of the transmembrane domains from the group consisting of the CD8 β transmembrane domain, the CD4 transmembrane domain, the CD3 zeta transmembrane domain, the CD28 transmembrane domain, the CD134 transmembrane domain, or the CD7 transmembrane domain, respectively.

Intracellular activation domain

The intracellular activation domains useful in the engineered signaling polypeptides of the invention typically induce the production of one or more interleukins upon activation; increase cell death; and/or increase CD8⁺T cell, CD4⁺Proliferation of T cells, NKT cells, gamma T cells and/or neutrophils. The activation domain may also be referred to herein as an activation domain. The activation domain may be used in the CARs provided herein or in the lymphoproliferative component.

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 may have at least 80%, 90%, or 95% sequence identity with the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains as described below, or may have 100%.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the invention include intracellular signaling polypeptides comprising an immunotyrosine-based activation motif (ITAM). ITAM element is YX₁X₂L/I, wherein X₁And X₂Independently any amino acid. In some cases, the intracellular activation domain of the engineered signaling polypeptide comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular activation domain, where the first and second instances of the ITAM motif are from 6 to 8 amino acids from each other (e.g., (YX)₁X₂L/I)(X₃)_n(YX₁X₂L/I), wherein n is an integer from 6 to 8, and from 6 to 8X₃Each of which can be any amino acid). In some cases, the intracellular activation domain of the engineered signaling polypeptide comprises 3 ITAM motifs.

Suitable intracellular activation domains may be, for example, an ITAM motif-containing portion derived from an ITAM motif-containing polypeptide.a suitable intracellular activation domain may be an ITAM motif-containing domain from any protein that contains an ITAM motif.thus, a suitable intracellular activation domain need not contain the entire sequence of the protein from which it is derived.examples of suitable ITAM motif-containing polypeptides include, but are not limited to, CD3Z (CD3 ζ), CD3D (CD3), CD3E (CD3), CD3G (CD3 γ), CD79A (antigen receptor complex-associated protein α chain), CD79B (antigen receptor complex-associated protein β chain) 12, and FCERlG (Fc receptor I γ chain).

In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD 3-zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, suitable intracellular activation 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 the following sequence or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa 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 in any of the following amino acid sequences (2 isoforms):

MKWKA L FTAAI L QAQ L0 PITEAQSFG L1L 2DPK L3 CY L4L 5DGI L6 FIYGVI L7 TA L8F L9 RVKFSRSADAPAYQQGQNQ L [ YNE L0N L1 GRREYDV L2 ] DKRRGRDPEMGGKPRRKNPQEG L4 [ YNE L6 YNE L ] YNE L [ YNE L ] YNE L [ YNE L9 ] HMQA YNE L PPR (SEQ ID NO:11) or MKWKA YNE L1 FTAAI YNE L2 QAQ YNE L3 YNE L DPK YNE L CY YNE L5 DGI YNE L YGFIVI YNE L TA YNE L F YNE L [ YNE L N YNE L GRREYDV YNE L ] YNE L [ YNE L ] HMQALPPR (SEQ ID NO:12), wherein ITAM elements are bracketed.

Likewise, suitable intracellular activation domain polypeptides can include a portion of the full-length CD3 ζ amino acid sequence that includes an ITAM motif. Thus, suitable intracellular activation 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 the following sequence, or to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa 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 in any of the following amino acid sequences:

RVKFSRSADAPAYQQGQNQ L [ YNE L N L0 GRREYDV L1 ] DKRRGRDPEMGGKPRRKNPQEG L2 [ YNE L3 QKDKMAEAYSEI ] GMKGERRRGKGHDG L4 [ YQG L5 STATKDTYDA L6 ] HMQA L7 PPR (SEQ ID NO: 13); RVKFSRSADAPAYQQGQNQ L8 [ YNE L9N L GRREYDV L0 ] DKRRGRDPEMGGKPQRRKNPQEG L1 [ YNE L2 QKDKMAEAYSEI ] GMKGERRRGKGHDG L3 [ YQG L4 STATKDTYDA L5 ] HMQA L6 PPR (SEQ ID NO: 81); NQ L7 [ YNE L8N L GRREYDV L ] DKR (SEQ ID NO: 14); EG L [ YNE L QKDKMAEAYSEI ] GMK (SEQ ID NO: 15); or L [ YQG L STATKDTYDA L ] HMQ (SEQ ID NO:16) with brackets bracketing the ITAM DG motif.

In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 chain (also known as CD3D, CD3-, T3D, CD3 antigen, subunit, CD3, CD3d antigen, polypeptide (TiT3 complex), OKT3, chain, T cell receptor T3 chain, T cell surface glycoprotein CD3 chain, and the like). Thus, suitable intracellular activation 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 the following sequence, or to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa 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 in any of the following amino acid sequences:

MEHSTF L SG L V L1 AT L2L 4SQVSPFKIPIEE L5 EDRVFVNCNTSITWVEGTVGT L3 SDITR L D L GKRI L DPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVE L DPATVAGIIVTDVIAT L0A L GVFCFAGHETGR L SGAADTQA LL RNDQV [ YQPLRDRDDAQYSHL ] GGNWARNK (SEQ ID NO:17) or

MEHSTF L SG L V L0 AT L1L SQVSPFKIPIEE L EDRVFVNCNTSITWVEGTVGT LL SDITR L D L GKRI L DPRGIYRCNGTDIYKDKESTVQVHYRTADTQA LL RNDQV [ YQPLRDRDDAQYSHL ] GGNWARNK (SEQ ID NO:18) with brackets bracketing the ITAM motif.

Thus, a suitable 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 of DQV [ YQP L RDRDDAQYSH L ] GGN (SEQ ID NO:19), with the ITAM motif bracketed.

In some cases, the intracellular activation domain is derived from a T cell surface glycoprotein CD3 chain (also known as CD3e, T cell surface antigen T3/L eu-4 chain, T cell surface glycoprotein CD3 chain, AI504783, CD3, CD3, T3e, etc.) thus, suitable intracellular activation domains may comprise 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 of the following sequence or to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa 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 the following amino acid sequence:

MQSGTHWRV L G L C L1L 2SVGVWGQDGNEEMGGITQTPYKVSISGTTVI L TCPQYPGSEI L WQHNDKNIGGDEDDKNIGSDEDH L S L KEFSE L EQSGYYVCYPRGSKPEDANFY L Y L RARVCENCMEMDMSVATIVIVDICITGG L0 VYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD [ YEPIRKGQRDLYSGL ] NQRRI (SEQ ID NO:20) with brackets bracketing the ITAM motif.

Thus, a suitable 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 of NPD [ YEPIRKGQRD L YSG L ] NQR (SEQ ID NO:21) with the ITAM motif bracketed.

In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD 3-gamma, T3G, gamma polypeptide (TiT3 complex), etc.). Thus, suitable intracellular activation 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 the following sequence or to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa 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 in the following amino acid sequence:

MEQGKG L AV L I L0 AII L1L 2QGT L4 AQSIKGNH L5 VKVYDYQEDGSV L3 TCDAEAKNITWFKDGKMIGF L6 TEDKKWN L7 GSNAKDPRGMYQCKGSQNKSKP L QVYYRMCQNCIE L NAATISGF L FAEIVSIFV L AVGVYFIAGQDGVRQSRASDKQT LL PNDQ L [ YQP L KDREDDQYSHL ] QGNQLRRN (SEQ ID NO:22), with the ITAM motifs bracketed.

Thus, a suitable 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 of DQ L [ YQP L KDREDDQYSH L ] QGN (SEQ ID NO:23) bracketed with the ITAM motif.

In some cases, the intracellular activation domain is derived from CD79A (also referred to as B cell antigen receptor complex-associated protein α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; Ig- α; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.) thus, suitable intracellular activation 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 of the following sequences or to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or a stretch of about 150aa to about 160 aa:

MPGGPGV QA PATIF 0F 25 SAVY 6GPGCQA 7WMHKVPAS 8MVS 91 23 FCAVVPGT 4G 8[ YEG 0N 1] SRG 2 NIGDVQ 6EKP (SEQ ID NO:24) or MPGGPGV 7QA 8PATIF F5 SAVY 9GPGCQA WMHVPAS 0MVS 12 3FCAVVPGT [ ] (SEQ ID NO:25) with brackets bracketing the ITAM motif.

Thus, a suitable 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 of EN L [ YEG L N L DDCSMYEDI ] SRG (SEQ ID NO:26), with the ITAM motif bracketed.

In some cases, an intracellular activation domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; P L OS L; DNAX activating protein 12; KAR related protein; TYRO protein tyrosine kinase binding protein; killing activating receptor related protein; etc.) suitable intracellular activation domains may include 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 of the following sequence or to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or to a stretch of about 150aa to about 160aa of any of the following amino acid sequences (4 isoforms), for example:

MGG EPCSR P0 1 AVSG 3 AGIVMGD 7V 8TV 9IA 0AVYF 1GR 2[ YQE 34 ] NTQRPYK (SEQ ID NO:27), MGG 5EPCSR 4P 5AVSG 6AGIVMGD V0 TV 1IA 2AVYF 3GR 4[ YQE 56 ] NTQ (SEQ ID NO:28), MGG 7EPCSR 8P 8 AGIVMGD 1V 2TV 3IA 4AVYF 5GR 6 [ YQE 7 ] NTQRYK (SEQ ID NO:29) or MGG 9EPCSR 7P 0 AGIVMGD V IA [ ] NTQRPYK (SEQ ID NO:30) with the ITAM motif bracketed.

Thus, a suitable 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 of ESP [ YQE L QGQRSDVYSD L ] NTQ (SEQ ID NO:31), with the ITAM motif bracketed.

In some cases, the intracellular activation domain is derived from FCERlG (also known as FCRG; Fc receptor Igamma chain; Fc receptor gamma chain; Fc-RI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin receptor subunit gamma; immunoglobulin E receptor, high affinity gamma chain, etc.). For example, suitable intracellular activation 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 of the following sequence, or to a contiguous stretch of about 50 amino acids to about 60 amino acids (aa), about 60aa to about 70aa, about 70aa to about 80aa, or about 80aa to about 88aa of the following amino acid sequence:

MIPAVV LLLLLL VEQAAA L GEPQ L0 CYI L DAI L F L YGIV L T L L YCR L KIQVRKAAITSYEKSDGV [ YTGLSTRNQETYETL ] KHEKPPQ (SEQ ID NO:32) wherein the ITAM element is bracketed.

Thus, a suitable 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 of DGV [ YTG L STRNQETYET L ] KHE (SEQ ID NO:33), with the ITAM motif bracketed.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the invention include DAP10/CD 28-type signaling chains. An example of the DAP10 signaling chain is amino acid SEQ ID NO 34. In some embodiments, suitable intracellular activation domains may include 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 of SEQ ID No. 34.

An example of a CD28 signaling chain is amino acid SEQ ID NO 35. In some embodiments, suitable intracellular activation domains may include 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 of SEQ ID No. 35.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the invention include ZAP70 polypeptides, for example, suitable intracellular activation 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 of SEQ ID No. 36 or to a contiguous 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 amino acid sequence.

Regulatory domains

The regulatory domain may alter the effect of an intracellular activation domain in an engineered signaling polypeptide, including enhancing or inhibiting the downstream effects of the activation domain or altering the nature of the response. Regulatory domains suitable for use in the engineered signaling polypeptides of the invention include co-stimulatory domains. The regulatory domain suitable for inclusion bodies in the engineered signaling polypeptide may be about 30 amino acids to about 70 amino acids (aa) in length, for example, the regulatory domain may 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 length of the conditioning domain may be from about 70aa to about 100aa, from about 100aa to about 200aa, or greater than 200 aa.

Co-stimulatory domains generally enhance and/or alter the nature of the response of the activation domain co-stimulatory domains suitable for use in engineered signaling polypeptides of the invention are generally receptor-derived polypeptides in some embodiments, co-stimulatory domains homodimerize individual co-stimulatory domains may be the intracellular portion of a transmembrane protein (i.e., the co-stimulatory domains may be derived from a transmembrane protein). non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-lBB (CD137), CD27, CD28, CD L, ICOS, OX L, BT L, CD L, GITR and HVEM. for example, state-like co-stimulatory domains of the invention may be at least partially identical to 4-L (CD137), CD L for L ck binding (IC Δ) deletion, CD L, ICOS, L, CD 36137, CD L, CD 3695% identical to the co-stimulatory domains of the activation domain of the invention, CD L, CD 3695% or hvtr may be at least partially identical to the sequence of the co-stimulatory domains of the invention (CD L, CD 3695, CD L, CD 3695, or hvtr 95.

The co-stimulatory domain suitable for inclusion bodies in the engineered signaling polypeptide may be about 30 amino acids to about 70 amino acids (aa) in length, e.g., the co-stimulatory domain may 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 length of the co-stimulatory domain may be from about 70aa to about 100aa, from about 100aa to about 200aa, or greater than 200 aa.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD137 (also known as TNFRSF 9; CD 137; 4-lBB; CDwl 37; I L A, etc.) suitable co-stimulatory domains may include, for example, 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 of SEQ ID NO: 1. in some of these embodiments, the co-stimulatory domain is 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 length.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp 44). For example, suitable co-stimulatory 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 of SEQ ID NO. 2. In some of these embodiments, the co-stimulatory domain 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, or about 65aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 that is deleted for L ck binding (IC Δ.) suitable co-stimulatory domains may include, for example, 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 of SEQ ID NO. 3. in some of these embodiments, the co-stimulatory domain is 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 some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane proteins ICOS (also known as AI L IM, CD278, and CVIDl.) for example, suitable co-stimulatory 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 of SEQ ID NO. 4. in some of these embodiments, the length of the co-stimulatory domain is 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 70 aa.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGPl L) OX40 contains a P85 PI3K binding motif at each residue 34 to 57 of SEQ ID NO. 504 and a TRAF binding motif at residues 76 to 102. in some embodiments, the co-stimulatory domain may comprise a P85 PI3K binding motif of OX 40. in some embodiments, the co-stimulatory domain may comprise a TRAF binding motif of OX 40. the lysines corresponding to amino acids 17 and 41 of SEQ ID NO. 504 are potential regulatory sites that serve as part of a targeted ubiquitin motif. in some embodiments, one or both of these lysines in the co-stimulatory domain of OX40 are mutant spermine or another amino acid.in some embodiments, a suitable co-stimulatory domain may comprise a sequence that is at least about 20, about 70, about 20, about 80, about 20 to about 20, about 80% to about 20, about 20% or about 80% as long, about 20, about 70, about 20% as about 70, about 20 to about 70, about 20% as long as about 70, about 75% as about 70, about 75% as about 75 to about 70, about 20, about 75% as about 70, about 20% as about 20, about 75% as about 70, about 75% as about 10, about 75% as about.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp 55). For example, suitable co-stimulatory 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 of SEQ ID NO 6. In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50 aa.

In some cases, co-stimulatory domains are derived from the intracellular portion of the transmembrane protein BT L A (also known as BT L Al and CD 272). for example, suitable co-stimulatory 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 of SEQ ID NO. 7.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, DlS166E, and Ki-1). For example, suitable co-stimulatory 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 about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, or about 160aa to about 185aa in SEQ ID NO 8.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane proteins GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, suitable co-stimulatory 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 of SEQ ID NO. 9. In some of these embodiments, the co-stimulatory domain 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, or about 65aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, L IGHTR, and TR 2). for example, suitable co-stimulatory 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 of SEQ ID NO. 10. in some of these embodiments, the co-stimulatory domain of the first and second polypeptides is from 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.

Connector sign

In some cases, the engineered signaling polypeptide includes a connector between any two adjacent domains. For example, the connector may be between the transmembrane domain and the first stimulation domain. As another example, the ASTR may be an antibody, and the linker may be between the heavy chain and the light chain. As another example, a connector may be between the ASTR and the transmembrane and co-stimulatory domains. As another example, the linker can be between the co-stimulatory domain and the intracellular activation domain of the second polypeptide. As another example, the connector may be between the ASTR and the intracellular signaling domain.

The linker peptide can have any of a variety of amino acid sequences. Proteins may be linked by spacer peptides, which are generally flexible in nature, without excluding other chemical bonds. 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 linker-encoding oligonucleotides. Peptide linkers having a certain degree of flexibility may be used. The linker peptide may have virtually any amino acid sequence, given that a suitable linker will have a sequence that results in a generally flexible peptide. The use of smaller amino acids, such as glycine and alanine, is for creating flexible peptides. The creation of such sequences is conventional to those skilled in the art.

Suitable linkers can be readily selected and can be any of suitable different 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 can be 1, 2, 3, 4, 5,6, or 7 amino acids.

Exemplary flexible connectors include glycine polymer (G)_nGlycine-serine polymers (including, for example, (GS)_n、GSGGS_n、GGGS_nAnd GGGGS_nWhere n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible connectors known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore can serve as neutral chains between the 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, rev. comparative chem.11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGGGSGGGGSGGS (SEQ ID NO:53), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:54), GGGGSGGGSGGGGS (SEQ ID NO:55), GGSG (SEQ ID NO:56), GGSGG (SEQ ID NO:57), GSGSG (SEQ ID NO:58), GSGGG (SEQ ID NO:59), GGGSG (SEQ ID NO:60), GSSSG (SEQ ID NO:61), and the like. The ordinarily skilled artisan will recognize that the design of the peptide to be incorporated into any of the components described above may include a connector that is fully or partially flexible, such that the connector may include a flexible connector as well as one or more portions that impart a less flexible structure.

Combination of

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

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 also includes 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 also includes one or more of: 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, the one or more engineered signaling polypeptides are expressed under a T cell specific promoter or a general promoter under the same transcript, 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 (IRE) or one or more protease cleavage 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 a first antigen, and a first intracellular signaling domain, but not a co-stimulatory domain, and a second engineered signaling polypeptide comprises a second extracellular antigen-binding domain capable of binding VEGF, and a second intracellular signaling domain, such as the 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), e.g., an antibody or fragment thereof specific for PSCA, PSMA, or BCMA. In one 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 VEGFR 3. In a certain embodiment, the VEGFR is VEGFR 2.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein a first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, and a 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 co-stimulatory molecule signaling motifs may include, for example, a co-stimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1 BB.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein a first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, and a 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 4-1BB in another embodiment, the first signaling polypeptide or the second signaling polypeptide also has a T cell survival motif, in some embodiments, the T cell survival motif is the intracellular signaling domain of I L-7 receptor (I L-7R), the intracellular signaling domain of I L-12 receptor, the intracellular signaling domain of I L-15 receptor, the intracellular signaling domain of I L-21 receptor, or the intracellular signaling domain of transforming growth factor β (TGF β) receptor or TGF β decoy receptor (TGF- β -negative receptor (ii) or the like derived from TGF- β -negative receptor (rii).

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 I L-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 remainder 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 some embodiments, the tumor-associated or tumor-specific antigen is Her, Prostate Stem Cell Antigen (PSCA), PSMA (prostate-specific membrane antigen), B Cell Maturation Antigen (BCMA) -fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA-9, calretinin, MUC-1, epithelial membrane protein (EMA), Epithelial Tumor Antigen (ETA), tyraminidase, melanoma-associated antigen (MAGE), CD117, chromogranin, cytokeratin, desmin, Glial Fibrillary Acidic Protein (GFAP), macrocystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melanin A (melanoma antigen recognized by T lymphocytes; MART-1), myosin-D, muscle-specific actin (MSA), neuropilin, neuron-specific enolase (NSE), placental phosphatase, vesicular protein, thyroglobulin, thyroid transcription factor-1, thyroid-isozyme (NY-D), rat-11, mouse epithelial-derived protein (CD-rat), rat-derived from EphA + 2, rat-mouse epithelial-derived from EphA, rat-6, rat-mouse, rat, mouse, rat, mouse, rat, mouse, and human liver, and animal.

In some embodiments, the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain that binds a first antigen, and a first intracellular signaling domain; and a second engineered signaling polypeptide comprising 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 one 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 second 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 comprise a transmembrane domain. In a certain embodiment, the first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, such as 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 to 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 an alarm (alarmin)). In another embodiment, DAMP is a heat shock protein, chromatin-associated protein high mobility group box 1(HMGB1), 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 certain embodiments, hypoxia is induced by activation of hypoxia inducible factor-1 α (HIF-1 α), HIF-1 β, HIF-2 α, HIF-2 β, HIF-3 α, or HIF-3 β.

In some embodiments, expression of one or more engineered signaling polypeptides is modulated by a control component disclosed in more detail herein.

Additional sequences

An engineered signaling polypeptide, such as a CAR, can further comprise one or more additional polypeptide domains, wherein such domains include, but are not limited to, a signal sequence, an epitope tag, an affinity domain, and a polypeptide whose presence or activity can be detected (detectable tag), e.g., by antibody assay or as a result of its being a polypeptide that produces a detectable signal. Non-limiting examples of additional domains for any of the aspects or embodiments 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.

Suitable signal sequences for use in an individual CAR (e.g., the first polypeptide of an individual 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 can be, for example, the CD8 signal sequence MA L PVTA LLL P L a LLL HAARP (SEQ ID NO: 74).

Suitable epitope tags include, but are not limited to, prothrombin (HA; e.g., YPYDVPDYA; SEQ ID NO:37), F L AG (e.g., DYKDDDDK; SEQ ID NO:38), c-myc (e.g., EQK L ISEED L; SEQ ID NO:39), and the like.

Affinity domains include His5 (hhhhhhh; SEQ ID NO:40), HisX6 (hhhhhhhhh; SEQ ID NO:41), C-myc (k L isod L; SEQ ID NO:39), Flag (dykddkdd; SEQ ID NO:38), Strep tag (WSHPQFEK; SEQ ID NO:42), prothrombin (e.g., albumin-tagged HA (YPYDVPDYA; SEQ ID NO:37)), GST, thioredoxin, fibrin-binding domain, RYIRS (SEQ ID NO:43), Phe-Thr (e.g., albumin-tagged HA (YPYDVPDYA; SEQ ID NO:37)), GST, thioredoxin, fibrin-binding domain, phyrin-binding domain, calcineurin binding protein B (e.g., calponin binding domain, calponin binding protein C3544), calponin-binding protein binding domain, calponin binding protein C3525, calponin-binding protein C binding domain, calponin-binding protein C binding protein, e.g., calponin binding protein C3525, calponin-binding protein C, calponin-binding protein C, treponin, tretin binding protein C, tretin binding protein, treponin, tretin binding protein C, tretin binding protein, such as exemplified by a peptide, peptide binding protein, peptide, such as those from the peptide, peptide containing coding sequences exemplified as histone peptide, calcium binding protein.

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

Suitable fluorescent proteins include, but are not limited to, Green Fluorescent Protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), Enhanced GFP (EGFP), Enhanced CFP (ECFP), Enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, unstable EGFP (dEGFP), unstable ECFP (dECFP), unstable EYFP (dEYFP), mCFpm, Cerulean, T-Sapphire, CyPet, ypet, mKO, HcRed, t-HcRed, DsRed2, DsRed-monomer, J-Red, dimer 2, t-dimer 2(12), mRFP1, coral, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliprotein and phycobiliprotein conjugates, including B-phycoerythrin, R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydev, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al (2005) nat. methods 2:905 and 909), and the like. Any of a variety of fluorescent and colored proteins from Anthozol species are suitable for use, as described, for example, in Matz et al (1999) Nature Biotechnol.17: 969-973.

Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), Alkaline Phosphatase (AP), β -galactosidase (GA L), glucose-6-phosphate dehydrogenase, β -N-acetylglucosaminidase, β -glucuronidase, invertase, xanthine oxidase, firefly luciferase, Glucose Oxidase (GO), and the like.

Identifying and/or eliminating domains

Any of the replication-defective recombinant retroviral particles provided herein may comprise a nucleic acid encoding a recognition domain or an abrogation domain 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 may comprise a recognition domain or an elimination domain. For example, any of the CARs disclosed herein can include a recognition domain or an elimination domain. Furthermore, the recognition domain or ablation domain may be expressed with, or even fused to, any of the lymphoproliferative components disclosed herein. The recognition domain or the ablation domain is expressed on T cells and/or NK cells, but not on replication-defective recombinant retroviral particles.

In some embodiments, the recognition domain or the abrogation domain may be derived from the herpes simplex virus-derived enzyme thymidine kinase (HSV-tk) or inducible caspase 9. In some embodiments, the recognition domain or the elimination domain may comprise a modified endogenous cell surface molecule, for example as disclosed in U.S. patent 8,802,374. The modified endogenous cell surface molecule can be any cell surface associated receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin, or modified Cluster of Differentiation (CD). In some embodiments, the modified endogenous cell surface molecule is a truncated tyrosine kinase receptor. In one aspect, the truncated tyrosine kinase receptor is a member of the Epidermal Growth Factor Receptor (EGFR) family, e.g., ErbB1, ErbB2, ErbB3, ErbB 4. In some embodiments, the recognition domain can be a polypeptide recognized by an antibody that recognizes an extracellular domain of an EGFR member. In some embodiments, the recognition domain can be at least 20 contiguous amino acids of an EGFR family member, or between, for example, 20 and 50 contiguous amino acids of an EGFR family member. For example, SEQ ID No. 78 is an exemplary polypeptide that is bound by an antibody that recognizes the extracellular domain of an EGFR member and that recognizes under appropriate conditions. Such extracellular EGFR epitopes are sometimes referred to herein as etags. In illustrative embodiments, such epitopes are recognized by commercially available anti-EGFR monoclonal antibodies.

Epidermal growth factor receptors, also known as EGFR, ErbB1, and HER1, are cell surface receptors for members of the epidermal growth factor family of extracellular ligands. Alterations in EGFR activity have been implicated in certain cancers. In some embodiments, the gene encoding an EGFR polypeptide comprising human Epidermal Growth Factor Receptor (EGFR) is constructed by removing a nucleic acid sequence encoding a polypeptide comprising a membrane distal EGF binding domain and a cytoplasmic signaling tail, but retaining the extracellular membrane proximal epitope recognized by the anti-EGFR antibody. Preferably, the antibody is a known commercially available monoclonal antibody against EGFR, such as cetuximab (cetuximab), matuzumab (matuzumab), nimustizumab (necitumumab), or panitumumab (panitumumab).

Others have shown that biotin-labeled cetuximab conjugated to anti-biotin microbeads for application in immunomagnetic selection successfully enriched T cells that have been transduced with EGFRt-containing constructs from as low as 2% of the population lentiviruses to greater than 90% purity without observable toxicity to the cell preparation. Furthermore, others have shown that constitutive expression of this inert EGFR molecule does not affect T cell phenotype or effector function as directed by the co-expressed Chimeric Antigen Receptor (CAR), CD 19R. In addition, others have shown that EGFR successfully serves as T cell transplantation in mice via flow cytometry analysisTracking markers in vivo in the plant. In addition, has passed

The mediated antibody-dependent cellular cytotoxicity (ADCC) pathway demonstrates that EGFR has suicide gene potential. The inventors of the present invention have successfully expressed eTag in PBMC using lentiviral vectors, and have found that PBMC exposed to cetuximab express eTag in vitro, providing an effective mechanism for elimination of PBMC. Thus, EGFR can be used as a non-immune gene selection tool, a tracking marker, and a suicide gene for transduced T cells with immunotherapeutic potential. EGFR nucleic acids can also be detected by methods well known in the art.

In some embodiments provided herein, the EGFR is expressed as part of a single polypeptide that also includes a CAR or as part of a single polypeptide that includes a lymphoproliferative component, in some embodiments, the amino acids encoding the EGFR recognition domain can be separated from the amino acids encoding the chimeric antigen receptor by a cleavage signal and/or ribosome skipping sequence the ribosome skipping and/or cleavage signal can be any ribosome skipping and/or cleavage signal known in the art, without being limited by theory, the ribosome skipping sequence can be, for example, T2A (also referred to as 2A-1) having the amino acid sequence GSGEGRGS LL TCGDVEENPGP (SEQ id no:77), other examples of cleavage signals and ribosome skipping sequences include fm2A (F2A), equine rhinovirus type a 2A (abbreviated E2A), porcine tesla virus 12A (P2A), and pseudoplusia (thoseasia) virus 2A (T2A), in some embodiments, the polynucleotide sequence encoding the CAR recognition domain can be identical to the polynucleotide sequence encoding the lymphoproliferative component, or the lymphoproliferative component can enter the lymphoproliferative component via the ribosome transcription site, either internally or the proliferative component.

Such constructs provide the advantage of occupying less genomic space on the RNA genome than the polypeptide alone, particularly in combination with the other "space saving" components provided herein, in one illustrative embodiment, the eTag is expressed as a fusion polypeptide fused to an I L7R α mutant, as demonstrated experimentally herein.

Chimeric antigen receptors

In some aspects of the invention, the engineered signaling polypeptide is a Chimeric Antigen Receptor (CAR) or a polynucleotide encoding a CAR, referred to herein for simplicity as a "CAR". the CAR of the invention comprises a) at least one antigen-specific targeting region (ASTR), b) a transmembrane domain, and c) an intracellular activation domain.

The CARs of the invention 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 a eukaryotic cell, the CAR of the invention is activated in the presence of one or more target antigens (which, under certain conditions, bind ASTR). The target antigen is a second member of the specific binding pair. The target antigen of the specific binding pair can be a soluble (e.g., not bound to a cell) factor; factors present on the surface of cells such as target cells; an agent present on the surface of a solid; factors present on the lipid bilayer; and the like. When the ASTR is an antibody and the second member of the specific binding pair is an antigen, the antigen can be a soluble (e.g., not bound to a cell) antigen; antigens present on the surface of cells such as target cells; an antigen present on the surface of a solid; an antigen present on the lipid bilayer; and the like.

In some cases, the CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, increases the expression of at least one nucleic acid in the cell. For example, in some cases, a CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, 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 10-fold compared to the level of transcription of the nucleic acid in the absence of the one or more target antigens.

As an example, a CAR of the invention can include an intracellular signaling polypeptide comprising an immunoreceptor tyrosine-based activation motif (ITAM).

For example, a CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can increase production of one or more interleukins 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 more than 10-fold as compared to the amount of interleukins produced by the cell in the absence of the one or more target antigens, as compared to the amount of interleukins produced by the cell in the absence of the one or more target antigens.

In some cases, a CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can result in both increased transcription of nucleic acids in the cell and increased production of interleukins by the cell.

In some cases, a CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, produces cytotoxic activity of the cell toward the target cell expressing the antigen on its cell surface 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., an NK cell or a cytotoxic T lymphocyte), the CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, increases the cytotoxic activity of the cell towards the target cell expressing the one or more target antigens on its cell surface. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, the CAR of the invention, when present in the plasma membrane of the eukaryotic cell and activated by one or more target antigens, results in 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 10-fold increase in the cytotoxic activity of the cell as compared to the cytotoxic activity of the cell in the absence of the one or more target antigens.

In some cases, the CARs of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can produce events associated with the activation of other CARs, such as proliferation and expansion (due to increased cell division or anti-apoptotic response).

In some cases, a CAR of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can produce events associated with the activation of other CARs, such as intracellular signaling regulation, cellular differentiation, or cell death.

In some cases, the CARs of the invention are limited by the microenvironment. This property is typically a result of the microenvironment-restricted nature of the ASTR domain of the CAR. Thus, the CARs of the invention can have a lower binding affinity or, in illustrative embodiments, can have a higher binding affinity for one or more target antigens under conditions of a microenvironment than under conditions of a normal physiological environment.

Lymphoproliferative component

Despite the continuous increase in cells, the number of peripheral T lymphocytes remains at a very stable level throughout adulthood due to the removal from the thymus and the proliferation encountered in response to antigen, and due to the depletion of cells that remove antigen-specific effects following antigen clearance (Marrak, P. et al. Nat Immunol 1: 107-111; Freitas, A.A. et al. 2000.Annu Rev Immunol 18: 83-111). The size of the peripheral T cell compartment is regulated by a number of factors that affect both proliferation and survival. However, in the context of lymphopenia, T lymphocytes divide independently of the cognate antigen, due to an "acute constant proliferation" mechanism that maintains the size of the peripheral T cell compartment. Conditions for lymphopenia have been established in individuals or patients during adoptive cell therapy by proliferating T cells in vivo and introducing them into lymphodepleted individuals, resulting in engraftment of the metastasized T cells and enhanced antitumor function. However, lymphatic depletion in an individual is undesirable because it can cause serious side effects, including immune disorders and death.

Some interleukins such as I L-7 and I L-15 are known to mediate antigen-independent proliferation of T cells and are therefore capable of inducing stable proliferation in a non-lymphopenic environment.

Many aspects provided herein include lymphoproliferative component, or nucleic acids encoding same, typically as part of an engineered signaling polypeptide, thus, in some aspects of the invention, an engineered signaling polypeptide is a lymphoproliferative component (L E), such as a chimeric lymphoproliferative component (C L E), typically, L E comprises an ectodomain, a transmembrane domain, and at least one intracellular signaling domain that drives proliferation, and in illustrative embodiments, a second intracellular signaling domain as exemplified herein (see, e.g., examples 11 and 12), wherein there are first and second intracellular signaling domains of C L E, the first intracellular signaling domain being positioned between a membrane-associated motif and the second intracellular domain — in illustrative embodiments, the first intracellular domain having two or more than two intracellular domains in either the intracellular domain of L E or L E is not a functional intracellular domain from an ITAM-containing intracellular domain, e.g., from CD 463, CD 465, CD 585, CD 8624, CD 3655, CD 3679, CD A, CD 8624, CD 3655, CD 8624, CD 3679, CD 593, CD 8624, CD A, CD L A, CD27, CD 8624, CD L A, CD27, CD 3679, CD 3655, CD 593, CD 3679, CD 3659, CD reel 3679, CD reel 3659, CD reel.

C L E does not contain both ASTR and an activation domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, or zap 70. without being limited by theory, the ectodomain and transmembrane domains of salty messenger play a supporting role in L E, ensuring that the intracellular signaling domain is in an effective conformation/orientation/location for driving proliferation. therefore, the ability of salty messenger L E to drive proliferation is provided by the intracellular domain of L E, and salty messenger ectodomain and transmembrane domain play a minor role relative to the intracellular domain.

For example, for embodiments comprising replication-deficient recombinant retroviral particles, there is a limit to the length of a polynucleotide that can be packaged into the retroviral particle such that L E having a shorter amino acid sequence can be advantageous in certain illustrative embodiments, in some embodiments, L E may have a total length of between 3 and 4000 amino acids, e.g., 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, when present to form extracellular and transmembrane domains, 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, or between 4 and 400 amino acids, or between 4 and 200 amino acids, between 4 and 100 amino acids, between 4 and 50 amino acids, between 4 and 20 amino acids, or between 100 and 100 amino acids, such as for example, between 100 and 50 amino acids, between 100 and 100 amino acids, or between 100 and 100 amino acids, or between 100 and 100 amino acids in this embodiment, and 100 amino acids, or 100 amino acids, and 100 amino acids, or more typically between 5, and 100 amino acids, or 100 amino acids of the signal-conducting signal from a certain amino acid sequence in this invention, or 100 amino acids, or 100, 10, or 100 amino acids, or more specific amino acids of the invention, or 100 amino acids of the extracellular domain, or 100 amino acids of the invention, and 10, or 100 amino acids of the invention, or 100 amino acids of the invention may be provided for example, or 100 amino acids of the extracellular domain, or 100 amino acids of the invention, or 100 amino acids, or 100 amino acids of the invention, or 100 amino acids of the invention, or 10, or 100 amino acids of the invention, or 10, or 100 amino acids of the invention, or 10, or 100 amino acids of the invention, or.

The C L E identified in examples 10, 11 and 12 herein facilitates proliferation of PBMC cell cultures transduced with lentiviral particles encoding C L E between day 7 and day 21, 28, 35 and/or 42 after transduction these examples provide a test and/or standard which can be used to identify any test polypeptide, including L E or L E test domains, such as the first or second intracellular domains or both the first and second intracellular domains, which are indeed L E or L E effective intracellular domains, or particularly effective intracellular domains of L E or L E, or a control lymphocyte, if such a test is performed using the same or other example, or a test polypeptide encoding a lentiviral vector or a regulatory protein encoding a lentiviral vector, or a regulatory protein encoding a retroviral gene construct, or a regulatory protein encoding a lentiviral regulatory protein encoding a retroviral gene encoding a lentiviral regulatory protein encoding a retroviral gene encoding a lentiviral regulatory protein expressing a retroviral gene encoding a lentiviral regulatory protein expressing a retroviral gene encoding a retroviral protein expressing a lentiviral regulatory protein expressing a retroviral protein expressing a regulatory protein expressing a regulatory protein expressing a protein expressing.

In some embodiments, this test for improved properties of a putative or test lymphoproliferative component is performed by conducting replication and/or conducting statistical tests. The skilled person will recognise that a number of statistical tests may be used for this lymphoproliferative component test. It is contemplated that this test in these embodiments will 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. 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, L E provides, is capable of providing, and/or possesses the property (or cells genetically modified and/or transduced with L E are capable of providing, are suitable for, are capable of providing, and/or are modified for) that, in the absence of exogenously added interleukins, between day 7 and day 21, day 28, day 35, and/or day 42 of in vivo culture, when transduced with an anti-CD 19 CAR that comprises a CD3 ζ intracellular activation domain but does not comprise a costimulatory domain, 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 of pre-activated PBMC transduced with a nucleic acid encoding L E, or between 2-fold and 20-fold amplification, or between 2-fold and 15-fold amplification, or between 5-fold and 25-fold amplification, or between 5-fold and 20-fold amplification, or between 5-fold and 15-fold amplification, or between 5-fold amplification, or between 0.1.1.5-fold and 15-fold amplification, or between the test for example, where the cells are tested at a statistical match is required, or under some other examples, such as a test that the P1.1.1.1.11 is performed.

For any of the lymphoproliferative component tests provided herein, the number of test cells is compared to the number of control cells between day 7 and day 14, 21, 28, 35, 42, or 60 post-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, e.g., with a hemocytometer 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, test cells may be seeded in one or more wells, flasks or containers, and control cells may be seeded in one or more flasks or containers. In some embodiments, the test and control cells may be seeded into the wells or flasks individually, e.g., one cell per well. In some embodiments, the number of test cells to control cells can be compared using the level of enrichmentIn some embodiments, the enrichment level of a 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 at day 7 for each construct in some embodiments, the enrichment level of a test or control construct can be calculated by dividing the number of cells at a time point (day 14, day 21, day 28, day 35, or day 45) by the number of cells at that time point for untransduced cells 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, L E encoded in the test construct provides (or genetically modified and/or transduced cells with retroviral particles having a genome encoding L E (e.g., lentiviral particles) can provide, be adapted to, possess the following properties and/or modified for at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 8-fold increase between the normalized enrichment level and the normalized enrichment level can be measured between a single 5-fold, or five-fold measurement, when the normalized enrichment level is more than the normalized enrichment level, the normalized enrichment level can be measured between a single time point, or between 5-fold, the normalized enrichment level is measured, or the normalized enrichment level is found between at least 1.5-fold, or the normalized enrichment level is found to meet the normalization value of the normalization of the test construct₂((normalized count data on test day + 1)/(normalized count data on day 7 + 1)).

As demonstrated in examples 10, 11, and 12, C L E was identified from a library of constructs comprising constructs encoding a test chimeric polypeptide (designed to comprise an intracellular domain that is believed to induce proliferation and/or survival of lymphoid or myeloid cells) and an anti-CD 19 CAR (comprising an intracellular activation domain but not a co-stimulatory domain), in a commercial medium comprising PBMCs, lymphocytes (intact OpTmizer)^TMCTS^TMT-cell expansion SFM), recombinant humanPre-activation was performed in a pre-activation reaction mixture of interleukin-2 (100IU/ml) and anti-CD 3 Ab (OKT3) (50ng/ml), which was performed overnight at 37 ℃. After pre-activation, transduction was performed at a multiplicity of infection (MOI) of 5 overnight at 37 ℃ after addition of the test and control lentiviral particles to the pre-activation reaction mixture. Some control lentiviral particles contain a construct encoding a polypeptide having an extracellular domain and a transmembrane domain, but no intracellular domain. In contrast, the test lentiviral particles comprise a construct encoding a polypeptide having an extracellular domain and a transmembrane domain, and one or two intracellular domains. After transduction, the intact OpTsizer will be added^TMCTS^TMT-cell amplification SFM to dilute the reaction mixture 5-fold to 20-fold, and culture cells at 37 ℃ for up to 45 days after day 7 post-transduction, cultures were "fed" or not ("not fed") with additional untransduced donor-matched PBMC after the transduction reaction mixture was initially formed, no additional interleukins (e.g., I L-2, I L-7, or I L-15 and no other lymphomitotic agents) were added to these cultures that were not present in commercial media amplification was measured by analyzing the enrichment of cell counts that were actually counted as the nucleic acid sequence counts of the unique identifier for each construct in the mixed culture cell population, such that the enrichment was positive, calculated as the base 2 logarithm of the ratio between the normalized counts of the last day of the analysis plus one and the counts of day 7 plus one.

As shown in examples 10, 11, and 12, the construct was identified as C L E because C L E induced proliferation/amplification in the fed or unfed cultures without the need to add an interleukin (such as I L-2) between day 7 and day 21, day 28, day 35, and/or day 42, for example, example 10 identified effective C L E by identifying test C L E that provided increased amplification of the in vitro cultures, as compared to a control construct that did not include any intracellular domains, between day 7 and day 21, day 28, day 35, and/or day 42 after transduction, regardless of whether there was a non-transduced pbmc fed or not, example 10 revealed that at least one and typically more than one test C L E including an intracellular domain from the test gene, as opposed to each control construct (not including an exemplary intracellular domain) present on day 7 after transduction, provided more amplifications, as opposed to the case of which was fed or unftransduced, example 10 further provided that the test C L E including an intracellular domain from the test gene, was used to calculate a statistical score greater than 0, or less than 0, as calculated for the first test negative for the test construct.

As shown by examples herein, in exemplary aspects and embodiments including L E (which typically includes a CAR), such as the methods for genetically modifying and/or transducing, genetically modifying and/or transducing cells and uses thereof provided herein, the genetically modified cells are modified so as to possess new properties that the cells did not pre-own prior to genetic modification and/or transduction, such properties may be provided by genetic modification using a nucleic acid encoding CAR or L E (and in illustrative embodiments, both CAR and L E), for example, in certain embodiments, in the absence of added I L-2 or in the absence of added interleukins (such as I L-2, I L-15, or I L-7), and in certain illustrative embodiments, in the presence of an antigen recognized by the CAR, the genetically modified and/or transduced cells are capable of, suitable for possessing, modified and/or for, live and/or live in culture and for at least one of the live, or live at, 7, 21, 14, or 60 days after the live and/or 60 days of the live cell activation of the cell in vitro and/or on the cell.

In certain embodiments, the cells that are genetically modified and/or transduced exhibit, can be supplied, are adapted to possess, or are modified for improved survival or expansion in culture media ex vivo or in vitro culture, by virtue of the one or more interleukins added to the culture media (such as I L-2, I L-15 or I L-7) or the lymphokine medium is added to the cells that are genetically modified and/or transduced, as compared to control cells that are genetically modified and/or transduced prior to their genetic modification and/or transduction or the same control cells transduced with retroviral particles (as tested retroviral particles comprising L E or putative L E but not having the endodomain of L E or L E), and the cells that are genetically modified and/or transduced exhibit, can be supplied, are adapted to, possess, or are modified for improved survival or expansion in culture media in vitro or ex vivo culture, wherein the one or more interleukins added to the culture media (such as I L-2, I L-15 or I L-7) or the lymphokine medium is added to the culture media that is not present, and the cells that are cultured in culture media that are not cultured in culture media that are genetically modified and/or cultured, and the cells that are not cultured, or cultured, the cells that are not cultured, and the cells that are not cultured, or cultured, the cells that are not cultured, or cultured, and that the exogenous interleukin(s) are not cultured, and the cells that are not cultured, or are not cultured, as compared to the cells that are not cultured, and that are not cultured, the cells that are not cultured, or that are not cultured, and that are not cultured, or are not cultured, and that are not cultured, the cells that are not cultured, and that are not cultured, and that are not cultured, the exogenous, are not cultured.

In some embodiments, improved or enhanced survival, amplification and/or proliferation may be displayed as an increase in the number of cells determined by sequencing DNA from cells transduced with a retroviral particle having a genome encoding C L E (e.g., a lentiviral particle) and counting the occurrence of sequences present in a unique identifier from each C L E. in some embodiments, the improved survival and/or improved amplification may be determined by directly counting the cells at each time point with a hemocytometer or cell counter. in some embodiments, the improved survival and/or improved amplification and/or enrichment may 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 at day 7 for each construct. in some embodiments, the cells may be counted by a hemocytometer or cell counter. in some embodiments, the cells may be counted using the nucleic acid counts or cell counts determined for each particular test as a normalized level between the modified level (i.e., having the same normalization between the modified level of the modified construct and the modified domain, i.e., the normalized level of enrichment provided for the normalized construct, the normalized or enriched for the normalized level of the construct, between the normalized level of the modified construct, i.5 and the normalized level of the normalized gene encoding the modified domain, or enriched for the normalized construct, or for the normalized level of the normalized construct, between the normalized level of the normalized expression of the normalized gene encoding the normalized gene found between the normalized level of the normalized gene found at least 10 fold of the normalized gene found at 635 fold of the normalized level found at the normalized.

In illustrative embodiments herein, one or more lymphoproliferative components are introduced into T cells and/or NK cells by genetically modifying and/or transducing T cells with replication-defective recombinant retroviral particles, the genome of the replication-defective recombinant retroviral particles encodes a lymphoproliferative component as part of an engineered signaling polypeptide, the lymphoproliferative component can comprise a signaling endodomain or can comprise more than one endodomain, or can comprise two endodomains in certain illustrative embodiments, the lymphoproliferative component comprises two endodomains in tables 8-25 as provided herein and can be recognized in examples 10, 11 and 12, the intracellular domains provide one endodomain for a C E and provide two endodomains for a C E as recognized by the experimental RB method provided herein, the first endodomains domain is between the transmembrane domain and the second endodomains, or 1E, the "endodomain" signaling domain "referred to as 2E, which is found to be capable of signaling between the intracellular domain of CD 21, 7, or a CD 21, R12, 7I 12, or a CD 21, R12, 7I, 7, or CD 21, R7, or a CD 75, or CD I or CD7, which has at least one or 7 signal transduction effect in at least one or a CD 75-7, 60, 7, 60, 7, 60, 7, 10, 60, 7, or 7, 60, 7, 60, 7, 10, or 7, or 7, respectively, 7, or 7, or 7, or a chimeric, 7, or a chimeric, 7, or 12, 7, or a transient, 7, or 12, 7, or 12, 7, or 12, or a transient, or 12, 7, or a transient, 7, or a transient, 7, 120, 7, or a transient, or 12, or a transient, or 7, or 12, or a transient, or 12, 7, 2, 7, 120, or 12, 120, 7, 2, 7, 2, 120, or 12, 120, or a transient, 7, 2, 120, or 12, or a transient, or 7, or 12, or a transient, or 7, or a transient, or 12, or a transient, respectively, or 7, or a transient, 120, or a transient, or 12, 120, or a transient, or 12, or 10, or 7, or 12, or a transient, or 7, or a transient response, or a transient response, respectively, or a transient response, or 7.

In some illustrative embodiments, the lymphoproliferative component may be or may comprise an interleukin, or in further illustrative embodiments, an interleukin receptor, or a fragment comprising a signaling domain thereof, that activates the STAT3 pathway, the STAT4 pathway, or even in further illustrative embodiments, the Jak/STAT5 pathway. Thus, in a non-limiting example, the lymphoproliferative component can be an interleukin receptor or an active fragment comprising a signaling domain thereof, such as the interleukin receptor, or an active fragment comprising a signaling domain thereof that activates STAT 5. Lymphoproliferative component a lymphoproliferative component is therefore a polypeptide that promotes proliferation and optionally survival (anti-apoptosis) and optionally provides a costimulatory signal that enhances the differentiation state, proliferative potential, or resistance to cell death of lymphocytes. In illustrative embodiments, lymphoproliferative component a lymphoproliferative component is a polypeptide that induces proliferation of T cells and/or NK cells. Illustrative lymphoproliferative component lymphoproliferative components induce proliferation by activating STAT 5. Thus, this lymphoproliferative component, fragment of the lymphoproliferative component, in illustrative embodiments, retains the ability to induce proliferation of T cells and/or NK cells by activating STAT 5.

In illustrative embodiments, the lymphoproliferative component is capable of promoting lymphocyte proliferation/expansion and is optionally present in vitro or ex vivo in culture during 6 days, 7 days, 14 days, 21 days, or 35 days of culture in the absence of exposure of a cell to an interleukin (such as I L-15, I L-7, or in illustrative embodiments, I L-2), in some illustrative embodiments, further in the absence of a target of an ASTR of a CAR expressed by the cell, or in certain illustrative embodiments, in the presence of an antigen recognized by the CAR (wherein the method comprises using a retroviral particle that has on its surface a pseudo-differentiation component and optionally an isolated or fusion activation domain, and typically does not require pre-activation), when present in genetically modified PBMCs, lymphocytes, or genetically modified T cells and/or NK cells.

In some of the methods and compositions presented herein, lymphoproliferative component a lymphoproliferative component is used to promote proliferation or expansion of a genetically modified T cell in vivo without having to lymphodeplete the individual. Thus, non-limiting illustrative embodiments of the methods provided herein (including inserting a lymphoproliferative component into resting T cells and/or NK cells of an individual, typically by transducing such T cells and/or NK cells) can be performed without lymphodepleting the individual prior to, during, and/or after performing the method, or without lymphodepleting the individual prior to, during, and/or after collecting blood from the individual prior to performing the method, or without lymphodepleting the individual prior to, during, and/or after genetically modifying T cells and/or NK cells from the individual ex vivo, and/or prior to, during, and/or after reintroducing the genetically modified T cells and/or NK cells into the individual. Factors that promote proliferation in living T cells include interleukins and their receptors, which typically include a ligand binding domain and a signaling domain. In some embodiments, the lymphoproliferative component used in the methods and compositions disclosed herein is an interleukin and/or an interleukin receptor. The interleukin may be interleukin, and the interleukin receptor may be an interleukin receptor. Lymphoproliferative component the lymphoproliferative component can be a functional fragment of an interleukin and/or a functional fragment of an interleukin receptor (such as a signaling domain thereof), wherein the fragment is capable of promoting T cell proliferation, for example, by activating STAT 5.

In some examples, the interleukin lymphoproliferative component of the methods and compositions herein includes one or more of interleukin-7 (I L-7) or its receptor (I L-7R), or its signaling domain, interleukin-12 (I L-12) or its receptor (I L-12R), or its signaling domain, interleukin-23 (I L2-23) or its receptor consisting of I L3-12R L and I L-23R, or its signaling domain, interleukin-27 (I L-27) or its receptor (I L-27R), or its signaling domain, interleukin-15 (I L-15) or its receptor (dni L-15R), or its signaling domain, interleukin-21 (I L-21) or its receptor (I L-21R), or its receptor shifting domain, or its receptor for Transforming Growth Factor (TGF) receptor ii) or its receptor (TGF I465-5) receptor (I468-7R), or its receptor (I465-7R), or its signaling domain, or its receptor shifting receptor (TGF-7R), or its signaling domain, or its receptor (I468-7R), or its signaling domain, in some embodiments, TGF-7 receptor (I-7) receptor (I468-7R), or its signaling domain, TGF 7 receptor (I-7) receptor (I465), or negative receptor (TGF 7) receptor (I).

I L-7 binds to the I L-7 receptor (a heterodimer consisting of I L-7R α and the common gamma chain.) binding produces a signaling cascade essential for T cell development and survival in the periphery within the thymus, binding of I L-7 to the I L-7 receptor is known to activate the Jak/STAT5 pathway.

I L-12 relates to the differentiation of primary T cells into Th1 cells (Hsieh CS et al 1993.science.260(5107):547-9) and is referred to as T cell stimulating factor I L-12 binds to the I L0-12 receptor (which is a heterodimeric receptor formed by I L-12R-L and I L-12R- β 2). I L312 can act by activating STAT4, but is shown to activate STAT5(Ahn, H. et al 1998.J.Immun.161:5893 5900) in T cells as well, the I L-12 family consists of interleukins I L-12, I L-23 and I L-27. the receptor for I L-23 consists of I L-12R β and I L-23 R.I 5953-27 consists of two different genes (Epin-843-3) and the receptor for the I368627-EB8627 is induced by the mutual action of the genes of Epstein-863-3.

I L-15 is a T cell and NK cell stimulatory factor structurally and functionally similar to I L-2 both interleukins induce proliferation of T cells and their shared function is thought to be generated by two receptors using I L0-2/I L-15R L1 and the common gamma chain the signaling pathway of I L-15 begins by binding to the I L-15R α receptor and subsequent presentation to surrounding cells bearing the I L-15R β yc complex on their cell surface upon binding I L-15 β the subunits activate Janus kinase 1(Janus kinase 1; Jak1) and the yc subunit Janus kinase 3(Jak3) which leads to phosphorylation and activation of STAT3 and STAT 5.

I L-21 is expressed in activated human CD4+ T cells and NK T cells, and I L-21 is up-regulated in the Th2 and Th17 subsets of T helper cells I L-21 receptor (I L-21R) is expressed on the surface of T cells, B cells and NK cells and is similar in structure to the receptors of other type I interleukins like I L-2R or I L-15I L-21R needs to dimerize with the common gamma chain (γ c) in order to bind I L-21, when bound to I L-21, the I L-21 receptor acts via the Jak/STAT pathway, activating STAT1, STAT3 and STAT 5.

TGF β decoy receptor (TGF- β -dominant-negative receptor ii (DNRII)) blocks TGF β signaling by competing with the natural receptor for TGF β binding TGF β -DNRII is a truncated form of kinase-inactive RII that contains the extracellular TGF β binding domain and transmembrane domain of RII TGF β -DNRII binding ligand but does not phosphorylate and activate RI, which thereby reduces or eliminates Smad phosphorylation.

Mutations that have been identified for I L-7R α in individuals with B-and T-cell acute lymphoblastic leukemia (B-A LL and T-A LL) (Zenatti PP et al 2011.Nat Genet 43: 932-939; Snochat, C. et al 2011.J Exp Med 208: 901-908; McElroy, C.A. et al 2012.PNAS 109(7): 2503-2508.) mutations include insertions and deletions in the N-terminal region of I L-7R α TMD, where almost all sequences contain additional Cys residues, and S165 to C165 mutations-cysteine results in constitutive activation of receptors.

Thus, in some embodiments, the lymphoproliferative component is a mutated I L-7 receptor, in other embodiments, the mutated I L-7 receptor is constitutively active, thereby activating the JAK-STAT5 pathway in the absence of an interleukin ligand, in yet other embodiments, the mutated I L0-7 receptor comprises a1 to 10 amino acid insertion at a position between 237 and 254, the insertion comprising a cysteine residue comprising the ability to constitutively activate the STAT5 pathway, in some embodiments, the mutated I L-7 receptor is I L-7R α -insPPC L (represented by SEQ ID NO: 82), further, in some chimeric lymphoproliferative component (C L E) embodiments provided herein, one or more (but not all) of the domains are from I L-7R α -insPPC L.

In some embodiments, the lymphoproliferative component is a chimeric interleukin receptor, such as, but not limited to, an interleukin that is linked to its receptor that typically constitutively activates the same STAT pathway as a corresponding activated wild-type interleukin receptor (such as STAT3, STAT4, and in the illustrative embodiment, STAT 5). in some embodiments, the chimeric interleukin receptor is an interleukin or a fragment thereof that is linked or covalently linked to its cognate receptor via a connector.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component that is an interleukin or an interleukin receptor polypeptide, or a fragment thereof that includes a signaling domain, the lymphoproliferative component can include a moiety covalently linked to its cognate interleukin receptor polypeptide via a linker. Typically, this portion of the cognate interleukin receptor includes functional portions capable of binding the extracellular and transmembrane domains of the interleukin. In some embodiments, the intracellular domain is an intracellular portion of a cognate interleukin receptor. In some embodiments, the intracellular domain is the intracellular portion of a different interleukin receptor that is capable of promoting lymphocyte proliferation. In some embodiments, the lymphoproliferative component is a interleukin polypeptide covalently linked to its full length cognate interleukin receptor polypeptide via a linker.

In the illustrative embodiments of any of the methods and compositions provided herein that include lymphoproliferative components, the intracellular domain may be derived from the intracellular portion of the transmembrane protein CD the CD protein contains several binding sites for TRAF proteins without being limited by theory, the binding sites for TRAF, TRAF and TRAF are localized to the membrane distal domain of the intracellular portion of CD and include the amino acid sequence PXQXT (SEQ ID NO:522), wherein each X may be any amino acid (corresponding to amino acids 35 to 39 of SEQ ID NO: 416) (Elgueta et al, Immunol Rev.2009 5.p.; 229(1):152-72) TRAF is also shown to bind to the consensus sequence SXXE (SEQ ID NO:523), wherein each X may be any amino acid (corresponding to amino acids 57 to 60 of SEQ ID NO: 416) (Elgueta et al, wherein each X may be any amino acid (corresponding to amino acids 57 to 60 of SEQ ID NO: 15) (Elguet R.70, R.70 to 60) or about 20% of the amino acid sequence of the extracellular domain of the polypeptide, such as found in the intracellular portion of the CD receptor CD70, NO: 14, NO: 23, NO: 14, NO:8, NO: 14, NO:8, or about 20, NO: 14, or about 20, NO:8, or about 10% by the length of the amino acid sequence of the polypeptide recognized by the cell receptor, or about the amino acid sequence of the cell receptor (see the amino acid sequence of the extracellular domain of the amino acid sequence of the polypeptide which is found in the cell receptor (see the amino acid sequence of the cell receptor identified by the amino acid sequence of the cell receptor found in the cell receptor identified by the amino acid sequence of the amino acid sequence.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of CD 27. The serine at amino acid 219 of full-length CD27 (corresponding to the serine at amino acid 6 of SEQ id no: 413) was shown to be phosphorylated. 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 SEQ ID No. 413 or a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID No. 413. In some embodiments, the intracellular domain derived from CD27 has a length of about 30 amino acids (aa) to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of CSF2 RB. Full-length CSF2RB contains a Box1 motif at amino acids 474 to 482 (corresponding to amino acids 14 to 22 of SEQ ID NO: 421). The tyrosine at amino acid 766 of full-length CSF2RB (corresponding to the tyrosine at amino acid 306 of SEQ ID NO: 421) is shown to be phosphorylated. In some embodiments, suitable endodomains can 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: 421. In some embodiments, the intracellular domain derived from CSF2RB 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 150aa, about 150aa to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, about 250aa to 300aa, about 300aa to 350aa, about 350aa to about 400aa, or about 400aa to about 450 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of I L2 RB full length I L2 RB contains a Box1 motif at amino acids 278 to 286 (corresponding to amino acids 13 to 21 of SEQ ID NO: 448). 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: 448. in some embodiments, the intracellular domain derived from I L2 RB 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 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 125aa, about 250aa to about 250aa, or about 175aa to about 250 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include lymphoproliferative components, the intracellular domain may be derived from the intracellular portion of I L ST full length I L ST contains the Box1 motif at amino acids 651 to 659 (corresponding to amino acids 10 to 18 of SEQ ID NO: 455), amino acid 661, amino acid 667, amino acid 782, amino acid 789, amino acid 829, and serine at amino acid 839 (corresponding to amino acid 20, amino acid 26, amino acid 141, amino acid 148, amino acid 188, and serine at amino acid 198, respectively) are shown to be phosphorylated.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of I L17 RE, full-length I L17 RE contains a TIR domain at amino acids 372-495 (corresponding to amino acids 13-136 of SEQ ID NO: 473), in some embodiments, a suitable intracellular 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 a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:473, in some embodiments, the intracellular domain derived from I L17 RE 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 125aa to about 150aa, about 100aa to about 150aa, about 175aa to about 175aa, or about 175aa to about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of I L2 RG full length I L2 RG contains a box1 motif at amino acids 286 through 294 (corresponding to amino acids 3 through 11 of SEQ ID NO: 449) 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:449, in some embodiments, the intracellular domain derived from I L2 RG 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, about 60aa to about 65aa, about 65aa to about 70aa, or about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include lymphoproliferative components, the intracellular domain may be derived from the intracellular portion of I L18R 1 full-length I L18R 1 contains a TIR domain at amino acids 222 through 364 (corresponding to amino acids 28 through 170 of SEQ ID NO: 474). 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: 474. in some embodiments, the intracellular domain derived from I L18R 1 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, or about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include lymphoproliferative components, the intracellular domain may be derived from the intracellular portion of I L27 RA full length I L27 RA contains a box1 motif at amino acids 554 to 562 (corresponding to amino acids 17 to 25 of SEQ ID NO: 481.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:481 or SEQ ID NO: 482. in some embodiments, the intracellular domain derived from I L27 RA 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, or about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the endodomain can be derived from the intracellular portion of IFNGR 2. Full-length IFNGR2 contains a double leucine internalization motif at amino acids 276 to 277 (corresponding to amino acids 8 to 9 of SEQ ID NO: 438). In some embodiments, suitable endodomains can 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 438. In some embodiments, the intracellular domain derived from IFNGR 21 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, or about 65aa to about 70 aa.

In the illustrative embodiments of any of the methods and compositions provided herein comprising a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of MyD88, the MyD88 protein has an N-terminal death domain (corresponding to amino acids 29 to 106 of SEQ ID NO: 492) mediating the interaction with other death domain-containing proteins, an intermediate domain (corresponding to amino acids 107 to 156 of SEQ ID NO: 492) interacting with I L-1R-related kinase and a C-terminal TIR domain (corresponding to amino acids 160 to 304 of SEQ ID NO: 492) (Biol Res.2007; 40(2) 97-112) MyD88 also has a canonical nuclear localization and an output motif point mutation has been identified in MyD88, and comprises a functional deletion mutation L P and R193C (corresponding to about L P and R93P in SEQ ID NO:492, or about 100% to 260, 100% to about 100% of the length of the mature polypeptide of the CD 72, mature polypeptide of the mature polypeptide of.

The illustrative examples of methods and compositions comprising lymphoproliferative components provided herein include the amino acid substitutions in the intracellular domain of transmembrane protein MP (120) and the observed effect of the polypeptide fragments.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from a portion of the transmembrane protein CD79B, also known as B29; IGB; agm6. cd79b contains the ITAM motif at residues 193 to 212 (corresponding to amino acids 16 to 30 of SEQ ID NO:419) CD79B has two tyrosine amino acids Y196 and Y207 (corresponding to Y16 and Y27 of SEQ ID NO:419) known to be phosphorylated in some embodiments, the intracellular portion of the transmembrane protein CD79B may include the ITAM motif described herein and the known phosphorylation site in CD79B motif and phosphorylatable tyrosine amino acids known in the art, and the skilled person will be able to recognize the corresponding motif and phosphorylatable tyrosine amino acids in a similar CD79B polypeptide and in some embodiments, suitable intracellular domains may include at least 10, 15, 20, or 100% of the amino acids found in SEQ ID No. 75, or all of the amino acids in some embodiments described herein as having a sequence identity with at least 10, 15, 35, 30, 35, 75, 95, or 75, 95% of the amino acids in the amino acids of the amino acids in the amino acids of the similar CD79 amino acids of the amino acid sequence of the amino acids of the.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the endodomain can be derived from a portion of the transmembrane protein OSMR. OSMR contains the Box1 motif at amino acids 771 to 779 of isoform 3 (corresponding to amino acids 16 to 30 of SEQ ID NO: 502). OSMR has two serines at amino acids 829 and 890 of the known phosphorylated isoform 3 (serines at amino acids 65 and 128 of SEQ ID NO: 502). In some embodiments, the intracellular portion of the protein OSMR may comprise the box1 motif described herein and known phosphorylation sites. The motifs of OSMR and phosphorylatable tyrosine are known in the art and the skilled person will be able to identify the corresponding motifs and phosphorylatable serine in similar OSMR polypeptides. In some embodiments, suitable endodomains can 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. 502. In some embodiments, the OSMR-derived intracellular domain 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 150aa, about 150aa to about 175aa, about 175aa to about 200aa, or about 200aa to about 250 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from a portion of transmembrane protein PR L R PR L R contains a growth hormone receptor binding domain at amino acids 185 to 261 of isoform 6 (corresponding to amino acids 28 to 104 of SEQ ID NO: 503), the growth hormone receptor binding domain of PR L R is known in the art, and one of skill will be able to identify the corresponding domain in a similar PR L R polypeptide, in some embodiments, suitable intracellular domains may include a domain 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 amino acids in SEQ ID NO:503, in some embodiments, the intracellular domain derived from PR L R has a length of from about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 70, or about 100aa to about 100aa, about 70aa to about 300aa, about 200aa to about 300aa, about 300aa to about 300aa, about 200aa to about 300aa, about 100aa to about 100aa, about 100aa, about aa to about 100aa, about 100aa, about 175aa, about 200aa, about 200aa, about 200 aa.

In illustrative embodiments of the methods, uses and composition comprising a lymphoproliferative component provided herein, the intracellular domain may be, the lymphoproliferative component may comprise, or a fragment thereof, an intracellular domain from the following signaling domain, wherein the intracellular domain may comprise, or a fragment thereof, the signal transduction domain of CSF2, CR 2, CSF3, EPOR, GHR, IFNAR, IFNGR, IFN R, I01R, I001 RAP, I011R 021, I031R 042, I052 RA, I072 RB, I083 RA, I095 RA, I16 ST, I117 RA, I129 RA, I1410, RB I1511 RA, I1612 RB, I1712 RB, I3 RA, I1913 RA, I215 RA, I2017 RB, I7 RC, I7 RD, I8R, I2418 RA, I RA, I1 RA, I1 RB 1310, I1 RA, I3 RA, I R, I1 RA, I1R, I1R, I1R, I, R1R, R1R, R1R, R1R, R.

In some embodiments, the lymphoproliferative component can comprise the sequence: s054, S057, S058, S059, S062, S063, S064, S069, S072, S077, S081, S082, S083, S084, S085, S086, S087, S098, S099, S100, S101, S102, S103, S104, S105, S106, S109, S115, S116, S117, S120, S121, S126, S129, S130, S135, S136, S137, S138, S141, S142, S143, S145, S147, S148, S154, S155, S156, S157, S158, S161, S165, S168, S169, S170, S171, S174, S175, S176, S177, S180, S183, S186, S189, S190, S191, S193, S198, S199, S202, or any of its signal fields (S202, S) as shown in the signal field. Each of these portions is present at position P3 on at least one construct of the first 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component can comprise the sequence: s057, S058, S059, S064, S069, S072, S084, S085, S099, S100, S101, S102, S104, S106, S115, S116, S126, S130, S135, S137, S138, S142, S143, S148, S158, S165, S168, S169, S170, S171, S174, S175, S176, S177, S186, S190, S191, S192, S193, S197, S198 or S199 (as shown in table 7), or any fragment thereof including a signaling domain. Each of these portions is present at the P3 position on at least one construct that does not have an endodomain in the P4 position and in the first 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component can comprise the following sequences as shown in table 7: s057, S062, S063, S064, S081, S084, S105, S106, S117, S129, S138, S149, S161, S168, S169, S170, S186, S190, S191, S192, S194, S196, S197, S198, S199, or S202 (table 1), or any segment thereof that includes a signaling domain. Each of these fractions was significantly enriched at the P3 position of the repeat spanning pool 3.1.

In illustrative embodiments of any of the methods, uses, and composition aspects provided herein that include a lymphoproliferative component, the endodomain can include an endodomain comprising a signaling domain from: CD3D, CD3E, CD3G, CD27, CD28, CD40, CD79A, CD79B, FCER1G, FCGR2C, FCGRA2, ICOS, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14 or TNFRSF 18. Each of these genes has an endodomain that is present at position P4 on at least one construct of the first 100 hits of at least one of the repeats of library 3.1. In some embodiments, a lymphoproliferative component can include an endodomain or a fragment thereof comprising a signaling domain from: CD40, CD79B, FCGR2C or FCGRA 2. In some embodiments, a lymphoproliferative component can include an endodomain or a fragment thereof comprising a signaling domain from: CD3D, CD3G, CD40, CD79A, ICOS, TNFRSF8 or TNFRSF 9. Each of these genes has an endodomain present at the P4 position on at least one construct that does not have an endodomain in the P3 position and in the first 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component can comprise an endodomain or fragment thereof comprising a signaling domain from CD 40. In some embodiments, a lymphoproliferative component can include an endodomain or fragment thereof that includes a signaling domain from a TNF receptor family member, and in illustrative embodiments as a second intracellular signaling domain, such as a TNF receptor family member as set forth in table 2. In some embodiments, a lymphoproliferative component can include an endodomain or a fragment thereof comprising a signaling domain from: CD27, CD40, CD79B, TNFRSF4, TNFRSF8, TNFRSF9, or TNFRSF18 (table 2). Each of these genes had an endodomain that was significantly enriched at the P4 position of the repeat spanning pool 3.1 (P < 0.1). In some embodiments, a lymphoproliferative component can include an endodomain or fragment thereof comprising a signaling domain from CD40 or CD 79B. In some embodiments, the lymphoproliferative component can comprise the sequence: s037, S038, S039, S047, S048, S049, S050, S051, S052, S053, S074, S075, S076, S080, S211, S212, S213, S214, S215, or S216 (as shown in table 7), or any fragment thereof including a signaling domain. Each of these portions is present at position P4 on at least one construct of the first 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component can comprise the sequence: s037, S039, S050, S051, S052, S080, S212, or S213 (as shown in table 7), or any fragment thereof including a signaling domain. Each of these portions is present at the P4 position on at least one construct that does not have an endodomain in the P3 position and in the first 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component can comprise the following sequences as shown in table 7: s047, S050, S051, S053, S211, S212, S213, or S215 (table 2), or any fragment thereof including a signaling domain. Each of these fractions was significantly enriched at the P4 position of the repeat spanning pool 3.1.

In some embodiments, the interleukin receptor may be a CD, CR F, CSF2, CSF3, EPOR, GHR, IFNAR, IFNGR, IFN R, I01R, I11 RAP, I21R 31, I41R 52, I62 RA, I82 RB, I92 RG, I3 RA, I04 15RA, I26 ST, I47 RA, I69 RA, I810 RB, I RA, I12 RB, I013 RA, I213 RB, I315 RA, I517 RA, CSF I617 RB, I717 RC, I817 RE, I918R, I18, I RA, I120 RB, I423 RA, I527 RB, I51 RA, I213 RA, I517 RA, CSF 1RA, I717 RA, I817 RA, I120 RA, I423 RA, I527 RA, I51 RA, I213 RA, I51 RA, I52 RA, I51 RA, ii 51 RA, iii 52RA, iii 51 RA, iii 52RA, iii 51, iii 51, iii 51, iii 7, iii 7, iii.

For 8 pool 3.1 repeats, when analyzing all constructs not related to part P, table 1 provides an identifier of the most significant first intracellular signaling domain (P) portion and the corresponding gene when analyzing the most significant first intracellular signaling domain (P) portion of the polypeptide or the corresponding gene, in some embodiments the lymphoproliferative component may comprise the first intracellular signaling domain or its variant or its fragment in the case P134, or its fragment 170, or the lymphoproliferative domain thereof comprises the signal transduction domain of the polypeptide or its fragment P85, or its fragment 170, or the signal transduction domain of the polypeptide or its fragment S134, or its fragment S168, or its signal transduction domain 170, or its fragment 170, or its signal transduction domain, including the signal transduction domain of the polypeptide or protein.

For the 8 pool 3.1 repeats in example 10, when analyzing all constructs not related to the P3 portion table 2 provides an identifier for the most effective second intracellular signaling domain (P4) portion and corresponding gene when the second intracellular signaling domain portion is identified in the constructs shown to be most effective by this statistical analysis (P <0.1 or P <0.05), wherein the numbers are in parentheses CD4 (S047), CD4 (S051) CD79 4 (S053), TNFRSF4(S211), TNFRSF4 (S4), CD79 4 (S053), TNFRSF4(S211), TNFRSF4 (S212), TNFRSF4 (S213) and TNFRSF4 (S215) when the second intracellular signaling domain portion is represented by a second intracellular signaling domain (S99) or a second intracellular signaling domain (S97) or a second coding domain equivalent region (S) or a second coding domain equivalent of a coding domain of which is smaller than the first intracellular signaling domain (P) or a coding sequence number of the second coding domain (CD 4) when the coding sequence is represented by the second intracellular coding domain (P4, when the coding domain is smaller than the coding region of the coding sequence of the first coding sequence of the coding sequence 4, the coding sequence of the coding sequence shown as the coding sequence of the coding sequence 4, the coding sequence of the coding sequence 4, the coding sequence of the coding sequence 4, the coding sequence of the coding sequence 4, the coding sequence of the coding sequence of the coding sequence of the sequence of the coding sequence of the coding sequence 4 or coding sequence of the coding sequence of the sequence of sequence 4 or the sequence of the sequence of.

The constructs are statistically analyzed to determine which portions are most effective for specific portions of CD 053, and the results are set forth in Table 3, the cells transduced with the polynucleotides encoding the lymphoproliferative components are shown in example 10, the intracellular signaling domains derived from the intracellular signaling domains of CSF2 and TNFRS 032 and CD 032, the CD 053, and the CD 05186, and the CD 053, the CD 05186 and the CD 032, the CD 053, the CD 05186 and the CD 053, the CD 05186, and the CD 053, the CD 05186, the CD 053, the CD 052, the CD 05186, the CD 052, the CD 054, the CD 053, the CD 054, the CD 05186, the CD 053, the CD 054, the CD 05186, the CD 054, the CD 05186, the CD 054, the CD 05186, the CD 054, the CD 05186, the CD 054, the CD 054, the CD 054, the CD 054, the CD 05186, the CD 054, the CD 054, the CD 05186, the CD 054, the CD 054, the CD 05186, the CD 05186, the CD 054, the CD 053, the CD 05186, the CD 053, the CD 053, the CD 053, the CD 053, the CD 053, the CD 053, the CD 054, the CD 053, the CD 053, the CD 053, the CD 053, the CD 05186, the CD 053, the CD 053, the CD 053, the CD.

In certain illustrative embodiments, the lymphoproliferative component comprises an interleukin receptor or fragment thereof comprising a signaling domain that activates the Jak/STAT5 pathway for example, this lymphoproliferative component can comprise the intracellular domains of I L21R, I L27 3527 27R, I L31 RA, L IFR, and OSMR as shown in the experiments of examples 11 and 12 and tables 8-18, chimeric lymphoproliferative components comprising the intracellular domains of these genes were found in candidate chimeric polypeptides that induced the highest degree of proliferation in PBMCs cultured in the absence of exogenous interleukins such as I L-2.

In some embodiments, lymphoproliferative component the lymphoproliferative component can comprise an endodomain that is a interleukin or a interleukin receptor and is part of the optimal construct identified in libraries 1A, 1.1A, 2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B in examples 11 and 12 (tables 8-18). In some embodiments, lymphoproliferative component the lymphoproliferative component can comprise an endodomain that is an interleukin receptor and is part of the optimal construct identified in libraries 1A, 1.1A, 2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B in examples 11 and 12 (tables 8-18). In some embodiments, lymphoproliferative component the lymphoproliferative component can comprise an intracellular domain that includes at least one ITAM motif and is part of the optimal construct identified in banks 1A, 1.1A, 2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B of examples 11 and 12 (tables 8 to 18). In some embodiments, the lymphoproliferative component can comprise one of the following endodomains that are part of a roof construct in at least one of the banks 3A, 3.1A, 3B, 3.1B, 4B, or 4.1B.

In illustrative embodiments, lymphoproliferative component the lymphoproliferative component may comprise an intracellular domain from I L7R, I L12 RB1, I L15 RA, or I L27 RA, which is present in constructs that show particularly noticeable enrichment (i.e., cause the highest degree of hyperplasia) in the initial and repeat screens as detailed in examples 11 and 12 (tables 20-24).

In illustrative embodiments, the lymphoproliferative component may comprise an intracellular domain from an interleukin receptor selected from the group consisting of CD27, CD40, CR L F2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR2, I L1R L, I L RA 22, I L RG, I L RA 55, I L3677L RB, I L RA, I L RB L, I L RA L, I L RB 315, I L RB 20RB, I L RA L, I L RA 27RA, I L RA, I L RA, MP 72, MP L, I L R L, I L RB 618, I L RB L, I L RB L, TNFRSF 3624, and other screening constructs as shown in particular in the initial screening constructs and in the detailed in the TNFR.

In illustrative embodiments, lymphoproliferative component the lymphoproliferative component may comprise an intracellular domain from CD3D, CD3E, CD3G, CD79A, CD79B, FCER1G, FCGR2A, or FCGR2C, which includes at least one ITAM motif and is present in constructs that show particularly noteworthy enrichment in primary and repeat screens as detailed in examples 11 and 12 (tables 20-24).

In some embodiments, the lymphoproliferative component of this paragraph, which is shown in examples 11 and 12 to be active in a construct having only a single endodomain, can be an endodomain of a lymphoproliferative component having two or more endodomains, or in illustrative embodiments, a single endodomain (i.e., the lymphoproliferative component does not comprise two or more endodomains). in illustrative embodiments, the endodomains in a lymphoproliferative component comprise domains from CD, CR F, CSF2, CSF3, FCGR2, IFNAR, IFNGR, I1R, I03 RA, I17 RB 210RA, I311 RA, I RB 412, I RA, I618 RAP, I731 RA, MP 8, MYD, TNFRSF or TNFRSF, which are present in a construct shown in the initial and repeat screening assays in example 12 (tables 23 and 24) as a construct which includes at least one of the lymphoproliferative component with a proliferative component endoplasmic receptor proliferative component (IRF) from the initial screening domain, IRR 23, IRRA, or TNFRSF, which are present in illustrative embodiments, and IRRA, which are present in an EPCR 23, which are present in embodiments, which are present in which are shown in an example as a construct, which is shown in an EPR 2, an EPCR 23, an EPR 23, an EPCR 23, which is shown in an example, an EPR 23, which is shown in an EPR 23, an EPR-enriched for example, an EPR-LRP-enriched for which is shown in which is an EPR-23, an EPR-LRP-23, an EPR-23, or a cell-enriched for example, or an EPR-enriched for example, or a cell-enriched for which is shown in which is.

In illustrative embodiments, the lymphoproliferative component comprises a co-stimulatory domain from CD27, CD28, OX40 (also known as TNFRSF4), GITR (also known as TNFRSF18), or HVEM (also known as TNFRSF14) present in constructs that show particularly noteworthy enrichment in initial and repeated screening as detailed in examples 11 and 12 (tables 20 to 24.) in some embodiments, the lymphoproliferative component comprising a co-stimulatory domain from OX40 does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BT 24, GITR, or HVEM in some embodiments, the lymphoproliferative component comprising a co-stimulatory domain from GITR does not comprise an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, CD BT 2, or HVEM in some embodiments, the lymphoproliferative component comprising a co-stimulatory domain from CD28 does not comprise an intracellular domain from CD3, CD28, CD-1 BB, ICOS, CD 8672, CD 368672, the co-stimulatory domain from CD 368653, CD 368672, or HVEM comprises a co-stimulatory domain from CD 368672 in some embodiments, CD 368653, CD 368672, CD 368653, CD 8672, CD 368672, CD 8672, or CD 8653.

In some embodiments, the lymphoproliferative component may be one of the following constructs, M024-S190-S047, M025-S050-S197, M036-S170-S047, M012-S045-S048, M049-S194-S064, M025-S190-S050, M025-S190-S05, E013-T041-S186-S051, E013-T028-S186-S051, E014-T015-S186-S051, E011-T016-S186-S050, E011-T073-S-S or E013-T011-S186-S211, all of which stimulate the proliferation of resting lymphocytes after transduction as shown in example 13 (FIGS. 19 to 20). in certain embodiments, the lymphoproliferative component comprises a My CD 3635, I R, I L, ICOS 2, ICOS 4628, ICOS 2, CD L, CD 3635, or CD 3635 domain from My CD 387 or My 3, I3535.

In some embodiments, the lymphoproliferative component can be the construct E013-T041-S186-S051, which stimulates proliferation of resting lymphocytes after use of the replication deficient recombinant retroviral particles displaying UCHT1 scfvffc-GPI as shown and analyzed in example 16 (fig. 22A and 22B.) in other embodiments, the lymphoproliferative component is I L7-I L7 RA-I L2 RB, as shown and analyzed in example 16.

As detailed in example 11, constructs with domains from the interleukin receptors CD27, I L1R L1, I L6R, I L31 RA, TNFRSF4, or TNFRSF18 for both libraries 1A and 1.1A (table 20) show particularly noteworthy enrichment in both libraries 1A and 1.1A.

As detailed in example 11, constructs with domains from the interleukin receptors CD40, IFNGR2, GHR, I L10 RB, I L11 RA, I L13 RA2, I L17 RB, I L22 RA1, TNFRSF14, or TNFRSF9 showed particularly noteworthy enrichment in both libraries 2B and 2.1B (table 21) for libraries 2B and 2.1B.

As detailed in example 12, constructs with domains from the interleukin receptors CD27, CD40, CSF2RA, MP L, OSMR, TNFRSF4, or TNFRSF18 for both libraries 3A and 3.1A (table 22) show particularly noteworthy enrichment.

As detailed in example 12, constructs with domains from the interleukin receptors CD, CR F, CSF2, CSF3, EPOR, IFNAR, IFNGR, I1R 01, I12 RG, I23 RA, I35 RA, I46 57 69 RB, I811 RA, I912 RB, I13 RA, I013 RA, I115 RA, I18 RAP, I20 RB, I27 RA, I31 RA, EPR, MP, OSMR, PR, TNFRSF, or TNFRSF for both libraries 3B and 3.1B (table 23) showed particularly noteworthy enrichment.

As detailed in example 12, constructs with domains from the interleukin receptors CD27, CD40, CR L F2, CSF2RA, CSF3R, EPOR, IFNAR2, IFNGR2, I L1R 1, I L02 RA, I L13 RA, I L22 RG, I L36R, I L7R, I L10 RB, I L11 RA, I L31 RA, I L13 RA1, I L13 RA2, I L18R 1, MP L, OSMR, TNFRSF4, TNFRSF9, or TNFRSF18 showed particularly notable enrichment in both libraries 4B and 4.1B (table 24).

In certain illustrative embodiments, lymphoproliferative component the lymphoproliferative component comprises the intracellular domains of CD40, MP L, and I L2 Rb, which are demonstrated in the examples herein to promote PBMC proliferation.

In some embodiments, the lymphoproliferative component may not be an interleukin receptor. In some embodiments, a lymphoproliferative component other than an interleukin receptor may comprise an intracellular signaling domain from: CD2, CD3D, CD3G, CD3Z, CD4, CD8RA, CD8RB, CD28, CD79A, CD79B, FCER1G, FCGR2A, FCGR2C or ICOS. Exemplary embodiments of chimeric lymphoproliferative components comprising these recited genes are provided in examples 11 and 12 and the tables cited therein.

In some embodiments, C L E is not I L-15 linked to the I L-2/I L-15 receptor.

In some of the methods and compositions disclosed herein, expression of the lymphoproliferative component is induced by, and may even rely on, binding of a compound to a control component (as discussed in WO2017/165245a2, WO2018/009923a1, and WO2018/161064a 1), which in non-limiting embodiments is a riboswitch.

For example, I L-7R, which binds I L7 more strongly in a neoplastic environment than in a normal physiological environment, can be used.

In some embodiments, the lymphoproliferative component is fused to a recognition domain or an ablation domain. Such identification or elimination domains are disclosed in greater detail herein. This fusion provides the advantage that, especially when using truncated or otherwise mutated lymphoproliferative component lymphoproliferative components, less polynucleotide is required in the retroviral genome. This is important in the illustrative embodiments provided herein because it helps to allow more nucleic acid encoding a functional component to be included in the retroviral genome and because it adds a mechanism by which cells expressing the lymphoproliferative component can be killed if their proliferation is no longer required or is otherwise detrimental to the organism.

In some illustrative embodiments, the lymphoproliferative component (including C L E) comprises an intracellular activation domain as disclosed herein above, in some illustrative embodiments, the lymphoproliferative component is C L E comprising an intracellular activation domain comprising an ITAM-containing domain, and thus, C L E may comprise an intracellular activation domain having at least 80%, 90%, 95%, 98%, or 100% sequence identity to a CD3Z, CD3D, CD3E, CD3G, CD79 DAP G, FCERlG, FCGR 2G, DAP G/CD G, or ZAP G domain provided herein, wherein the C G E does not comprise an astr, in certain illustrative embodiments, the intracellular activation domain is from an ITAM-containing domain from CD3G, CD3, CD G, CD 3679, or a CD 3679 cell comprising an icam-containing domain that is effective in promoting proliferation in certain illustrative embodiments when the cell culture is performed in a cell comprising an intracellular activation domain from CD3, CD G, fce G, CD 3679, and an exogenous C3619, CD G, CD 3679, or a cell comprising an intracellular activation domain provided herein.

In some embodiments, one or more domains of a lymphoproliferative component is fused to a regulatory domain (such as a co-stimulatory domain) and/or an intracellular activation domain of the CAR. In some embodiments, one or more endodomains of the lymphoproliferative component can be part of the same polypeptide as the CAR or can be fused or optionally functionally linked to some component of the CAR. In yet other embodiments, the engineered signaling polypeptide may include an ASTR, an intracellular activation domain (such as a CD3 zeta signaling domain), a costimulatory domain, and a lymphoproliferative domain. Additional details regarding co-stimulatory domains, intracellular activation domains, ASTRs, and other CAR domains are disclosed elsewhere herein.

In the illustrative examples herein, the T cell and/or NK cell survival module is typically introduced into resting T cells and/or resting NK cells by transducing the resting T cells and/or resting NK cells with a replication-defective recombinant retroviral particle whose genome encodes the T cell and/or NK cell survival module as part of an engineered signaling polypeptide. Lymphoproliferative component in some embodiments, the lymphoproliferative component is also a T cell and/or NK cell survival component. As discussed above, lymphoproliferative components some of the lymphoproliferative components not only promote proliferation, but they also promote cell survival. In some embodiments, the T cell and/or NK survival cell motif is not a lymphoproliferative component, a lymphoproliferative component. In some embodiments, the T cell and/or NK cell survival motif can be a CD 28T cell survival motif or a CD137 cell survival motif. These T cell survival motifs can be found in engineered signaling polypeptides including ASTRs (such as scFv). In an illustrative embodiment, the T cell survival motif is a CD 28T cell survival motif or a CD137 motif linked to an scFv via a CD8a transmembrane domain or a CD28 transmembrane domain. In certain embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM). In one embodiment, the polypeptide sequence is a CD3 zeta signaling domain.

In some embodiments, the lymphoproliferative component is not a polypeptide, but instead comprises an inhibitory RNA in some embodiments, methods, uses, compositions, and products of processes according to any aspect herein include a lymphoproliferative component comprising an inhibitory RNA and a lymphoproliferative component that is an engineered signaling polypeptide in embodiments in which the lymphoproliferative component is an inhibitory RNA, the inhibitory RNA can be a miRNA that stimulates activation of STAT5, typically by using degradation or down-regulation of a negative regulator in the SOCS pathway, in some embodiments, the miRNA refers to an mRNA encoding a protein that affects proliferation, such as but not limited to ABCG1, SOCS1, TGFbR2, SMAD2, cCB L, and pd1. in illustrative embodiments, as exemplified herein, this inhibitory RNA (e.g., miRNA) can be positioned in a packaging cell and/or replicating recombinant virion genome and/or in an intron vector, and/or in teaching embodiments, such as described herein, this inhibitory RNA (e.g., miRNA) can be positioned in a packaging cell and/or replicating recombinant virion particle genome and/or a retroviral vector, thus, the expression of a single miRNA-containing promoter can be expected to increase the activity of a single miRNA, such as a miRNA-containing promoter, or miRNA, e.g., a miRNA-expressing promoter in a cell, such as a retrovirus-expressing a cell (e.g., a retrovirus) in a cell, such as a cell, a cell expressing a retrovirus vector, e.g. comprising no cell expressing a gene, e.g., a gene, e.g., a cell expressing a retrovirus vector, a gene, such as a cell, a cell expressing a retrovirus vector, e.g., a cell expressing a cell.

ABCG1 is an ATP-binding cassette transporter that down-regulates thymocyte and peripheral lymphocyte proliferation (Armstrong et al 2010.J Immunol 184(1): 173-183).

SOCS1 is a member of the SOCS (inhibitor of interleukin signaling) family that inhibits negative regulators of interleukin signaling of the Jak/Stat pathway, such as Stat 5. SOCS1 is also known as JAB (Janus kinase binding protein), SSI-1 (Stat-induced Stat inhibitor-1), and TIP3(Tec interacting protein).

TGFbR2 is a member of the serine/threonine protein kinase family that binds to TGF- β, forming a complex that phosphorylates proteins, which then enter the nucleus and regulate transcription of genes associated with hyperplasia.

SMAD2 mediates signals that convert growth factor (TGF) - β and regulates a number of cellular processes such as cell proliferation, apoptosis, and differentiation.

cCB L is an E3 ubiquitin ligase that inhibits TCR signaling by dephosphorylating and inactivating ZAP-70 and internalizing through the TCR.

PD1(CD279) is a cell surface receptor expressed on T cells and ProB cells PD-1 binds two ligands, PD-L1 and PD-L2 act to prevent cell activation by signaling through PD-1.

In some embodiments, a lymphoproliferative component herein is a polypeptide comprising an intracellular region of any one of the genes of table 19. in some embodiments, a lymphoproliferative component comprises or is an intracellular domain identified in a chimeric polypeptide of table 19. in these embodiments, a lymphoproliferative component can be a polypeptide that is a chimeric polypeptide (see C L E, infra), or is not C L E, but comprises or is an intracellular domain of a gene identified in the P3 (first intracellular domain) position of table 19. table 19 identifies a C L E construct that promotes proliferation of PBMCs between day 7 and the absence of a second intracellular domain on the construct.

In some embodiments, the lymphoproliferative component comprises MP L or is MP L or a variant or fragment thereof, including the intracellular domain of MP L with or without the transmembrane domain and/or extracellular domain of MP L0, and/or a variant or fragment having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the intracellular domain of MP L with or without the transmembrane domain and/or extracellular domain of MP L, wherein the variant and/or fragment retains the ability to promote cell proliferation of PBMCs (and, in some embodiments, T cells). in illustrative embodiments, the lymphoproliferative component comprises the intracellular domain of MP L or a variant or fragment thereof, including at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the intracellular domain of MP 635 or a variant or fragment thereof, including at least 75%, 85%, 90%, 95%, 96%, 97%, or 100% of the intracellular domain of MP L, including the variants or fragments of the lymphoproliferative component of the interleukin L, including the variants or fragments thereof, including the variants of the variants or fragments of the variants of the interleukin 6395%, 95%, 97%, 98%, or 100% of the intracellular domain of the lymphoproliferative component of the intracellular domain of the cell containing the herein described herein, 95%, including the thrombopoietin, 95%, or 100% of the thrombopoietin, 95%, or 100% of the intracellular domain of the cell containing the intracellular domain of the cell, 95%, or 100% of the.

In some embodiments (which provide separate aspects of the invention), provided herein are chimeric polypeptides that are chimeric lymphoproliferative component lymphoproliferative components (C L E) and isolated polynucleotides and nucleic acid sequences encoding the same C L E may include any of the domains and/or domains derived from particular genes discussed in this section similarly, isolated polynucleotides and nucleic acid sequences encoding C L E may encode any of the domains and/or domains derived from particular genes discussed in this section as part of C L E exemplary C L E is illustrated in fig. 15-18C L E herein promotes proliferation of T cells and/or NK cells and may also promote survival of T cells and/or NK cells in the case of a T cell and/or NK cell, some C L E promotes proliferation and may also promote survival of other types of PBMC (e.g., B cells) in the case of a CAR provided herein is referred to as a convenient design C L E in embodiments and may include at least one of the C L domains found in a chimeric polypeptide from a chimeric polypeptide of the same family as a chimeric polypeptide, and/or a chimeric polypeptide that is not a chimeric polypeptide that is found in the case of a third, a chimeric polypeptide, a chimeric.

Without being limited by theory, none of these C L es are designed to promote proliferation and optionally cell survival of B cells, NK cells, and/or T cells in a constitutive manner (i.e., ligand binding is not required for activation.) none of C L es are found in nature, and many C L es have components that are not normally expressed in vivo in B cells, T cells, and/or NK cells, and/or some candidate C L es are not generally known to specifically promote cell proliferation and/or cell survival signaling of B cells, T cells, and/or NK cells.

In addition, separate aspects of the invention are provided that specifically include C L E, including, for example, isolated chimeric lymphoproliferative component polypeptides, isolated polynucleotides and nucleic acids encoding the same, and vectors including plasmids, viruses and retroviral vectors (including these nucleic acid sequences or isolated polynucleotides), including methods for transducing or transfecting PBMCs with the isolated polynucleotides and vectors comprising the same (such as B cells, or specifically T cells and NK cells), these cells may be isolated unmodified cells, or they may be modified cells, such as genetically modified cells, such as CAR-expressing cells or TCR cells.

As examples, the lymphoproliferative component as provided herein generally comprises a transmembrane domain, the transmembrane domain may have 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with any of the transmembrane domains from the following genes and representative sequences, CD8 β (SEQ ID NO:47), CD4(SEQ ID NO:48), CD3 zeta (SEQ ID NO:49), CD28(SEQ ID NO:50), CD134(SEQ ID NO:51), CD7(SEQ ID NO:51), CD2(SEQ ID NO:322), CD 32 (SEQ ID NO:323), CD 32 (SEQ ID NO:324), CD3 RB 72(SEQ ID NO:325), CD3 RB 72(SEQ ID NO:326), CD2 (2) NO: 78), SEQ ID NO:78 (36NO: 78), SEQ ID NO: 78) or a transmembrane No. (36NO: 2), the transmembrane No. (36NO: 33 (36NO: 33), the polypeptide as disclosed herein, the TNFRNO: 2 receptor ID NO: 2, the TNFRNO: 2, the TNFRID NO (36NO: 2, the TNFRNO: 36NO: 150) receptor (36NO: 150), the TNFRNO: 36NO (36NO: 150, the TNFRNO: 36NO: 150, the TNFRNO: 150 receptor ID NO: 36NO: 150, the polypeptide as shown in SEQ ID NO (36NO: 150) as shown in SEQ ID NO: 150, the TNFRID NO: 2, the TNFRNO: 36NO: 150, the TNFRNO: 36NO: 150, the TNFRNO (36NO: 150, the invention as shown in SEQ ID NO: 36NO: 150, the TNFRNO: 36NO: 150, the TNFRNO: 36NO: 150, the TNFRNO: 36NO: 150, the antisense NO: 150, the TNFRNO: 36NO: 150, the accessory NO: 36NO: 150, the TNFRNO: 36NO: 150, the accessory NO: 36NO: 150, the accessory 36NO: 150, the TNFRNO: 150, the accessory 36NO: 150, the.

In some embodiments, C L E includes both an extracellular portion and a transmembrane portion from the same moiety (in the illustrative embodiment, the same receptor), one of which is a mutant in the illustrative embodiment, thus forming an extracellular domain and a transmembrane domain.

One aspect of the transmembrane domains provided herein that utilize receptor mutants is an isolated polynucleotide comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

(a) an extracellular domain and a transmembrane domain from an interleukin receptor or a hormone receptor, wherein at least one of the extracellular domain and the transmembrane domain comprises a mutation found on a constitutively active mutant of the interleukin receptor, and wherein the extracellular sequence does not bind a ligand of the interleukin receptor; and

(b) a first endodomain of a gene selected from the group consisting of genes having a first endodomain and optionally a second endodomain of selected polypeptides identified in tables 8 to 12, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells.

The extracellular region of this extracellular domain and transmembrane domain in these embodiments is typically long enough to form a connector, in illustrative embodiments a flexible connector between the transmembrane domain and another functional peptide region, such as a gap domain, which in some embodiments is connected to the amino terminus of the extracellular region. Thus, the extracellular region, when present to form the extracellular and transmembrane domains, may be between 1 amino acid and 1000 amino acids in length, and typically between 4 amino acids and 400 amino acids, between 4 amino acids and 200 amino acids, between 4 amino acids and 100 amino acids, between 4 amino acids and 50 amino acids, between 4 amino acids and 25 amino acids, or between 4 amino acids and 20 amino acids. In one embodiment, the extracellular region is GGGS for the extracellular and transmembrane domains of this aspect of the invention.

As discussed in more detail below, the transmembrane domain is typically at least long enough to cross the plasma membrane, whether as part of an embodiment comprising an extracellular domain and a transmembrane domain (as one part shown, for example, as banks 1A, 1.1B, 2B, and 2.1B of example 11) or as part of an embodiment comprising an extracellular dimerization motif (as shown as banks 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B of example 12). Thus, the transmembrane region of the transmembrane domain or extracellular and transmembrane domains may be between 10 and 250 amino acids, and more typically is at least 15 amino acids in length, and may for example be between 15 and 100 amino acids, between 15 and 75 amino acids, between 15 and 50 amino acids, between 15 and 40 amino acids or between 15 and 30 amino acids.

Exemplary ectoand transmembrane domains of C L E, including embodiments of these domains (in the illustrative embodiment, ectodomains) are typically less than 30 amino acids from the membrane proximal ectodomain together with transmembrane domains from mutant receptors reported to be constitutive, which do not require ligand binding for activation of the relevant ectodomain in the illustrative embodiment, these ectoand transmembrane domains include I L RA Ins PPC L, CR L F2F 232C, CSF2RB V449E, CSF3R T640N, EPOR L C I252C, GHRE260C I270, I L RA F523C, and MP L s505n additional non-limiting examples of such ectoand transmembrane domains are provided in table 7 and exemplified in examples 11 and corresponding tables, in some embodiments, the ectoand transmembrane domains are not included in the sequence with the ectoand/or some of the ectoand/or transmembrane domains of the same portion of the ectoand/or transmembrane domains as the ectodomain of I L RA or mutant thereof in examples 11 and corresponding tables, some embodiments, the ectoand transmembrane domains are not included in the sequence of seq id 5, 15, or more than the ectodomain of PPC 15 and/or 15.

Additional exemplary transmembrane domains are provided in example 12. These transmembrane domains in the illustrative examples are from type I transmembrane proteins.

Accordingly, provided herein in one aspect is an isolated polynucleotide comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first endodomain of a gene selected from the group consisting of endodomains of selected polypeptides identified in tables 13 to 18, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells.

In illustrative embodiments, the chimeric polypeptides promote cell proliferation of PBMCs (e.g., B cells and/or NK cells, and/or in illustrative embodiments, T cells). as demonstrated in example 12, these chimeric polypeptides are capable of promoting cell proliferation of PBMCs in the absence of exposure of PBMCs to exogenous interleukins (such as I L-2, I L-15, or I L-7) during culture (e.g., in the absence of culture medium added I L-2 to PBMCs expressing nucleic acids encoding the chimeric polypeptides), for these embodiments, as illustrated in example 12, I L-2 may be added during transduction of PBMCs but in subsequent culture, for example, the chimeric polypeptides disclosed herein are lymphoproliferative component lymphoproliferative components, as they are capable of promoting cell proliferation and optionally survival of PBMCs (and in illustrative embodiments, T cells) after transduction with nucleic acids encoding the chimeric lymphoproliferative components, and optionally after culture for example, the cells are transduced at day 1, day 2, and optionally the proliferative component proliferation activity of these PBMCs is measured by a method of proliferating cell expression assay for proliferative cells on day 13.

In one embodiment of this aspect, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In illustrative embodiments of this aspect, the ectodomain 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.

In embodiments of any of these aspects in which the transmembrane domain is a type I transmembrane protein, the transmembrane domain may be a type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF family receptor. In one embodiment 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 interleukin receptor, a hormone receptor, a T cell receptor, or a TNF family receptor.

In some embodiments, the transmembrane domain is from CD4, CD8RB, CD40, CR L F2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, I L1R 1, I L01 RAP, I L RG 12, I L RA, I L ST, I L RA, I L RB, I L711 RA, I L RA L, I L RA 917RA, I L RB, I L017 RC, I L RE, I L R L, I L318, I L RA, I L PR 72R L, I L PR 72RA, I L PR 72R, I L PR 72R, or a transmembrane domain that, when provided as a transmembrane signaling construct, or a transmembrane signaling promoter, or a transmembrane domain, or a transmembrane domain, a transmembrane signaling construct, or a transmembrane domain from a cell.

Exemplary transmembrane domains from this modality are shown as P2 in tables 13 to 18 for pool 3A, when found at transmembrane domain position (P2) of candidate chimeric polypeptide modules herein, transmembrane domains from CD40, CD8B, CR L F2, CSF2RA, GCGR2C, ICOS, IFNAR C, IFNGR C, I C RB, I C R C, I C RAP, I C RA, C EPR and PR C R (P C) or mutants thereof known to promote constitutive signaling activity in certain cell types when these mutants are present in the transmembrane domain constructs provided in example 12, when considering in combination the data of all constructs having a transmembrane domain derived from this gene, promoting proliferation of PBMCs between day 7 and day when the data of a transmembrane domain having a structural domain derived from this gene is provided in the screening examples 11 and 12, the screening constructs provided for example 1, or a recombinant dna construct from a cell type of which is capable of promoting proliferation in the screening CD1, or a cell type of a cell type which is not capable of providing a proliferation in a cell type of proliferation, or a cell type of proliferation in which is capable of cell type of cell proliferation when cell type of cell proliferation, cell type of proliferation, cell type of proliferation, cell type of cell type no cell type of proliferation, cell type of proliferation, cell type of cell type P C, P363, P C, P.

In some embodiments of any aspect herein, the chimeric polypeptide comprises a transmembrane domain identified in any one of the constructs shown in tables 13 to 18 in addition to transmembrane domain mutant V449E of CSF2 RB.

In some embodiments, the ectodomain and transmembrane domain are viral protein MPs or mutants and/or fragments thereof, MP is a multi-spanning transmembrane protein known to activate cell signaling independently of ligand when targeting lipid rafts or when fused to CD (Kaykas et al, EMBO j.20:2641(2001)) fragments of 0MP are typically long enough to cross the plasma membrane and activate the linked intracellular domain, for example, 1MP 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 3 include AT least one or more of the transmembrane domains thereof that when attached to a transmembrane receptor d, CD T, CD receptor d, and a T, CD receptor d, and a, which are typically comprise one or a receptor d, a receptor d, a.

In other embodiments of C L E provided herein, the extracellular domain comprises a dimeric portion, many different dimeric portions disclosed herein may be used in these embodiments, in illustrative embodiments, the dimeric portion is capable of homodimerization, without being limited by theory, the dimeric portion may provide an activation function for the intracellular domain connected thereto via the transmembrane domain.

For example, for leucine zipper dimers, these leucine zippers are capable of forming dimers because they retain leucine motifs that are 7 residues apart along α, however, the leucine zipper moieties of certain embodiments of C L E provided herein may or may not retain their DNA binding function.

An exemplary extracellular domain of this type is a leucine zipper domain from Jun moieties (such as c-Jun). For example, the ectodomain may be a variant of c-Jun found in NM _002228_3 (table 7). This extracellular domain was used in the constructs discussed in example 18.

Without being limited by theory, it is believed that the alanine spacer affects signaling of an intracellular domain linked to the extracellular domain of leucine zipper via a transmembrane domain by altering the orientation of the intracellular domain.

The first and second endodomains of the C E provided herein are intracellular signaling domains of genes known to be in at least some cell types to promote proliferation, survival (anti-apoptosis) and/or provide co-stimulatory signals that enhance differentiation status, proliferation potential or resistance to cell death, therefore, these endodomains can be the endodomains from lymphoproliferative component lymphoproliferative disorder and the co-stimulatory domains provided herein some of the endodomains of the known candidate chimeric polypeptides can activate JAK/JAK signaling, JAK, STAT, 54, from IFNAR, ifnr, IFN, I02 RB, I14 RB, I25 RB, I36 RB, I57 RB, I69 RA, I710 RA, CSF 927 RA, 0IFR, and statr the endodomains from ifra 1, stacsf 2, CSF3, CSF R, EPOR, epora 23, 0IFR, mr 821 RA, mr 13, mr 821, mr 18, mr 7, mr.

Exemplary intracellular domains include those from the constructs listed in tables 8 to 12 exemplary intracellular domains from these genes empirically determined to be active in the experiments provided in example 11 are provided in the first and second intracellular domain (P) positions of the tables provided in example 11 and as further listed in this section below illustrative intracellular domains identified in the best hits screened for example 11 include CD, CSF2, IFNAR, I1 RAP, I4 ST, I111 RA, I212 RB, I317 RA, I417 RD, I517 RE, I618R, I721 823 9 and MyD88 illustrative intracellular domains identified in the best hits screened for example 12 include CD, EPR, MyD, ifr, MP 0, I118R, I213 RA, I310 RB, I23R or ifra in some illustrative embodiments the first illustrative intracellular domain is provided as a CD, ifr, CD 19 or non-proliferative domain linked to the first or second intracellular domain in these examples 13, including non-proliferative domains provided in the non-proliferative components of these examples 19, including CD 19, CD 19 or CD 19.

In certain illustrative embodiments, the second endodomain is from CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4, and TNFRSF8, or a mutant thereof known to promote signaling activity in certain cell types when such mutants are present in the construct provided in example 12. In certain illustrative embodiments, the second intracellular domain is from CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including in illustrative examples the intracellular domains of CD40, CD79B, and CD 27. For these certain illustrative embodiments, non-limiting exemplary first intracellular domains (P3) are those linked to a gene of CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including in illustrative examples the intracellular domains of CD40, CD79B, or CD27 in tables 13-18, including (as non-limiting examples) the first intracellular domain and/or the second intracellular domain of those genes provided in these tables. For these certain illustrative embodiments, other non-limiting exemplary first intracellular (P3) domains are those linked to genes of CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, the intracellular domains of CD40, CD79B, and CD27 second intracellular domains of the tables including these genes as P3 provided in example 12 in the illustrative examples, including (as non-limiting examples) the first intracellular domain and/or the second intracellular domain of those genes provided in these tables.

In illustrative embodiments, the first intracellular domain is from MP L and the second intracellular domain is from OX40, CD40, CD79A or cd79b. as shown in example 10, the intracellular domains from these genes are shown to significantly increase proliferation in at least one of pool 3.1 or pool 4.1 when the second intracellular domain which is a chimeric lymphoproliferative component which contains MP L as a first intracellular domain comprises a chimeric intracellular domain of the intracellular signaling domain which is shown to significantly increase proliferation in at least one of pool 3.1 or pool 4.1, in illustrative embodiments, the first intracellular domain is from MP L and the second intracellular domain is from SEQ 40, CD40 or cd79b. in some embodiments, the first intracellular domain contains a sequence from S186(SEQ ID NO:491) or a variant or fragment thereof which promotes signaling, and the second intracellular signaling domain contains a signal from SEQ ID No. 26 or SEQ ID No. 27 or No. 2 as a chimeric signal domain which is shown to No. 26, or No. 2 as a chimeric signal domain which is shown to No. 2, or No. 5 as a chimeric signal domain which contains a signal domain which is shown to No. 5 or No. 5 or No. 3 or No. 1 or No. 3 or No. 1 or No. NO.

The detailed information (including sequence information) about the exemplary intracellular domains of the above genes identified in examples 11 and 12 in the construct promoting cell survival and proliferation is provided in table 7 the intracellular domains that can be included in the embodiment of C L E include mutants and/or fragments thereof that promote proliferation, survival (anti-apoptosis) and/or provide a co-stimulatory signal that potentiates differentiation status, proliferation potential or resistance to cell death, the intracellular domains include at least one of the chimeric domains that promote proliferation, the intracellular signaling domains that promote proliferation, the intracellular signaling domains that promote, the intracellular proliferation, or the intracellular signaling domains that promote proliferation, the intracellular signaling domains that promote, the proliferation, the intracellular domains that promote, the proliferation, or the intracellular domains that promote proliferation, the intracellular signaling domains that promote the proliferation, the intracellular domains that promote the proliferation, the intracellular domains that promote the proliferation, the intracellular domains that promote the proliferation, the intracellular domains that promote the proliferation, or the intracellular domains that promote the proliferation, the intracellular domains that promote the proliferation, or the proliferation, the intracellular domains that promote the proliferation, the intracellular domains that promote the intracellular, or the intracellular, the intracellular domains that promote the intracellular, or the intracellular domains that promote the intracellular domains of the intracellular fusion, or the intracellular fusion, or the intracellular fusion, comprise at least one or the intracellular fusion, or the intracellular fusion, or the intracellular fusion, or the intracellular fusion, or the intracellular fusion.

In some embodiments, all of the domains of C L E are not the I L-7 receptor or mutants thereof, and/or fragments thereof having at least 10, 15, 20, or 25 contiguous amino acids of the I L-7 receptor, or are not the I L-15 receptor or mutants thereof, and/or fragments thereof having at least 10, 15, 20, or 25 contiguous amino acids of the I L-15 receptor.

In illustrative embodiments, C L E comprises an identification domain and/or an elimination domain the details regarding identification domains and/or elimination domains are provided in other sections herein any of the identification domains and/or elimination domains provided herein may be part of C L E.

In illustrative embodiments, as illustrated in fig. 15 and 16, C L E provided herein is co-expressed with a CAR that can be designed according to any of the CAR embodiments provided herein or otherwise known in the art.

Some embodiments provided herein are isolated polynucleotides and nucleic acid sequences encoding any of the C L E provided herein, as exemplified in fig. 15-18, these isolated polynucleotides may include any components known in the art for providing expression of a transcript, such as a transcript encoding C L E, for example, the isolated polynucleotides may include a promoter component that is active in B cells, T cells, and/or NK cells, these promoters that are suitable for these embodiments are known in the art, some of which are recognized in other embodiments of the invention, for example, the skilled artisan will understand as discussed in more detail in examples 11 and 12, how to relate to isolated polynucleotides and vectors containing the same, for expressing any of the C850E embodiments provided herein, for example, the Kozak sequence is provided within 10 nucleotides of the ATG initiation site, the initiation site encodes C L E1 or is located on the same upstream of the multiple nucleotide sequence of the multiple terminator sequences of the sequence of the C850E embodiments, the multiple terminator sequences may be provided in multiple nucleotide sequences disclosed in embodiments, the multiple nucleotide sequences disclosed in multiple embodiments, including multiple nucleotide sequences upstream terminator sequences of the multiple nucleotide sequences disclosed in multiple embodiments, including multiple nucleotide sequences disclosed in multiple embodiments, multiple nucleotide sequences upstream of the upstream terminator sequences of the multiple nucleotide sequences of the gce 5, multiple nucleotide sequences disclosed in multiple embodiments, including multiple nucleotide sequences of the upstream terminator sequences of the multiple nucleotide sequences disclosed herein, the upstream terminator sequences of the multiple nucleotide sequences of the invention, the multiple nucleotide sequences of the multiple nucleotide sequences disclosed in multiple embodiments, the invention, including multiple embodiments, the multiple nucleotide sequences of the multiple nucleotide sequences of the upstream terminator sequences of the multiple embodiments, No. 5, No. 8, No. 5, No. 8, multiple embodiments, No. 5, multiple embodiments include multiple nucleotide sequences disclosed in multiple embodiments, multiple nucleotide sequences of the multiple nucleotide sequences include multiple nucleotide sequences of the multiple embodiments, multiple nucleotide sequences of the multiple embodiments, multiple nucleotide sequences of the multiple nucleotide sequences disclosed in multiple nucleotide sequences of the multiple nucleotide sequences disclosed in multiple embodiments, multiple nucleotide sequences of the multiple embodiments, multiple nucleotide sequences of the upstream terminator sequences of the multiple nucleotide sequences of the multiple embodiments, multiple nucleotide sequences of the upstream terminator sequences of the multiple nucleotide sequences of the promoter, multiple nucleotide sequences of the upstream terminator sequences of the multiple nucleotide sequences of the promoter, multiple nucleotide sequences of the.

In addition, polynucleotides comprising nucleic acid sequences encoding C L E provided herein typically also comprise signal sequences to directly express the plasma membrane exemplary signal sequences are typically provided herein in other sections components can be provided on the transcript such that both CAR and C L E are expressed from the same transcript.

For pool 1A (which includes constructs encoding chimeric polypeptides and CARs), the 172 top candidate chimeric polypeptides are identified (see table 8), promote PBMC proliferation between day 7 and the last day when cultured in the absence of I L-2 for pool 1A (which includes constructs encoding chimeric polypeptides and not including CARs), the 167 top candidate chimeric polypeptides are identified (see table 9), promote PBMC proliferation between day 7 and the last day when cultured in the absence of I L-2, certain illustrative C L E and polynucleotides and nucleic acid sequences encoding the same, and methods including any of these include on constructs provided in tables 8-12 intracellular domains from genes having matched domains (e.g., exemplary transmembrane genes and first and optionally second intracellular genes) and (in non-limiting examples) from constructs provided in tables 8-12 having the same intracellular domains as those provided in exemplary tables 8-12 (e.g., transmembrane genes and first and optionally second intracellular genes) and wherein certain of the matched constructs provided in tables 8-041, 048, and optionally second intracellular domains have the same characteristic score in the first and second intracellular domains as those provided in examples, particularly in the examples 048-048, 048-2, 048-2, especially when the same complement of the first and second intracellular domains as those provided in examples, e.g., C-200, the first and second intracellular domains, with the same score in the third, e.g., the first and third, the third, C-200, and fourth, C-3, and fourth, C-200, and fourth, particularly the C-200, and fourth, C-2, and fourth, C-3, and fourth, C-3, and fourth, C, and fourth, C-3, and fourth, and sixth, and fifth, C, and sixth, C.

Further information on the first and second endodomains in constructs with particularly noteworthy enrichments in the screen of both library 1A and library 1.1A is provided in table 20, including the first and second endodomains derived genes, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif as shown in example 17, constructs with endodomains from I L R, MYD88, CD27, MYD88, TNFRSF18 or I L RA when present in the first endodomains (P3) and constructs with endodomains from CD 7378, I L1R L, CD8A, TNFRSF4 or MYD88 when present in the second endodomains (P4), constructs with enrichments from both library 1A and 1.1A showing particularly noteworthy enrichments in constructs from table 1A 4620 (P5920) and tnfr 4624 when present in constructs with intracellular domains from tnfr 585, tnfr 465 or tnfr 6331 (P5920) are present in the second endodomains from table 5 and 5).

For both libraries 2B and 2.1B, constructs M007-S049-S051, M007-S050-S039, M012-S050-S043, M012-S161-S213, M030-S142-S049, M001-S145-S130, M018-S085-S039, M018-S075-S053, M012-S135-S074, and M007-S214-S077 had particularly noteworthy enrichments in both screenings. Constructs with particularly noteworthy enrichment for the purpose of repeat screening were log₂Those constructs having a value of greater than 2 (normalized count data on the last day + 1)/(normalized count data on day 7 + 1).

Further information about the first and second endodomains in constructs with particularly noteworthy enrichment in screening for both sink 2B and sink 2.1B is provided in table 21, including whether the first and second endodomains are derived from a gene, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif when constructs with endodomains from CD28, CD40, I40 RA 40, I40 RB, IFNGR 40, FCGR2 40, I36111 RA or TNFRSF 40 are present in first endodomains constructs (P40), and constructs with endodomains from CD40, CD3 40, CD8 40, TNFRSF 40, CD40, I36210, CD79, CD40, fcr 1 or GHR 40 (with both ifra domains from a cell receptor domains are present in a cell, particularly when constructs with two ifra domains from a cell rich ifra 40, a cell receptor ifra 40, a cell with ifra 40, a cell having a receptor motif (e) from a cell, a cell with ifra 40, a cell, and a cell having a cell with a cell receptor motif from a cell characteristic of a cell, a cell receptor B40, a cell receptor epitope from a cell (e receptor B40, a cell) when constructs with two receptor domains from a cell, a cell exhibiting cell, a cell receptor epitope from cell (e receptor epitope from cell) when a cell) from cell, a cell exhibiting cell, a cell receptor epitope from cell receptor 7, a cell exhibiting cell, a cell receptor epitope 40, a cell exhibiting cell receptor epitope from cell, a cell (P40, a cell receptor epitope 40, a cell exhibiting cell (e 40, a cell) and a cell receptor epitope from a cell receptor epitope 40, a cell exhibiting a cell receptor epitope from a cell exhibiting cell, a cell exhibiting cell receptor epitope from a cell receptor agonist (P40, a cell receptor epitope from a cell receptor.

In an initial metaphase assay, for pool 3A (which includes constructs encoding a chimeric polypeptide and a CAR), 126 top candidate chimeric polypeptides were identified (see table 13), when cultured in the absence of I L-2, promoted PBMC proliferation between day 7 and the last day of transduced PBMCs stimulated with non-transduced PBMCs as described in example 12. for an initial metaphase assay of pool 3B (which includes constructs encoding a chimeric polypeptide and a CAR), 127 top candidate chimeric polypeptides were identified (see table 14), when cultured in the absence of I L-2, promoted PBMC proliferation between day 7 and the last day of transduced PBMCs not stimulated with non-transduced PBMCs as described in example 12. for pool 4B (which includes constructs encoding a chimeric polypeptide but not a CAR), 154 top candidate chimeric polypeptides were identified (see table 17), when cultured in the absence of I L-2, promoted proliferation between day 7 and the last day of PBMCs not stimulated with non-transduced PBMCs as described in example 12.

After further decoding, which provides the deep decoding, in banks 3A and 3B, which comprise constructs encoding the chimeric polypeptide and CAR and transduced PBMCs supplemented with (bank 3A) or without (bank 3B) fresh transduced PBMCs, 134 top candidates were identified for bank 3A on day 21, 124 top candidates were identified for bank 3A on day 35, and 131 top candidates were identified for bank 3B on day 21 (see table 13 and table 14, respectively) when cultured in the absence of I L-2 (i.e., I L-2 was not added to the culture medium during culture after initial transduction), I L-7, or any other exogenous interleukins, PBMC proliferation was promoted between day 7 and the last day.

Certain illustrative C L E and polynucleotides and nucleic acid sequences encoding the same and methods comprising any of these include intracellular domains from genes with matched domains (e.g., transmembrane genes and first and optionally second intracellular genes) on the constructs provided in tables 13-18 and, in non-limiting examples, the particular matched domains provided on these tables, some of which are described in the exemplary embodiments section herein, it is contemplated in the illustrative C L E example that the intracellular domains identified in the data provided in examples 11 and 12 from the first or second intracellular domain location may be interchangeably used as first or second intracellular domains in the illustrative C L E example, in illustrative embodiments, the constructs with particularly noteworthy degrees of transmembrane screening (e.g., libraries 3A and 3.1A) from the first and repeated screening of the constructs with the same transmembrane domains P1, P2, P3 and P4 (e.g., libraries 3A and 3.1A) for the first and repeated screening of the genes with the genes from the matched domains (e.g., libraries 3A-011, 3.1A-007) and the first and second intracellular domains are worth of T-053-007, such as T-053A-186S-186E-186-3-186E-3-186E-3-186-3-186E-186-3-186-3E-186E-186-3-186E-3-186S051, E006/E011-T031-S186-S211, E006/E011-T011-S186-S050, E006/E011-T011-S186-S047, E007/E012-T001-S186-S050, E006/E011-T041-S186-S051, E008/E013-T028-S186-076, E009/E014-T029-S199-S053, E009/E014-T062-S186-S216, E007/E012-T006-S058-S051, E009/E014-T076-S186-S211 and E007/E012-T001-S186-S047 have particularly noteworthy enrichments in both screenings, where the portions of P1 separated by the slashes herein include the different labels of libraries 3A and 3.1A as shown in tables 7, 13 and 15. Constructs with particularly noteworthy enrichment for the purpose of repeat screening were log₂Those constructs having a value of greater than 2 (normalized count data on the last day + 1)/(normalized count data on day 7 + 1).

Additional information about the first and second endodomains in constructs with particularly noteworthy enrichments in the screen of both library 3A and library 3.1A is provided in table 22, including the genes from which the first and second endodomains are derived, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif when a construct with endodomains from MP L, OSMR or CSF2RA is present in the first endodomains (P3), and a construct with endodomains from CD40, TNFRSF4, CD79B, CD27, FCGR2A or TNFRSF18 is present in the second endodomains (P4), particularly noteworthy enrichments (table 22) are shown in both libraries 3A and 3.1A when a construct with endodomains from cellular receptors MP L, OSMR 2 or TNFRSF 2 is present in the second endodomains (P3527), particularly noteworthy enrichments from ifsa 3A construct with intracellular endodomains from cellular receptors L, OSMR 2 or tnfr 2 are present in second endodomains from table 3A 3, particularly noteworthy enriching construct with two ITAM 3A (P638) and CD9 when a construct with intracellular domains from tnfa 3, CD9, CD 3A, CD9, CD2, CD 3A, CD9, CD 3.1b 3b 3.3, CD9, and a construct with intracellular domains containing intracellular domains from tnfr 27, a construct with an ITAM domain containing.

For 3B and 3.1B, the constructs E007/E012-T017-S186-S051, E007/E012-T073-S186-S053, E008/E013-T028-S186-S047, E006/E011-T011-S186-S047, E007/E012-T082-S176-S214, E006/E011-T046-S186-S052, E008/E013-T029-S186-S052、E009/E014-T011-S186-S053、E008/E013-T032-S186-S039、E007/E012-T034-S186-S051、E007/E012-T041-S192-S213、E006/E011-T014-S069-S213、E006/E011-T022-S186-S053、E006/E011-T023-S115-S075、E006/E011-T029-S106-S213、E006/E011-T032-S155-S080、E006/E011-T041-S186-S216、E006/E011-T057-S135-S080、E006/E011-T072-S191-X002、E006/E011-T077-S186-S216、E006/E011-T080-S141-S080、E007/E012-T001-X001-S214、E007/E012-T007-S059-S211、E007/E012-T016-S186-S052、E007/E012-T031-S186-S053、E007/E012-T044-S102-S052、E007/E012-T044-S142-X002、E007/E012-T055-S069-S053、E007/E012-T063-S176-S216、E007/E012-T065-S157-S075、E008/E013-T008-S085-X002、E008/E013-T011-S085-S048、E008/E013-T021-S109-X002、E008/E013-T021-S168-S211、E008/E013-T032-S064-S214、E008/E013-T037-S170-S215、E008/E013-T038-S176-S048、E008/E013-T039-S137-S216、E008/E013-T041-S141-S053、E008/E013-T045-S177-S048、E008/E013-T048-S109-S074、E008/E013-T073-S199-S075、E009/E014-T001-S157-S074、E009/E014-T005-S196-S049、E009/E014-T011-S130-X002、E009/E014-T013-S155-X002、E009/E014-T017-S186-S076、E009/E014-T021-S142-S080、E009/E014-T023-S082-S076、E009/E014-T038-S196-S037、E009/E014-T055-S186-S052、E009/E014-T060-S175-S053、E009/E014-T070-S085-S212、E008/E013-T026-S054-S213、E009/E014-T007-S120-S053、E007/E012-T045-S186-S211、E008/E013-T073-S186-X002、E008/E013-T074-S186-X002、E007/E012-T055-S186-S053、E008/E013-T036-S186-S053、E007/E012-T017-S058-S053、E008/E013-T030-S189-S080、E006/E011-T029-S081-S047、E009/E014-T044-S194-S050、E006/E011-T028-S121-X002、E008/E013-T028-S186-S053、E009/E014-T078-S142-S213、E009/E014-T041-S186-S051、E008/E013-T006-S186-S050、E006/E011-T028-S186-S075、E006/E011-T040-S120-S038、E007/E012-T044-S115-S211、E009/E014-T039-S176-S075、E007/E012-T028-S186-S050、E008/E013-T031-S202-S050、E007/E012-T072-S192-S053、E006/E011-T065-X001-S051、E007/E012-T030-S062-X002、E007/E012-T073-S186-X002、E009/E014-T056-S186-S053、E008/E013-T046-S137-X002、E006/E011-T016-S136-S076、E007/E012-T032S142-S037, E007/E012-T065-S120-S215, E009/E014-T077-S186-S047, E009/E014-T001-S126-S051, E006/E011-T030-S121-S039, E008/E013-T006-S176-S213, E009/E014-T032-S130-S215, E008/E013-T041-S186-S039, E009/E014-T021-S186-S047, E008/E013-T026-S137-S214, E007/E012-T029-S116-S075, E008/E013-T026-S106-S049 and E012/E-T032-S168-S075 have, in particular, two screening tests with different sections marked by oblique lines P3 and B353, wherein the sections marked by distinct divisions of the two screening tests, as shown in tables 7, 14 and 16. Constructs with particularly noteworthy enrichment for the purpose of repeat screening were log₂Those constructs having a value of greater than 2 (normalized count data on the last day + 1)/(normalized count data on day 7 + 1).

Further information about the first and second intracellular domains in the constructs with particularly noteworthy enrichment in both repertoire 3B and repertoire 3.1B is provided in table 23, including genes derived from the first and second intracellular domains, whether the first and/or second intracellular domain is an interleukin receptor, and whether the first and/or second intracellular domain has at least one ITAM motif from MP L, L EPR, MYD L, EPOR, I L RA, I L RG, I L RAP, I L RA L, CSF 2L, I L R L, I L gr L, I L RA 713, I L RA, I36820 RB, IFNGR L, I3693 RA, I L RA, CSF3, I36031 RB 031, RB 031 RB, I L gr L, I L RA L, I L, when the constructs with two CD3, CD72, CD3, or CD72, CD3, a construct with an ifr, CD72, CD3, a CD3, a domain from an enriched, CD72, CD3, a CD3, CD72, CD3, a CD72, CD3, a CD72, a CD3, a CD72, CD3, a CD3, a CD72, a CD72, CD3, a CD72, a CD3, a CD L, a CD3, a CD72, CD3, CD L, a CD72, CD L, a CD3, a CD L, a CD3, a CD3, a CD72, a CD L, CD72, CD3, a CD72, CD3, CD7, CD L, a CD3, a CD7, a CD3, a CD72, a CD3, a CD72, a CD L, a CD72, a CD7, a CD72, a CD3, a CD72, CD3, CD72, a CD7, a CD 72.

For libraries 4B and 4.1B, constructs E007/E012-T078-S154-S047, E008/E013-T062-S186-X002, E008/E013-T055-S186-S050, E009/E014-T057-S186-S050, E007/E012-T077-S054, E007/E012-T034-S135-S211, E009/E014-T071-X001-S216, E009/E014-T011-S141-S037, E008/E013-T041-S186-S037, E006/E011-T038-S106-S039, E006/E011-T011-S121-X002, E007/E-T007-S085-S215, E006/E06-T008-S011-S186-S011, E I-S012-S011, E007-S186-S011, E006/E011-T041-S186-S047, E008/E013-T045-S186-S051, E008/E013-T003-S104-S216, E006/E011-T019-S186-S053, E008/E013-T071-S064-S080, E006/E011-T021-S054-X002, E006/E011-T003-S135-X002, E009/E014-T020-S199-S213, E008/E013-T027-S121-S211, E009/E014-T032-S195-X002, E009/E014-T050-S171-X002, E008/E013-T069-S002, E008/E-T026-S038, E014-S038-S014-S037, E014-S057-S012-S057, E008/E072-S057, E072-S057, E008/E013-T046-S142-S080, E006/E011-T065-S186-S076, E006/E011-T062-S069-X002, E007/E012-T047-S098-X002, E009/E014-T069-S099-S048, E008/E013-T039-S141-S050, E006/E011-T052-S130-S052, E008/E013-T041-S186-X002, E007/E012-T019-S120-X002, E008/E013-T045-S186-S053, E006/E011-T003-S170-S039, E007/E012-T047-S8-S051, E007/E013-T069-S06109-S006-S011-S170-S039, E012-S047-S011-S057, E007/E011-S054 and E011-S054, where the portions of P1 separated by diagonal lines herein include different labels for libraries 4B and 4.1B, as shown in tables 7, 17, and 18. Constructs with particularly noteworthy enrichment for the purpose of repeat screening were log₂Those constructs having a value of greater than 2 (normalized count data on the last day + 1)/(normalized count data on day 7 + 1). The screening of both library 4B and 4.1BFurther information on the first and second endodomains in the constructs of especially noteworthy abundance is provided in Table 24, including the genes from which the first and second endodomains are derived, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif when a construct with endodomains from I18R, MP, CR 0F, I111 RA, I213 RA, I32 RG, I47, CSF3, I52 RA, OSMR, MYD, I631 RA, IFNAR 813, I76, I RA, EPOR, I91R, I10 RB or I03 RA is present in the first endodomains (P), and when a construct with endodomains from CD, CD79, TNFRSF, GR 3, CD3, ICOS, TNFRSF, CD3, CD79, TNFRSF 2, CD GR, FCGR2, and when a construct with endodomains from CD3, CD79, and CD3, 2R 2, especially when a construct with two ITFR domains from the ITFR, CD3, 2R, 2, III, V, III, V.

In some demonstrative embodiments, C L E includes an endodomain from any one of the constructs of tables 19-23 having only a single endodomain (the other P3 or P4 being a linker or termination).

Information regarding various exemplary C L E domains provided herein is found in table 7 for example, the first C L E identified in table 8 is M008-S212-S075 for this C L E, the ectodomain and transmembrane domain (P1-2) is M008, the first endodomain (P3) is S212, and the second endodomain (P4) is S075 when considering the nucleic acid encoding it, e.g., M008, detailed information regarding the identifiers of these domains and modules is found in table 7 discloses that M008 is ECDTM-8, and more particularly is an interleukin 7 receptor α (I L7 RA) mutant with PPC L insertion and that the construct includes eTag.

For sink 1A, when found at the first intracellular domain position (P3) of a candidate chimeric polypeptide, the intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, I L2 RA, I L RA, I L RA2, TNFRSF8, and TNFRSF8, or mutants thereof having signaling activity, when data for all constructs having a first intracellular domain (P8) derived from that gene are considered in combination, promote the proliferation of PBMCs between day 7 and the last day, thus, exemplary embodiments of C8E provided herein have intracellular domains from CD 38, CD3 nar 72, CD 88, CD79 8, ifnr 8, I8 RA, tnfr 8RA, TNFRSF8, and TNFRSF8, as illustrative of their intracellular domain activities, include a second intracellular domain(s) signaling activity, or mutants thereof.

For sink 1A, when found at the second endodomain position (P4) of the candidate chimeric polypeptide, endodomains from CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B, CR L F2, FCGR2C, ICOS, I L2 RA, I L013 RA1, I L13 RA2, I L15 RA, TNFRSF9, and TNFRSF18, or mutants thereof known to have signaling activity, when the data for all constructs having the second endodomain (P4) derived from this gene are considered in combination, promoting proliferation of PBMCs between day 7 and the last day, examples of the second exemplary endodomains or portions and/or mutants thereof present in constructs promoting cell proliferation are provided in the table in example 11, thus, examples of C L E provided herein have intracellular signaling activity from CD3, CD72, CD3D, CD D, and tnfrs D include CD D, and tnfrs D, CD D, and tnfrs D.

For pool 3A, the first intracellular (P3) domain from CSF2RA, IFNAR1, I L1 RAP, I L4R, I L ST 06, I L RA, I L RB2, I L RA, I L RD, I L RE, I L R1, I L R, I L R, MP L and MyD88, or mutants thereof known to promote signaling activity in certain cell types when found at the first intracellular domain (P3) of the candidate chimeric polypeptide modules herein, when these mutants are present in the constructs provided in example 12, the results of counting the sequences of the constructs in the population of cells in mixed culture, when considered in combination with the data for all constructs with the first intracellular domain derived from this gene, the results between day 7 and the last day or between day 7 and day 21, provide a normalized ratio of the signal counts for these constructs in the pool of cells when the results of one CSF group are present in the first intracellular domain of the first cell type of the construct, or when the results of these constructs from the first intracellular domain of the construct in the pool are present in the normalized ratio of table 583, or the results of the first intracellular domain of the enrichment of these constructs in the pool 3, the first intracellular domain of the second map 3, the second intracellular domain of the second cell type of the second cell culture, the second cell of the.

For pool 3A, when found at the second intracellular domain (P4) of the candidate chimeric polypeptide modules herein, the second intracellular (P4) domains from CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF 4and TNFRSF8, or mutants thereof known to promote signaling activity in certain cell types when these mutants are present in the constructs provided in example 12, promote the proliferation of PBMCs between days 7 and 21 or between days 7 and the last as indicated on the table when considering the data of all constructs with the second intracellular domain derived from this gene in combination. This conclusion is based on enrichment of the sequence counts of the constructs in the mixed culture PBMC cell population such that when the results of all constructs are combined for one gene, the enrichment calculated as the log base 2 of the ratio between the normalized counts on the last day plus one and the normalized counts on day 7 plus one indicated on the table is at least 2 for pool 3A. Examples of second endodomains or parts and/or mutants thereof present in constructs promoting cell proliferation against pools 3A and 3B are provided in tables 13 to 18.

After an initial intermediate decoding assay, for sink 3A, when found at the first intracellular domain (P3) of the candidate chimeric polypeptide modules herein, the first intracellular (P3) domains from I L RD, I L RE, I L RAP, I L R and MP L, or mutants thereof known to promote signaling activity in certain cell types, when considered in combination with data from all constructs with the first intracellular domain derived from that gene, promote proliferation of PBMCs between day 7 and day 21, for sink 3B, when analyzed in this manner, the first intracellular domain or portions thereof from I L R, MP L or OSMR, or mutants thereof known to promote signaling activity in certain cell types, are best performed examples of the first intracellular domain or portions thereof and/or mutants present in the constructs promoting cell proliferation for sink 3A and 3B are provided in example 12, thus examples of intracellular domains from table 3A, 3B, which have activity from I3817R 73723, I8217R 73723, or I8217R 18R 23, or mutants thereof, are illustrated as having intracellular activity in examples of intracellular domains iv, I8217R 73723, I, R73723, R2R, or mutants thereof, which are provided herein as examples of intracellular domains which are illustrated as intracellular domains.

After an initial decoding assay, for pool 3A, when found at the second intracellular domain (P4) of the candidate chimeric polypeptide modules herein, the second intracellular (P4) domains from CD27, CD3G, CD40 and CE79B, or mutants thereof known to promote signaling activity in certain cell types, when considered in combination with the data for all constructs with the second intracellular domain derived from that gene, promote proliferation between day 7 and the last day indicated on the table for PBMC 3B, the second intracellular domain from CD40, or mutants thereof known to promote signaling activity in certain cell types, when analyzed in this manner, therefore, the embodiments of C L E provided herein have intracellular domains from CD27, CD3G, CD40 and CE79B, including mutants thereof that retain their signaling activity as the first intracellular domain (or in illustrative embodiments, the second intracellular domain).

As shown in tables 13-18, when found at the first endodomain (P3) of the candidate chimeric polypeptides herein, the first intracellular (P3) domain from MP L, L EPR, MYD88, IFNAR2, or in some cases a mutant thereof that retains signaling activity, when considering the first endodomain that most frequently occurs in the top candidate construct, promotes proliferation of PBMCs between day 7 and the end of the experiment examples of the first endodomain and/or its part and/or mutant present in constructs that promote cell proliferation for banks 3A, 3B, 3.1A, 3.1B, 4B and 4.1B are provided in the table in example 12.

As shown in tables 13-18, when found at the second endodomain (P4) of the candidate chimeric polypeptides herein, the second intracellular (P4) domain from CD40, CD79B, CD27, or in some cases a mutant thereof that retains signaling activity, when considering the first endodomain that most frequently occurs in the top candidate construct, promotes proliferation of PBMCs between day 7 and the last day indicated on the table examples of the first endodomain, or a portion thereof and/or mutant thereof, present in constructs that promote cell proliferation for libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B are provided in the table in example 12.

In illustrative embodiments, the construct of C E encodes an intracellular domain from MP at P and an intracellular domain from OX, CD79 or CD79 in examples 10, the intracellular domain from these genes shows an intracellular domain from chimeric lymphoid tissue proliferation component of P0 at P1 or library 4.1, the intracellular domain from P1 at P1 or library 4.1 shows an increased proliferation at 0.05 in examples 3.1 or library 4.1, the intracellular domain from MP1 at P and an intracellular domain from CD, CD or CD79 in examples 120, or the intracellular domain from chimeric P1, CD2 or library 3, or the intracellular domain from cell P1, or the intracellular domain from chimeric P1, or the proliferation promoting signal construct of SEQ ID NO 1, or library 3, the proliferation promoting signal construct of SEQ ID NO 1, or SEQ ID NO 2, or the proliferation promoting signal construct of SEQ ID NO 2, or the cell P2, or the proliferation promoting signal construct of SEQ ID NO 2, or SEQ ID No. 1, or 2, or No. 1, or 2, or a signal promoter, or 2, or a promoter, or a promoter, or a promoter, or a promoter, or.

In illustrative embodiments, the construct of C E encodes the intracellular domains or variants or fragments thereof from two of the following pairs of genes CSF2 and TNFRSF, CSF2 and CD, CSFR and CD79, IFNAR and TNFRSF, I1 RAP and CD79, I03 RA and CD, I110 RA and CD79, I211 RA and FCGRA, I313 RA and TNFRSF, I418 RAP and CD3, I527 RA and FCGRA, 6EPR and CD3, 7IFR and TNFRSF, MP 8 and CD9 and CD79, TNFRSF, MP 0 and CD3, MyD and CD79, and MyD and CDD, under a p of less than 0.1, the portion encoding the intracellular domain from these pairs of genes is most effective in example 10 (Table 3) and preferably the portions of the same or less than the same, preferably the portions of the same, preferably the portions of the intracellular domains or same, preferably the portions thereof, preferably the portions of the intracellular domains or the same, preferably the portions of the intracellular domains or the same, preferably the same, or the same, preferably the intracellular domains or the same, preferably the same, or the portions of the intracellular domains or the same, preferably the same, or the same, as the same, or the same, as the same, or the same, preferably or the same, preferably the same, or the same, preferably the same, or the same, as the same, preferably the same, as the same of the same, or the same of the same, preferably the same of the same, or the same, or the same of.

In notable embodiments of any of the isolated polypeptides or vector aspects and embodiments provided herein comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells (i.e., C L E) and a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, the first and second nucleic acid sequences may be separated by a ribosome skipping sequence in some embodiments, the ribosome skipping sequence is F2A, E2A, P2A, or T2A.

As disclosed herein, a lymphoproliferative component (as exemplified herein with C L E) may in some illustrative embodiments include a dimerization motif to help enhance and/or modulate its activity, for example to affect a signal in a cell, such as a signal that affects protein activity or gene expression.

In some embodiments of lymphoproliferative components including dimerizing agents and polynucleotides and nucleic acids encoding the same, and C L E, the dimerization motif may include amino acid sequences from transmembrane homodimeric polypeptides naturally occurring as homodimers.for example, the dimerization motif may be a leucine zipper polypeptide, such as a Jun polypeptide, as exemplified in example 12 herein. in some embodiments, these transmembrane homodimeric polypeptides may include the early activation antigen CD69(CD69), the transfer receptor protein 1(CD71), the B cell differentiation antigen (CD72), the T cell surface protein haptic (CD96), the endoglin (Cd105), the B member 1 of the killer lectin-like receptor subgroup (Cd161), the P-selectin glycoprotein ligand 1(Cd162), the glutaminyl peptidase (Cd162), the tumor necrosis factor receptor superfamily member 16(CD271), the cadherin-1 (epithelial cadherin) (Cd324) or active fragments thereof may be replaced with a homodimer loop terminator such as a rapamycin C homolog or a homolog of the rapamycin found in the aforementioned Ser 365935, or Ser 3655, the homolog of the homolog found in the aforementioned Ser 36598, the homolog 3691, or the homolog of the aforementioned, and the homolog of the homolog found in the homolog 3695, the homolog found in the aforementioned related analogs of the homolog 3695, the homolog found in the homolog 365, or the prodrug, the homolog of the homolog found in the homolog of the homolog found in the aforementioned, or the homolog 3695, or the homolog of the aforementioned, and the homolog of the homolog 3695-5, or the homolog of the homolog found in the homolog of the prodrug, or the homolog of the homolog found in the homolog of the homolog found in the prodrug, or the prodrug, or the analog of the prodrug, or the analog of the prodrug, the analog of the prodrug, or the analog of the prodrug, or the analog of the prodrug, wherein the prodrug, or the analog of the prodrug, or the prodrug, the analog of the prodrug, or the analog of the.

For example, a lymphoproliferative component comprising a leucine zipper-containing dimerization motif, or comprising a dimerization motif from a transmembrane homodimeric polypeptide comprising CD69, CD71, CD72, CD96, CD105, CD161, CD162, 249, CD271, CD324, active mutants thereof, and/or active fragments thereof, may be active in the absence of a dimerizing agent.

In embodiments of the methods that include a lymphoproliferative component (e.g., C L E) that includes a dimer for dimerizing a polypeptide that includes a dimerization motif, the dimer may be administered to a host (individual) using convenient means capable of producing the desired effect.

Those skilled in the art will readily appreciate that the dosage level may vary depending on the particular dimer, the severity of the symptoms, and the sensitivity of the individual to side effects. Preferred dosages for a given compound can be readily determined by one of skill in the art using a variety of means.

The dimer is administered to the subject using any available method and route suitable for drug delivery, including ex vivo methods of in vivo agents and systemic and local routes of administration.

In any aspect or embodiment in which the extracellular domain of C L E comprises a dimerization motif, the dimerization motif may be selected from the group consisting of polypeptides comprising leucine zipper motifs, CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, and mutants and/or active fragments thereof that retain dimerization capacity.

In illustrative embodiments of any aspect or embodiment wherein the extracellular domain of C L E comprises a dimerization motif, the extracellular domain may comprise a leucine zipper motif in some embodiments, the leucine zipper motif is from a jun polypeptide, e.g., C-jun.

In any of the aspects or embodiments provided herein that include a lymphoproliferative component, the lymphoproliferative component can be a polypeptide comprising a membrane targeting region, a dimeric (multimerizing) domain, a first intracellular domain and optionally a second intracellular domain and optionally a third intracellular domain and optionally a fourth intracellular domain, wherein the first intracellular domain and optionally the second intracellular domain, the third intracellular domain and the fourth intracellular domain are from any gene having an intracellular domain identified in tables 8-18, or are selected from any of the first intracellular domain and the second intracellular domain identified in tables 8-18, and wherein the internal dimeric and/or multimeric lymphoproliferative component promotes proliferation of PBMCs (and in illustrative embodiments, B cells, T cells and/or NK cells) in some embodiments, the polypeptide can be referred to as an internal dimeric and/or multimeric lymphoproliferative component, in some embodiments, a linker can be added between any of the domains in some embodiments, the first intracellular domain and the second intracellular domain of PBMC 355, the intracellular PBMC 2 or intracellular cd3, the intracellular cd3, cd.

The dimeric domain of this internal dimeric and/or multimeric polypeptide lymphoproliferative component in illustrative embodiments may be located between the membrane targeting region and the first intracellular domain or after the intracellular domain (see, e.g., Spencer et al, J Clin invest.2011, 4 months; 121(4):1524-34) or between intracellular domains. In certain embodiments, the dimeric (or multimeric) domain comprises a multimeric or dimeric ligand binding site, such as an FKBP region, e.g., FKBP 12. In some embodiments, the polypeptide may comprise an extracellular domain, such as any of the extracellular domains provided herein, e.g., in tables 8-12 of example 11 or in tables 13-18 of example 12, with or without a recognition domain and a gap domain. In certain embodiments, the membrane-targeting region is selected from the group consisting of myristoylation region, palmitoylation region, prenylation region, and transmembrane sequence of the receptor. In certain embodiments, the membrane targeting region is a myristoylation region. The internal dimeric and/or lymphoproliferative component is typically linked to the plasma membrane via a membrane targeting region, for example to a membrane with an N-terminal myristate. In an illustrative embodiment, the FKBP region binds to FK 506.

The internal dimeric and/or multimeric lymphoproliferative component of one embodiment is an integral part of a system using analogues of the lipid permeable dimeric immunosuppressant drug FK506, which lose its normal biological activity while gaining the ability to cross-link 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, we can stimulate signaling in a dimeric drug-dependent but ligand-and ectodomain-independent manner. This provides time control for the system, reversibility of using monomeric drug analogs, and enhanced specificity. The high affinity of the third generation AP20187/AP1903 dimer drug for its binding domain FKBP12 allows for specific activation of recombinant receptors in vivo without inducing non-specific side effects via endogenous FKBP 12. FKBP12 variants with amino acid substitutions and deletions (such as FKBP12V36) that bind to dimeric drugs may also be used. In addition, synthetic ligands are resistant to proteolytic cleavage, making them more effective at activating receptors in vivo than most delivered protein agents.

Pseudo-type component

Many of the methods and compositions provided herein include pseudotyped components. Pseudotyping of replication-deficient recombinant retroviral particles with heterologous envelope glycoproteins generally alters the tropism of the virus and facilitates transduction of host cells. A pseudotyped component as used herein may include: "binding polypeptides" which include one or more polypeptides (typically glycoproteins) that recognize and bind to a target host cell; and one or more "fusogenic polypeptides" that mediate the fusion of the retrovirus with the target host cell membrane, thereby allowing the retroviral genome to enter the target host cell. In some embodiments provided herein, the pseudotyped component is provided as a polypeptide/protein, or as a nucleic acid sequence encoding a polypeptide/protein.

In some embodiments, the pseudotyped component is a feline endogenous virus (RD114) envelope protein, an amphotropic envelope protein, a homophilic envelope protein, a herpetic oral virus envelope protein (VSV-G), a baboon reverse transcriptase envelope glycoprotein (BaEV), a murine leukemia virus envelope protein (Mu L V), and/or paramyxovirus measles envelope proteins H and F.

In some embodiments, the pseudotyped component may be a wild-type BaEV. Without being limited by theory, BaEV contains an R peptide that is shown to inhibit transduction. In some embodiments, the BaEV may contain a deletion of the R peptide. In some embodiments, after the nucleotide encoding amino acid sequence HA (referred to herein as BaEV Δ R (HA)), 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 Δ R (HAM)), the BaEV may contain a deletion of the inhibitory R peptide.

For example, the replication-defective recombinant retroviral particles in the methods and compositions disclosed herein can be pseudotyped by a fusion (F) polypeptide of Measles Virus (MV) and a prothrombin (H) polypeptide, as non-limiting examples, clinical wild-type strains of MV, and vaccine strains including Edmonston strain (MV-Edm) or fragments thereof without being limited by theory, it is believed that both the prothrombin (H) and fusion (F) polypeptides can function in entering a host cell, wherein the H protein binds MV to receptors CD46, S L AM and Nectin-4 on the target cell, and F mediates fusion of the retrovirus and the host cell membrane.

In some studies, lentiviral particles pseudotyped with truncated F and H polypeptides increased significantly in potency and transduction efficiency (Funke et al, 2008.Molecular therapy.16(8):1427-1436), (Frecha et al, 2008.blood.112(13): 4843-4852). The highest titer was obtained when the F cytoplasmic tail was truncated by 30 residues (also known as MV (Ed) -F.DELTA.30 (SEQ ID NO: 105)). For the H variants, the best truncation occurred in the deletion of 18 or 19 residues (MV (Ed) -H.DELTA.18 (SEQ ID NO:106) or (MV (Ed) -H.DELTA.19)), but variants with a truncation of 24 residues also gave the best titers in the deletion of residues with and without alanine substitution (MV (Ed) -H.DELTA.24 (SEQ ID NO:235) and MV (Ed) -H.DELTA.24 + A).

In some embodiments, including those directed to transduced T cells and/or NK cells, the replication-defective recombinant retroviral particles in the methods and compositions disclosed herein are pseudotyped with mutated or variant versions of the measles virus fusion (F) polypeptide and the hemagglutinin (H) polypeptide (in illustrative examples, cytoplasmic domain deleted variants of the 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 residue (or the encoding nucleic acid of the corresponding nucleic acid molecule encoding the protein) has been deleted. "H Δ Y" and "F Δ X" 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 F Δ 24 or F Δ 30 and/or the "truncated H protein" is selected from the group consisting of: h Δ 14, H Δ 15, H Δ 16, H Δ 17, H Δ 18, H Δ 19, H Δ 20, H Δ 21+ A, H Δ 24, and H Δ 24+4A, more preferably H Δ 18 or H Δ 24. In an illustrative embodiment, the truncated F polypeptide is mv (ed) -F Δ 30 and the truncated H polypeptide is mv (ed) -H Δ 18.

In some embodiments, the fusion polypeptide includes multiple components expressed as one polypeptide. In some embodiments, the binding and fusion polypeptides are translated from the same transcript but from separate ribosome binding sites; in other embodiments, the binding and fusion polypeptides are separated by a cleavage peptide site (which, without being limited by theory, cleaves after translation, as is common in the literature) or a ribosome skipping sequence. In some embodiments, translation of the binding polypeptide and the fusion polypeptide from the 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 between the lower end of the range of 1.5:1, 2:1, or 3:1 and the upper end of the range of 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1.

Activation assembly

Many of the present methods and composition aspects include an activating component (also referred to herein as a T cell activating component), or a nucleic acid encoding an activating component, the limitations associated with lentiviral (L V) transduction into resting T cells are due to a series of pre-and post-entry barriers and cell restriction factors (Strebel et al 2009 BMC Medicine 7:48), one limitation being that the envelope pseudotyped L V particles are unable to recognize potential receptors and mediate fusion with the cell membrane, however, under certain conditions, transduction of resting T cells by HIV-1 based lentiviral vectors is most likely after co-stimulation of the T Cell Receptor (TCR) CD3 complex and CD28 (Korin and zack.1998.journal of virology.72:3161-8, Maurice et al 2002.Blood99:2342-50), and via exposure to interleukins (caveieri et al 2003).

Cells of the immune system (such as T lymphocytes) recognize and interact with specific antigens via receptors or receptor complexes, which upon recognition or interaction with such antigens, allow the cells to activate and expand in vivo. An example of such a receptor is the antigen-specific T lymphocyte receptor complex (TCR/CD 3). The T Cell Receptor (TCR) is expressed on the surface of T lymphocytes. One component (CD3) is responsible for intracellular signaling after the TCR is occupied by a ligand. The T lymphocyte receptor directed against the antigen CD3 complex (TCR/CD3) recognizes antigenic peptides presented to it by proteins of the Major Histocompatibility Complex (MHC). Complexes of MHC and peptide are expressed 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. The TCR/CD3 complex plays an important role in the effector function and regulation of the immune system. Thus, the activation module provided herein activates T cells by binding to one or more components of a T cell receptor associated complex, for example by binding to CD 3. In some cases, the activation component may be activated alone. In other cases, activation requires activation via the TCR receptor complex in order to further activate the cell.

T lymphocytes also require a second, costimulatory signal to become fully active in vivo. In the absence of this signal, T lymphocytes are unresponsive to the antigen bound to the TCR, or become anergic.

However, transduction and expansion of T cells does not require a second costimulatory signal, such as provided by CD28, T lymphocyte proteins that interact with CD80 and CD86 on antigen-producing cells As used herein, functional extracellular fragments of CD80 retain their ability to interact with CD28 OX40, 4-1BB and ICOS (inducible costimulators) (other T lymphocyte proteins) provide costimulatory signals when bound to their corresponding ligands OX 40L, 4-1BB L and ICOS L G.

Activation of the T Cell Receptor (TCR) CD3 complex and co-stimulation by CD28 can occur by ex vivo exposure on 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 a solid surface coated ex vivo with anti-CD 3 and anti-CD 28. In other embodiments, resting T cells or NK cells (in illustrative embodiments, T cells) are activated by exposure to a soluble anti-CD 3 antibody (e.g., at 50ng/ml to 150ng/ml, or 75ng/ml to 125ng/ml, or 100 ng/ml). In this embodiment, which may be part of a method for genetically modifying or transducing (without prior activation in illustrative embodiments), such activation and/or contacting may be performed, for example, for 8 hours or less, 4 hours or less, or between 2 hours and 8 hours, between 2 hours and 4 hours, or between 2 hours and 3 hours.

In certain illustrative embodiments of the methods and compositions provided herein, polypeptides capable of binding CD3 and/or CD28 are present on the surface of the replication deficient recombinant retroviral particles in the methods and compositions disclosed herein as "activating components", which are also aspects of the invention, in some embodiments, the activating components on the surface of the replication deficient recombinant retroviral particles may comprise one or more polypeptides capable of binding OX40, 4-1BB or ICOS. in some embodiments, the activating components may be T cell surface protein agonists. the activating components may comprise co-stimulatory polypeptides that act as ligands for T cell surface proteins. in some embodiments, the co-stimulatory polypeptides may comprise one or more of OX 40L, 4-1BB L or ICOS L G. in some embodiments, one or typically multiple copies of these activating components may be expressed on the surface of the replication deficient recombinant retroviral particles as separate and distinct polypeptides from the pseudo-type components and different polypeptides, in some embodiments, the activating components may be expressed on the replication deficient recombinant viral particles as pseudo-envelope proteins, or may comprise one or more polypeptides that are found in the replication deficient recombinant retroviral particles as "activating components" or fusion proteins "activating components" expressed on the replication deficient recombinant retroviral particles ", e.g. as disclosed herein, it is found that the expression of a polypeptide found in the expression of a recombinant retroviral particle of a chimeric polypeptide encoding a chimeric polypeptide, e.g. a chimeric polypeptide encoding a chimeric polypeptide, a chimeric polypeptide encoding a chimeric gene encoding a human retrovirus, a chimeric gene encoding a chimeric gene.

In some embodiments, the activating component is a polypeptide capable of binding CD 3. In some embodiments, the polypeptide capable of binding CD3 is an anti-CD 3 antibody, or fragment thereof that retains the ability to bind to CD 3. In illustrative embodiments, the anti-CD 3 antibody or fragment thereof is a single chain anti-CD 3 antibody, such as, but not limited to, anti-CD 3 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD3 is an anti-CD 3 scfvffc.

A number of monoclonal antibodies against human CD3 and antibody fragments thereof are useful and can be used in the present invention, including, but not limited to, UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030(BW264/56), C L B-T3/3, CRIS7, YTH12.5, F111409, C L B-T3.4.2, TR-66, WT31, WT32, SPv-T3B, 11D8, XIII-141, XIII46, XIII-87, 12F 2 695695, T3/RW2-8C8, T3/24B 6, OKT3D, M-T301, SMC2, and F101.01.

In some embodiments, the activating component is a polypeptide capable of binding to CD 28. In some embodiments, the polypeptide capable of binding to CD28 is an anti-CD 28 antibody, or 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 CD80 means a fragment that is typically present outside of a cell in the standard cellular location of CD80 that retains the ability to bind to CD 28. In illustrative embodiments, the anti-CD 28 antibody or fragment thereof is a single chain anti-CD 28 antibody, such as, but not limited to, anti-CD 28 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD28 is CD80, or a fragment of CD80, such as an external fragment of CD 80.

anti-CD 28 antibodies are known in the art and may include, as non-limiting examples, monoclonal antibody 9.3, IgG2a antibody (dr. jeffery L edbeter, Bristol Myers Squibb Corporation, Seattle, Wash.), monoclonal antibody KO L T-2(IgG1 antibody), 15E8(IgG1 antibody), 248.23.2(IgM antibody), and EX5.3D10(IgG2a antibody).

In an illustrative embodiment, the activation module 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., single chain antibody fragment) — thus, an antibody fragment can 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 Δ C), a fragment variable region (Fv), a structure specific for adjacent epitopes of an antigen (CRAb), or a single domain antibody (VH or V L).

In any of the embodiments disclosed herein, the activation component or the nucleic acid encoding it can comprise a dimeric or higher order multimeric motif. Dimeric or multimeric motifs are known in the art and those skilled in the art will understand how to incorporate them into polypeptides for efficient dimerization or multimerization. For example, in some embodiments, an activating element comprising a dimerization motif may be one or more polypeptides capable of binding to CD3 and/or CD 28. 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 illustrative embodiments, the anti-CD 3 antibody or fragment thereof is a single chain anti-CD 3 antibody, such as (but not limited to) anti-CD 3 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD3 is an anti-CD 3 scfvffc, which in some embodiments is considered to be anti-CD 3 with a dimerization motif but without any additional dimerization motif, as the anti-CD 3 scfvffc construct is known to be capable of dimerization without the need for a separate dimerization motif.

In some embodiments, the dimeric or multimeric motif or nucleic acid sequence encoding the same can be an amino acid sequence from a transmembrane polypeptide that occurs naturally as a homodimer or multimer.

In some embodiments, the transmembrane homodimeric polypeptides may comprise the early activation antigen CD69(CD69), metastasis receptor protein 1(CD71), B cell differentiation antigen (CD72), T cell surface protein tactile sensation (CD96), endoglin (CD105), the B member 1 of the lectin-like receptor subfamily (CD161), P-selectin glycoprotein ligand 1(CD162), glutaminyl peptidase (CD249), tumor necrosis factor receptor superfamily member 16(CD271), cadherin-1 (epithelial cadherin) (CD324), or an active fragment thereof. In some embodiments, the dimerization motif and the nucleic acid encoding it may comprise amino acid sequences from transmembrane proteins that dimerize upon ligand (also referred to herein as a dimer or dimerizer) binding. In some embodiments, dimerization motifs and dimers may include (where the dimer is in parentheses after the dimer binding pair): FKBP and FKBP (rapamycin); GyrB and GyrB (coumaromycin); DHFR and DHFR (methotrexate); or DmrB and DmrB (AP 20187). As mentioned above, rapamycin may be used as a dimer. Alternatively, rapamycin derivatives or analogs can be used (see, e.g., WO96/41865, WO 99/36553, WO 01/14387; and Ye et al (1999) Science 283: 88-91). For example, analogs, homologs, derivatives, and other compounds structurally related to rapamycin ("rapamycin analogs") include, in addition to rapamycin, variants of rapamycin having one or more of the following modifications related to rapamycin: demethylation, elimination, or substitution of methoxy at C7, C42, and/or C29; elimination, derivatization, or substitution of hydroxyl groups at C13, C43, and/or C28; reduction, elimination, or derivatization of ketones at C14, C24, and/or C30; replacement of the 6-membered methylpiperidine ring with a 5-membered prolinacyl ring; and the cyclohexyl ring is substituted or replaced with a substituted cyclopentyl ring. Additional information is presented, for example, in U.S. patent nos. 5,525,610, 5,310,903, 5,362,718, and 5,527,907. Selective epimerization of the C-28 hydroxyl group is described (see for example WO 01/14387). Additional synthetic dimerizing agents suitable for use as replacements for rapamycin include those described in U.S. patent publication No. 2012/0130076. As mentioned above, coumaromycin may be used as a dimerizing agent. Alternatively, coumaromycin analogs can be used (see, e.g., Farrar et al (1996) Nature 383: 178-. As mentioned above, in some cases, the dimerizing agent is methotrexate, e.g., a non-cytotoxic, homobifunctional methotrexate dimer (see, e.g., U.S. patent No. 8,236,925).

In some embodiments, an activation module comprising a dimerization motif may be active in the absence of a dimerization agent when present on the surface of a replication-defective recombinant retroviral particle. For example, an activation module comprising a dimerization motif from a transmembrane homodimeric polypeptide comprising CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271, CD324, active mutants thereof, and/or active fragments thereof, may be active in the absence of a dimerization agent. In some embodiments, the activation component may be an anti-CD 3 single-stranded fragment and comprise a dimerization motif selected from the group consisting of: CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271, CD324, active mutants thereof, and/or active fragments thereof.

In some embodiments, the activation module 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. For example, activating components comprising dimerization motifs from FKBP, GyrB, DHFR or DmrB may be active in the presence of individual dimerizers or analogs thereof (e.g., rapamycin, coumaromycin, methotrexate, and AP20187), respectively. In some embodiments, the activation component may be a single chain antibody fragment directed against anti-CD 3 or anti-CD 28, or another molecule that binds CD3 or CD28, and the dimerization motif and dimerization agent may be selected from the group consisting of: FKBP and rapamycin or analogs thereof, GyrB and coumaromycin or analogs thereof, DHFR and methotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof.

In some embodiments, the activating component is fused to a heterologous signal sequence and/or a heterologous membrane adhesion sequence, both of which help direct the activating component onto the membrane. The heterologous signal sequence targets the activation component to the endoplasmic reticulum, wherein the heterologous membrane adhesion sequence is covalently linked to one or more fatty acids (also known as post-translational lipid modification) such that the activation component fused to the heterologous membrane adhesion sequence is anchored in a lipid raft of the plasma membrane. In some embodiments, post-translational lipid modification may occur via myristoylation, palmitoylation, or GPI anchoring. Myristoylation is a post-translational protein modification corresponding to the covalent attachment of a 14-carbon saturated fatty acid (myristic acid) to an N-terminal glycine of a eukaryotic or viral protein. Palmitoylation is a post-translational protein modification that corresponds to the covalent linkage of the acyl chain of C16 with cysteine, and less with serine and threonine residues of the protein. GPI-anchored refers to the attachment of glycosylphosphatidylinositol or GPI to the C-terminus of a protein during post-translational modifications.

In some embodiments, the heterologous membrane-linking sequence is a GPI-anchor linking sequence the heterologous GPI-anchor linking sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, HartGW. Glycosylphosphatidylinosol anchors. in: Varki A, Cummings RD, Esko JD et al, eds. Essentials of glycobiology. 2 nd edition. Cold Spring Harbor (NY): Cold Spring Harbor L absorbent Press; 2009. 11. in some embodiments, the heterologous GPI-anchor linking sequence is a GPI-anchor linking sequence derived from CD 64, CD16, CD48, CD55(DAF), CD 365631, CD 357, and CD 87. in some embodiments, the heterologous GPI-anchor linking sequence is derived from GPI-anchor linking sequence 16. in illustrative embodiments, GPI-anchor linking sequence is derived from GPI-anchor receptor gamma receptor (DAF) 16, or CD 35 55).

In some embodiments, one or both of the activation components include a heterologous signal sequence that helps direct the activation component to expression of the cell membrane. Any signal sequence that is active in the packaging cell line can be used. In some embodiments, the signal sequence is a DAF signal sequence. In an illustrative embodiment, the activation module is fused to the DAF loop sequence at its N-terminus and the GPI anchor linker sequence at its C-terminus.

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

Membrane-bound interleukins

Some embodiments of the methods and composition aspects provided herein include membrane-bound interleukins, or polynucleotides encoding membrane-bound interleukins. Interleukins are typically, but not always, secreted proteins. Naturally secreted interleukins can be engineered as membrane-bound fusion proteins. Membrane-bound interleukin 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 on its surface a membrane-bound interleukin fusion polypeptide capable of binding to and promoting proliferation and/or survival of T cells and/or NK cells. Typically, the membrane-bound 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 the polypeptide bound to the membrane of the transduced cell.

In some embodiments, the cytokine fusion polypeptide comprises I L-2, I L-7, I L-15, or an activated fragment thereof the membrane-bound cytokine fusion polypeptide is typically a cytokine fused to a heterologous signal sequence and/or a heterologous membrane-linking sequence in some embodiments, the heterologous membrane-linking sequence is a GPI-anchor linking sequence the heterologous GPI-anchor linking sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, hartgw. glycophospholipolysis anchors. in: Varki a, Cummings RD, Esko JD et al, eds. Essentials of glycobiology. version 2. Cold Spring Harbor (NY) Cold Spring Harbor L anchors; 2009. chapter 11 Press, 2009. chapter 11) in some embodiments, the heterologous CD-anchor sequence is derived from GPI 5634, GPI-2, GPI-anchor sequence, GPI-anchor binding sequence derived from GPI 3, GPI-anchor binding sequence derived from GPI-anchor CD9, GPI-anchor sequence derived from GPI-anchor binding sequence, GPI-anchor sequence.

In illustrative embodiments, the membrane-bound interleukin is a fusion polypeptide of an interleukin fused to DAF. DAF is known to accumulate in lipid rafts incorporated into the membrane of replication-defective recombinant retroviral particles that germinate from packaging cells. Thus, without being limited 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 a particular non-limiting illustrative embodiment, the fused interleukin polypeptide comprises, in order, the DAF signal sequence (residues 1-31 of DAF), I L-7 without its signal sequence, and residues 36-525 of DAF.

Packaging cell lines/methods for making recombinant retroviral particles

The present invention provides mammalian packaging cells and packaging cell lines for the production of replication-defective recombinant retroviral particles. Such cell lines that produce replication-defective recombinant retroviral particles are also referred to herein as packaging cell lines. A non-limiting example of this method is provided in example 1 herein. The cells of the packaging cell line can be adherent cells or suspension cells. In illustrative embodiments, the packaging cell line can 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 chemically defined media and/or serum-free media. In some embodiments, the packaging cell line can be a suspension cell line derived from an adherent cell line, e.g., HEK293 can be grown under conditions that produce a HEK293 cell line adapted for suspension according to methods known in the art. Packaging cell lines are typically grown in chemically defined media. In some embodiments, the packaging cell line medium can include serum. In some embodiments, the packaging cell line medium can include serum replacement, as is known in the art. In an illustrative example, the packaging cell line medium can be serum-free medium. The medium may be a chemically defined serum-free formulation manufactured in accordance with Current Good Manufacturing Practice (CGMP) regulations of the U.S. Food and Drug Administration (FDA). The packaging cell line medium may be xeno-free and intact. In some embodiments, packaging cell line media is cleared by regulatory agencies for ex vivo cell processing, such as FDA 510(k) clearing devices. In this context (where it is stated that a medium comprises a combination of a base medium and a medium supplement comprising the medium supplement's catalog number such as a1048501 or a 1048503), it is to be understood that the combination is intended to mean the combination of the medium and the added supplement. Typically, the manufacturing medium and supplement provide instructions regarding the volume of supplement added.

Thus, in one aspect, provided herein is a method of making a replication-defective recombinant retroviral particle comprising: A. culturing a packaging cell in a serum-free medium comprising a suspension, wherein the packaging cell comprises a nucleic acid sequence encoding a packagable RNA genome of a replication-defective recombinant retroviral particle, an REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyped component; and B, collecting the replication-defective recombinant retrovirus particles from the serum-free medium. In another aspect, provided herein is a method of transducing lymphocytes with a replication-defective recombinant retroviral particle comprising: A. culturing a packaging cell in a serum-free medium comprising a suspension, wherein the packaging cell comprises a nucleic acid sequence encoding a packagable RNA genome of a replication-defective recombinant retroviral particle, an REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyped component; B. collecting replication-defective recombinant retroviral particles from a serum-free medium; contacting the lymphocyte with a replication-deficient recombinant retroviral particle, wherein the contacting is performed for less than 24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours, or 2 hours (or between 1 hour, 2 hours, 3 hours, or 4 hours at the lower end of the range and 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 20 hours, or 24 hours at the upper end of the range), thereby transducing the lymphocyte.

In another aspect, provided herein is a retroviral packaging system comprising: a mammalian cell, comprising: a) a first trans-activator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the first ligand; b) a second trans-activator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of the second ligand; and c) a packagable RNA genome of the retroviral particle, wherein the first transactivator regulates expression of the second transactivator, and wherein the second transactivator regulates expression of a retroviral polypeptide comprised in the viral package, such as a gag polypeptide, a pol polypeptide, and/or a pseudotyping component and optionally other polypeptides that will be incorporated into or onto the replication deficient recombinant retroviral particle and are considered toxic to the packaging cell line, e.g., HEK-293. In certain aspects, the second transactivator is itself cytotoxic to the packaging cell line. Pseudotyped components are generally capable of binding to the cell membrane of a target cell and facilitating fusion thereof, as discussed in detail herein. Thus, without being limited by theory, the system provides the ability to accumulate certain polypeptides/proteins (e.g., non-toxic proteins) that cannot or cannot substantially inhibit or are considered to be unable to inhibit proliferation or survival of mammalian cells, while culturing a population of mammalian cells for a period of days or indefinitely, and controlling the induction of polypeptides (e.g., toxic polypeptides) that are desired for a retroviral product but that are or may have been reported to be inhibitory to survival and/or proliferation of mammalian cells, until a later time when replication-defective recombinant retroviral particles will be produced and harvested. The packagable RNA genome is typically encoded by a polynucleotide operably linked to a promoter (sometimes referred to herein as a third promoter for convenience), wherein the third promoter is typically inducible by either the first transactivator or the second transactivator. In illustrative embodiments, the packagable RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is induced by the second transactivator. Thus, the packagable RNA genome may be produced at a later point in time, close to when the replication-defective recombinant retroviral particle is to be harvested.

The skilled artisan will appreciate that many different trans-activators, ligands, and inducible promoters may be used in retroviral packaging systems the modification of inducible promoters for use in a second organism (e.g., first and second eukaryotes, first and second prokaryotes, etc.) derived from a first organism (e.g., first and second prokaryotes, etc.) are known in the art, and systems based on such inducible promoters but also including other control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I alcA) gene promoters, promoters responsive to alcohol trans-activator proteins (AlcR), etc.), tetracycline regulated promoters (e.g., promoter systems including TetATATATATTotorvas, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human hormone receptor promoter systems, retinoid promoter systems, thyroid promoter systems, cuticle promoter systems, promoter systems for TetGA regulatory genes, etc.), promoter systems having regulatory promoter regulatory sites for expression of a transgene promoter, promoter system such as promoter for the promoter system of a transgene promoter, promoter system for the promoter system of a promoter for the rat glucocorticoid receptor, promoter system of a promoter for expression of a transgene, a promoter, promoter system for controlling the promoter of a transgene, a promoter system, a promoter of a promoter system for controlling the expression of a promoter of a transgene, and a promoter of a promoter.

In some embodiments, either or both of the trans-activators may be separated into two or more polypeptides, in some embodiments, the two or more polypeptides may include a DNA binding domain and an activation domain capable of stimulating transcription on the individual polypeptides, such "activation domain" is not confused with "an activation component," such as a polypeptide that binds to CD3, which is capable of activating T cells and/or NK cells, and which is typically activated upon contact with such T cells and/or NK cells, as discussed in detail herein.

In some embodiments, the first transactivator regulates expression of a component to control nuclear export of transcripts containing a consensus sequence, such as HIV Rev, and the consensus sequence may be a Rev responsive component. In an illustrative embodiment, the target cell is a T cell.

In some embodiments, the pseudotyping component is a retroviral envelope polypeptide. The pseudotyped component generally includes a binding polypeptide and a fusion polypeptide for binding the target cell to the viral membrane and facilitating membrane fusion of the target cell to the viral membrane, as discussed in more detail herein. In some embodiments, the pseudotyped component is a feline endogenous virus (RD114) envelope protein, a tumor retrovirus amphotropic envelope protein, a tumor retrovirus hydrophile envelope protein, and/or a vesicular stomatitis virus envelope protein (VSV-G). In illustrative embodiments, the pseudotyped component includes a binding polypeptide and a fusion polypeptide derived from a different protein, as discussed in further detail herein. For example, in illustrative embodiments, particularly when the target cell is a T cell and/or NK cell, the binding polypeptide is a hemagglutinin (H) polypeptide of measles virus (e.g., the Edmonston strain of measles virus (Edmonston strain)) or a cytoplasmic domain deletion variant thereof, and the other fusion polypeptide is a fusion (F) polypeptide of measles virus (e.g., the Edmonston strain of measles virus) or a cytoplasmic domain deletion variant thereof. In some embodiments, a fusion polypeptide can include multiple components expressed as one polypeptide. In some embodiments, the binding and fusion polypeptides may be translated from the same transcript, but from different ribosome binding sites, or the polypeptides may be cleaved post-translation using a peptide cleavage signal or a ribosome skip sequence, as disclosed elsewhere herein, to produce the binding and fusion polypeptides. In some embodiments, when the binding polypeptide is a measles virus H polypeptide or a cytoplasmic domain thereof deletion and the fusion polypeptide is a measles virus F polypeptide or a cytoplasmic domain thereof deletion, translation of the F polypeptide and the H polypeptide from the isolated ribosome binding site produces a higher amount of the F polypeptide than the H polypeptide. In some embodiments, the ratio of F polypeptide (or cytoplasmic domain deletion thereof) to H polypeptide (or cytoplasmic domain deletion thereof) 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 first transactivator can modulate the expression of an activation module capable of binding to and activating a target cell (such as a T cell or NK cell). Any of the activation components disclosed herein may be expressed. For example, in these embodiments, the activation assembly may include: a.) a membrane-binding polypeptide capable of binding to and activating CD 3; and/or b.) a membrane-bound polypeptide capable of binding to and activating CD 28. In some embodiments, a membrane-bound polypeptide capable of binding to and activating CD3 may be an anti-CD 3 antibody. In other embodiments, the anti-CD 3 antibody can be anti-CD 3 scfvffc. In some embodiments, the membrane-binding polypeptide capable of binding to and activating CD28 is CD80, CD86, or a functional fragment thereof, such as the extracellular domain of CD 80.

In some embodiments, the second transactivator may modulate the expression of a packagable RNA genome, which may include an RNA encoding one or more target polypeptides, including by way of non-limiting example any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g., two or more) inhibitory RNA molecules. It should be noted that the aspects of the retroviral packaging system and the aspects of the method for preparing replication deficient recombinant retroviral particles are not restricted to the preparation of replication deficient recombinant retroviral particles for the transduction of T cells and/or NK cells, but also apply to any cell type that can be transduced by replication deficient recombinant retroviral particles. In some illustrative embodiments, the packagable RNA genome is designed to express one or more target polypeptides, including, as non-limiting examples, any one of the engineered signaling polypeptides disclosed herein and/or one or more (e.g., two or more) inhibitory RNA molecules that are oppositely oriented (e.g., encoded on opposite strands and in opposite directions) to retroviral components such as gag and pol. For example, the packagable RNA genome comprises 5 'to 3': a 5' long terminal repeat or an active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component; nucleic acid sequences encoding a first target polypeptide, and optionally a second target polypeptide, such as, but not limited to, engineered signaling polypeptides in opposite orientations that can drive a promoter in such opposite direction relative to a 5' long terminal repeat and cis-acting RNA packaging component, which promoter is referred to in some embodiments, for convenience only, as a "fourth" promoter (and sometimes referred to herein as a promoter active in T cells and/or NK cells) that is active in a target cell, such as a T cell and/or NK cell, but in illustrative examples, is not active in a packaging cell, or has only inducible or minimal activity in a packaging cell; and 3' long terminal repeats or active truncation thereof. In some embodiments, the packagable RNA genome can include a central polypurine region (cPPT)/Central Termination Sequence (CTS) component. In some embodiments, the retroviral cis-acting RNA packaging component can be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging component can be a Rev-responsive component. In exemplary embodiments, the engineered signaling polypeptide driven by a promoter oriented opposite the 5' long terminal repeat is one or more of the 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 WO2018/161064a 1.

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

In some embodiments, the engineered signaling polypeptide can include a first lymphoproliferative component. Suitable lymphoproliferative components are disclosed elsewhere herein. As a non-limiting example, a lymphoproliferative component can be expressed as a fusion with a recognition domain, 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 may 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 where the target cell is a T cell, a promoter active in the target cell is active in the T cell, as disclosed elsewhere herein.

In some embodiments, the engineered signaling polypeptide may comprise a CAR, and the nucleic acid sequence may encode any of the CAR embodiments provided herein. For example, the engineered polypeptide may 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 component. In some embodiments, wherein the target cell is a T cell or an 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, the packagable RNA genome may further comprise a riboswitch, as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1 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 'L TR and the 3' L TR.

In another aspect of this document there is provided a mammalian packaging cell line comprising a packageable RNA genome of a replication defective retroviral particle, wherein the packageable RNA genome comprises:

a 5' long terminal repeat or an activated fragment thereof;

b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c. a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acids encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acids encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain; and

a 3' long terminal repeat or an activated fragment thereof.

The inhibitory RNA molecules in the above aspects may include any of the inhibitory RNA molecules provided in other parts of the disclosure, as non-limiting examples, shRNA or miRNA.

In some embodiments of aspects of the mammalian packaging cell line, the polynucleotide of (c) may be in an opposite orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging component (b), the 5 'long terminal repeat (a), and/or the 3' long terminal repeat (d), e.g., relative to the orientation established by the 5 'L TR and the 3' L TR.

In some embodiments of the mammalian packaging cell line aspects, expression of the packagable RNA genome is driven by an inducible promoter active in the mammalian packaging cell line.

In illustrative embodiments, this promoter active in T cells and/or NK cells is located on the packagable RNA genome between nucleic acids encoding one (e.g., two) or more inducible RNAs and CARs and 3' L TR.

In any of the aspects involving a packageable cell or cell line encoding one or more inhibitory RNA molecules directed against one or more RNA targets, at least one inhibitory RNA molecule, and in some embodiments, all inhibitory RNA molecules, can comprise a5 'strand and a 3' strand that are partially or fully complementary to each other, wherein the 5 'strand and the 3' strand are capable of forming an 18 to 25 nucleotide RNA double helix. In some embodiments, the 5 'strand length can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and the 3' strand length can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the 5 'and 3' strand lengths may be the same or different. In some embodiments, the RNA duplex may include one or more mismatches. In alternative embodiments, the RNA duplex has no mismatches.

In any of the aspects provided above for the packageable cells or cell lines herein that encode inhibitory RNA molecules directed to one or more RNA targets, the inhibitory RNA molecule can be a miRNA or shRNA. In some embodiments, the inhibitory molecule may be a precursor of a miRNA, such as a Pri-miRNA or Pre-miRNA, or a precursor of a shRNA. In some embodiments, the one or more inhibitory RNA molecules can be an artificially-derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule can be dsRNA processed into siRNA (transcribed or artificially introduced) or siRNA itself. In some embodiments, the inhibitory RNA molecule can be a miRNA or shRNA having a sequence not found in nature, or having at least one functional segment not found in nature, or having a combination of functional segments not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules is miR-155.

In any of the aspects provided above for a packageable cell or cell line encoding an inhibitory RNA molecule directed to one or more RNA targets, the one or more inhibitory RNA molecules may, in some embodiments, comprise a5 'to 3' orientation: a 5' arm, a 5' stem, a loop, a 3' stem that is partially or fully complementary to the 5' stem, and a 3' arm. In some embodiments, at least one of the two or more inhibitory RNA molecules has this arrangement. In other embodiments, all of the two or more inhibitory RNA molecules have this arrangement. In some embodiments, the 5' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop length can be 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the 5 'arm, the 3' arm, or both are derived from a naturally occurring miRNA. In some embodiments, the 5 'arm, the 3' arm, or both are derived from a naturally occurring miRNA selected from the group consisting of: miR-155, miR-30, miR-17-92, miR-122 and miR-21. In illustrative embodiments, the 5 'arm, the 3' arm, or both are derived from miR-155. In some embodiments, the 5 'arm, the 3' arm, or both are derived from mus musculus miR-155 or homo sapiens miR-155. In some embodiments, the 5' arm has the sequence set forth in SEQ ID No. 256 or is a functional variant thereof, such as being the same length as SEQ ID No. 256, or is 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID No. 256, or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 256. In some embodiments, the 3' arm has the sequence set forth in SEQ ID No. 260 or is a functional variant thereof, e.g., the same length as SEQ ID No. 260, or is 95%, 90%, 85%, 80%, 75%, or 50%, or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less, of the length of SEQ ID No. 206; and a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 260. In some embodiments, the 3' arm comprises nucleotides 221 to 283 of the mus musculus BIC.

In another aspect, provided herein is a method for making a replication-defective recombinant retroviral particle comprising: culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed from a constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the first ligand, and wherein expression of a second transactivator is regulated by the first transactivator; incubating a population of packaging cells comprising the accumulated first transactivator in the presence of a first ligand to accumulate a second transactivator, wherein the second transactivator is capable of binding a second ligand and a second inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the second ligand; and incubating the packaging cell population comprising the accumulated second transactivator in the presence of a second ligand to induce expression of retroviral polypeptides involved in viral packaging, such as a gag polypeptide, pol polypeptide and/or pseudotyping module, and optionally other polypeptides thought to inhibit proliferation or survival of mammalian cells, which will be incorporated into or onto the replication-defective recombinant retrovirus, thereby producing the replication-defective recombinant retroviral particle. In illustrative embodiments, the packagable RNA genome is encoded by a polynucleotide operably linked to a promoter (sometimes referred to as a "third" promoter for convenience), wherein the third promoter is constitutively active or inducible by the first transactivator, or in illustrative embodiments by the second transactivator, to produce the replication-defective recombinant retroviral particle. The pseudotyped module is generally capable of binding to the cell membrane of the target cell and promoting fusion of the target cell membrane with the membrane of the replication-defective recombinant retroviral particle. The pseudotyped component can be any envelope protein known in the art. In some embodiments, the envelope protein may be a vesicular stomatitis virus envelope protein (VSV-G), a feline endogenous virus (RD114) envelope protein, a tumor retroviral amphotropic envelope protein, and/or a tumor retroviral hydrophile envelope protein. It will be understood by those skilled in the art that a variety of different transactivators, ligands, and inducible promoters may be used in the process for preparing replication-defective recombinant retroviral particles. Suitable transactivators, ligands, and inducible promoters are disclosed elsewhere herein, including above. The skilled artisan will further appreciate that the above teachings relating to the retroviral packaging system aspects provided herein also apply to methods of preparing replication-defective recombinant retroviral particle aspects, and vice versa.

In some embodiments, the expression of the first trans-activator regulatory element controls the nuclear export of transcripts containing a consensus sequence, such as HIV Rev, and the consensus sequence may be the Rev-responsive element (RRE). in illustrative embodiments, the target cell is typically a T cell.

In some embodiments, the pseudotyped component is a viral envelope protein. Pseudotyped analytical modules generally include binding and fusion polypeptides for binding the virus to a target cell membrane and promoting membrane fusion of the virus to the target cell membrane. In some embodiments, the pseudotyped component can be a feline endogenous virus (RD114) envelope protein, a tumor retrovirus amphotropic envelope protein, a tumor retrovirus hydrophile envelope protein, and/or a vesicular stomatitis virus envelope protein (VSV-G). In illustrative embodiments, the pseudotyped component includes a binding polypeptide and a fusion polypeptide derived from a different protein, as discussed in further detail herein. For example, in an illustrative embodiment, particularly where the target cell is a T cell and/or NK cell, the binding polypeptide can be a cytoplasmic domain deleted variant of a measles virus H polypeptide and the fusion polypeptide can be a cytoplasmic domain deleted variant of a measles virus F polypeptide. In some embodiments, a fusion polypeptide can include multiple components expressed as one polypeptide. In some embodiments, the binding and fusion polypeptides may be translated from the same transcript and from different ribosome binding sites, or the polypeptides may be cleaved post-translation using a peptide cleavage signal or a ribosome skip sequence, as disclosed elsewhere herein, to produce binding and fusion polypeptides. In some embodiments, translation of the binding polypeptide and the fusion polypeptide from the 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 first transactivator modulates expression of an activation module capable of binding to and activating a target cell, such as a T cell. In these embodiments, the activation assembly may include: a.) a membrane-binding polypeptide capable of binding to and activating CD 3; and/or b.) a membrane-bound polypeptide capable of binding to and activating CD 28. In some embodiments, the membrane-bound polypeptide capable of binding to and activating CD28 is CD80, CD86, or a functional fragment thereof. In some embodiments, the replication-defective recombinant retroviral particle may comprise an activating module on the retroviral membrane and the retroviral RNA within the nucleocapsid, thereby producing the replication-defective recombinant retroviral particle.

In some embodiments, the second transactivator modulates expression of an RNA comprising from 5 'to 3': a 5' long terminal repeat or an active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component; a nucleic acid sequence encoding a first target polypeptide and optionally a second target polypeptide (as non-limiting examples, one or two engineered signaling polypeptides); a promoter active in a target cell; and 3' long terminal repeats or active truncation thereof. In some embodiments, the RNA can include a cPPT/CTS component. In some embodiments, the RNA can include a primer binding site. In some embodiments, the retroviral cis-acting RNA packaging component can be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging component can be a Rev-responsive component. In any of the embodiments disclosed herein, the retroviral component on the RNA (including the RRE and Psi) may be located at any position, as will be understood by those of skill in the art. In illustrative embodiments, the engineered signaling polypeptide is one or more of the engineered signaling polypeptides disclosed herein.

In some embodiments, the engineered signaling polypeptide may comprise a first lymphoproliferative component suitable lymphoproliferative components are disclosed elsewhere herein, in some illustrative embodiments, the lymphoproliferative component is an I L-7 receptor mutant fused to a recognition domain, such as etag.

In some embodiments, the packagable RNA genome may further include a riboswitch, as discussed in WO2017/165245a2, WO2018/009923a1, and WO2018/161064a 1. In some embodiments, the nucleic acid sequence encoding the engineered signaling polypeptide may be in a reverse orientation. In other embodiments, the packagable RNA genome can further include a riboswitch, and optionally the riboswitch can be in a reverse orientation. In any of the embodiments disclosed herein, a polynucleotide comprising any of the components can comprise a primer binding site. In illustrative embodiments, a transcription blocker or polyA sequence may be placed near a gene to prevent or reduce unregulated transcription. In any of the embodiments disclosed herein, the nucleic acid sequence encoding Vpx may be located on the second transcription unit or on an optional third transcription unit, or on an additional transcription unit operably linked to the first inducible promoter.

In some embodiments of the packaging systems or methods for making the replication-defective recombinant retroviral particle state, the encoded RNA can include an intron, which can be, for example, transcribed from the same promoter used to express the target polypeptide. In certain illustrative embodiments, such introns may encode 1, 2, 3, or 4 mirnas. In these and other embodiments of the packaging systems or methods for making the replication-defective recombinant retroviral particle state, the packageable RNA genome size is 11,000KB or less, and in some examples 10,000KB or less.

For example, the first transactivator may induce expression of retroviral proteins Rev and Vpx in addition to the polypeptide to be transported to the cell membrane of the packaging cell, and the second transactivator may induce expression of retroviral proteins GAG, PO L, mv (ed) -F Δ 30, and mv (ed) -H Δ 18 or mv (ed) -H Δ 24, and expression of the lentiviral genome.

In another aspect, provided herein is a mammalian packaging cell comprising a) a first transcription unit in the genome of the mammalian packaging cell comprising a nucleic acid sequence encoding a first transactivator, wherein the first transcription unit is operably linked to a constitutive promoter, and wherein the transactivator is capable of binding to a first inducible promoter and affecting expression of the nucleic acid sequence operably linked thereto in the presence and absence of a first ligand, and wherein the first transactivator is capable of binding the first ligand, b) a second transcription unit and optionally a third transcription unit in the genome of the mammalian packaging cell comprising a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator, the second transactivator being capable of binding to a second inducible promoter and affecting expression of the nucleic acid sequence operably linked thereto in the presence and absence of a second ligand, wherein the second transactivator is capable of binding to the second ligand, and wherein the second transcription unit and optionally the third transcription unit are operably linked to a sixth transcription unit of a fusion polypeptide encoding a cellular membrane-inducible polypeptide, a targeting polypeptide, a targeting sequence encoding a polypeptide, a targeting polypeptide, a targeting sequence encoding a polypeptide, a polypeptide.

In another aspect, provided herein are methods for making a replication-defective recombinant retroviral particle comprising 1) culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise a) a first transcriptional unit in the genome of a mammalian packaging cell comprising a nucleic acid sequence encoding the first transactivator, wherein the first transcriptional unit is operably linked to a constitutive promoter, and wherein the transactivator is capable of binding to a first inducible promoter and in the presence or absence of the first ligand affecting expression of a nucleic acid sequence operably linked thereto, and wherein the first transactivator is capable of binding to the first ligand, b) a second transcriptional unit in the genome of a mammalian packaging cell and optionally a third transcriptional unit comprising a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator, the second trans activator is capable of binding to a second inducible promoter and in the presence or absence of a second transcriptional unit encoding a retroviral REV protein 355, wherein the second trans-inducible promoter is capable of binding to a second inducible promoter and in the presence or absence of a second ligand capable of binding to a target cell membrane-transducing polypeptide, wherein the second transcriptional unit encodes a chimeric polypeptide, a chimeric polypeptide comprising a chimeric retroviral TR 5, a chimeric polypeptide, a chimeric promoter capable of binding to a chimeric polypeptide, a chimeric polypeptide comprising a chimeric promoter, a chimeric polypeptide, a.

In one aspect provided herein, a retroviral packaging system can include a mammalian cell that includes 1) a first transactivator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the first ligand, 2) a second transactivator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of the second ligand, and 3) a packagable RNA genome of a reverse transcriptase particle, wherein the first transactivator modulates expression of the second transactivator, HIV REV, I L GPI DAF and an activating component, and wherein the second transactivator modulates expression of a GAG polypeptide, pol polypeptide, retroviral cis-acting RNA, and one or more envelope polypeptides in illustrative embodiments, the first transactivator can be placed adjacent to a p65 activating domain and a rapamycin domain of a rapamycin-inducible promoter, and a second retroviral regulatory region of a rapamycin-related polypeptide can be placed adjacent to a rapamycin-related promoter, and the second transactivatable promoter can be used to induce transcription of a rapamycin-related proteins including a tetracycline-related promoter, a retroviral gene, a retroviral regulatory polypeptide, 2, and a retroviral regulatory polypeptide can be used in illustrative embodiments, a retroviral regulatory region of a retroviral regulatory polypeptide, a retroviral regulatory region of a retroviral-related to induce a retroviral regulatory DNA, a retroviral regulatory polypeptide, a regulatory region of a retroviral regulatory polypeptide, a regulatory region of a regulatory polypeptide, a regulatory region of a regulatory polypeptide, a.

Some aspects of the invention include or are cells, in illustrative examples mammalian cells used as packaging cells to prepare replication-defective recombinant retroviral particles, such as lentiviral particles for transduction of T cells and/or NK cells. Any of a wide variety of cells can be selected to produce viruses or viral particles in vitro, such as recombinant retroviral particles 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 commonly used. In an illustrative example, the cell is a human cell. In other illustrative embodiments, the cells proliferate indefinitely, and thus are immortal. Examples of cells that can be advantageously used in the present invention include NIH3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells, and any cell derived from such cells, such as gpnlsllacZ

A cell derived from 293T cells. Highly transfectable cells, such as human embryonic kidney, may be used293T cells. By "highly transfectable" is meant that at least about 50%, preferably at least about 70%, and optimally at least about 80% of the cells express the gene of the introduced DNA.

Suitable mammalian cell lines include, but are not limited to, He L a cells (e.g., American Type Culture Collection (ATCC) No. CC L-2), CHO cells (e.g., ATCC No. CR L09618, CC L, CR L29096), 293 cells (e.g., ATCC No. CR L-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CR L-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CC L5965), PC12 cells (ATCC No. CR 631721), COS cells, COS-7 cells (ATCC No. CR L), RATl cells, mouse 356 cells (ATCC No. L I.3), HENK No. 15742 cells (ATCC No. CR L), human embryo L cells (ATCC No. CR 15727), and similar cell lines pG L, pG NO. H L, and similar cells.

In any of the embodiments disclosed herein, a method of making a replication-defective recombinant retroviral particle may comprise growing a mammalian packaging cell to 50%, 60%, 70%, 80%, 90% or 95% confluence or confluence, or to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density or peak cell density, and then dividing or diluting the cell. In some embodiments, a stirred tank reactor may be used to grow the cells. In some embodiments, the cells can be divided by at least about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, or 1:20 using methods that will be understood by the skilled artisan. In some embodiments, the cells may be diluted to a peak cell density of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, after dividing or diluting the cells, the cells can be grown for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, or 16 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days before adding the first ligand. In some embodiments, the cells are grown in the presence of a first ligand, which in illustrative embodiments can be rapamycin or a rapamycin analog, for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, or 28 days. In some embodiments, a second ligand can be added and the cells grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, or 28 days, in illustrative embodiments, the second ligand can be tetracycline or doxycycline. The culture conditions will depend on the cells and ligands used and the methods are known in the art. Specific examples of conditions for culturing and inducing HEK293S cells are shown in example 2.

As disclosed herein, replication-defective recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992)357: 455-460.) the ability of replication-defective recombinant retroviral particles to deliver unaligned 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, replication-defective recombinant retroviral particles can be derived from α retrovirus, β retrovirus, gammaretrovirus, retrovirus, lentivirus, or foamy virus there are a number of Retroviruses suitable for the methods disclosed herein, for example, murine leukemia virus (M L V), Human Immunodeficiency Virus (HIV), equine infectious Anemia Virus (AV), Mouse Mammary Tumor Virus (MMTV), Rous Sarcoma Virus (RSV), Fusarium sarcoma virus (FuSV), Mooney leukemia virus (Mo-M L V), FBR sarcoma virus (FBR V), murine sarcoma virus (MSR V), Rous sarcoma virus (MSV) (see, Genewe. J.: adenovirus Genome, murine leukemia virus, murine adenovirus virus NO. 76, murine adenovirus virus Genome, murine adenovirus virus NO. 76, murine adenovirus virus NO. 9, murine leukemia virus, murine adenovirus virus Genome, murine adenovirus No. 9, murine adenovirus No. 5-9, murine leukemia virus, murine adenovirus virus No. 5, murine adenovirus No. 5 virus, murine adenovirus virus No. 5 virus, murine adenovirus, murine leukemia virus, murine adenovirus No. 9 virus No. 9, murine leukemia virus.

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

In illustrative embodiments, the replication-defective recombinant retroviral particles provided herein contain a Vpx polypeptide that can be expressed in a packaging cell line following integration of a Vpx-encoding nucleic acid in its genome, e.g., as a cell membrane-binding protein incorporated into the retroviral membrane (Durand et al, J.Virol. (2013)87: 234-.

Without being limited by theory, Vpx polypeptide contributes to the transduction of resting cells by stimulating the efficiency of the reverse transcription process by degrading the limiting factor SAMHD 1. Thus, it is contemplated that in the methods provided herein, wherein Vpx is present in replication deficient recombinant retroviral particles used to transduce T cells and/or NK cells, Vpx is released into the cytoplasm of resting T cells or resting NK cells after transduction of the cells by the replication deficient recombinant retroviral particles containing Vpx. Vpx then degrades SAMHD1, which allows for an increase in free dntps, which in turn stimulates reverse transcription of the retroviral genome.

In some embodiments, the replication-defective recombinant retroviral particles provided herein comprise and/or contain a Vpu polypeptide. The Vpu polypeptide can be expressed from a plasmid or packaging cell line after integration of the Vpu-encoding nucleic acid in its genome, for example as a viral membrane protein incorporated into a retroviral membrane. Recombination formed by Vpu with its sequence codons optimized for expression in humans can be constructed and expressed such that free Vpu can be incorporated into viral particles. Vpu is an accessory protein found in HIV-1 and some HIV-1 related Simian Immunodeficiency Virus (SIV) isolates such as SIVcpz, SIVgsn and SIVmon, but not in HIV-2 or most SIV isolates. Vpu protein is involved in the down-regulation of CD 4and antagonism of tethered proteins for granule release. More importantly, Vpu shares structural similarity with viral porins, and in particular, the viral porin M2 from influenza a (iav), and as such, Vpu is shown to form cation selective ion channels and to permeate membranes in various models. It has been shown that IAV-M2 helps to acidify the particles and thus facilitates early release from the endosomal pathway for entry, reducing the amount of end-transported product.

Example 4 herein demonstrates that transduction of resting PBMCs by retroviral particles is potentiated by the presence of Vpu in the retroviral particles. Without being limited by theory, Vpu polypeptide aids in the transduction of resting cells by accelerating acidification of the lentiviral pseudotype, thereby allowing more capsids to reach the cytoplasm more quickly. Acidification of the virus interior leads to weakening of electrostatic interactions between viral structural proteins and thus helps to break down the capsid and ribonucleic acid protein (RNP) complexes. Thus, it is believed that in the methods provided herein, wherein Vpu is present in the membrane of replication-defective recombinant retroviral particles used to transduce T cells and/or NK cells, Vpu accelerates acidification of the lentiviral particles in the endosomal pathway upon transduction of the cells by the replication-defective recombinant retroviral particles containing Vpu, and facilitates release of the capsid into the cytoplasm of resting T cells and/or NK cells. Thus, in some embodiments, among others, a Vpu polypeptide comprising or being a fragment of Vpu that retains the ability to promote transduction of resting PBMCs (and in some embodiments, resting T cells). The fragment may comprise a fragment of Vpu having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to wild-type Vpu of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the Vpu polypeptide comprises a Vpu fragment that retains the ability to promote acidification of lentiviral particles in the endoplasmic pathway.

Retroviral genome size

In the methods and compositions provided herein, the recombinant retroviral genome (in a non-limiting illustrative example, a lentiviral genome) 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 is expressed from a single promoter. In some embodiments, the fusion polypeptide can have a cleavage signal to produce two or more functional polypeptides from one fusion polypeptide and one promoter. Furthermore, some functions that are not required after the initial ex vivo transduction are not included in the retroviral genome but are present on the surface of the replication defective recombinant retroviral particle via the packaging cell membrane. These different strategies are used herein to maximize the functional modules packaged within replication-defective recombinant retroviral particles.

In some embodiments, the recombinant retroviral genome to be packaged may comprise between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000 and 8,000 nucleotides at the lower 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 at the upper end of the range, the retroviral genome to be packaged comprises one or more polynucleotide regions encoding first and second engineered signaling polypeptides as disclosed in detail herein, in some embodiments, the retroviral genome to be packaged may comprise less than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides, packageable functions discussed elsewhere include targeting of a retroviral sequence for assembly and packaging, such as a retroviral targeting domain for expression of a chimeric targeting polypeptide encoding a targeting a targeting polypeptide, such as a targeting domain for targeting a targeting T3 TR 3, a targeting domain for targeting a targeting RNA targeting a targeting polypeptide, a targeting domain for targeting RNA targeting a targeting polypeptide, a targeting RNA targeting polypeptide, a targeting.

Recombinant retroviral particles

Recombinant retroviral particles are disclosed in the methods and compositions provided herein, for example, to transduce T cells and/or NK cells to produce genetically modified T cells and/or NK cells. Recombinant retroviral particles are themselves aspects of the present invention. Typically, a recombinant retroviral particle included in the aspects provided herein is replication-defective, meaning that the recombinant retroviral particle is incapable of replication when it leaves the packaging cell. In an illustrative embodiment, the recombinant retroviral particle is a lentiviral particle.

In some aspects, provided herein is a replication-deficient recombinant retroviral particle for transducing a cell, typically a lymphocyte and in illustrative embodiments a T cell and/or an NK cell, the replication-deficient recombinant retroviral particle may comprise any of the pseudotyping components discussed elsewhere herein in some embodiments, the replication-deficient recombinant retroviral particle may comprise any of the activation components discussed elsewhere herein in one aspect, provided herein is a replication-deficient recombinant retroviral particle comprising a polynucleotide comprising a transcription unit or units a, operably linked to a promoter active in a T cell and/or an NK cell, wherein the transcription unit or units encode a Chimeric Antigen Receptor (CAR), and b, a pseudotyping component on its surface and a T cell activation component, wherein the T cell activation component is not encoded by the polynucleotide in the replication-deficient recombinant retroviral particle, in some embodiments, the T cell activation component may be any of the activation components discussed elsewhere herein, in some embodiments, the T cell activation component may be an anti-CD 34 transcription component, in illustrative embodiments, a chimeric lymphocyte-c 3, in an illustrative embodiment, a chimeric lymphocyte-c.

In some aspects, provided herein is a replication-defective recombinant retroviral particle 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 a first polypeptide comprising a Chimeric Antigen Receptor (CAR) and a second polypeptide comprising a chimeric lymphoproliferative component (e.g., a constitutively active chimeric lymphoproliferative component). In illustrative embodiments, the chimeric lymphoproliferative component does not comprise an interleukin linked to its cognate receptor or to a fragment of its cognate receptor.

In some aspects, provided herein is a recombinant retroviral particle comprising (i) a pseudotyped component capable of binding to T cells and/or NK cells and promoting membrane fusion of the recombinant retroviral particle; (ii) a polynucleotide having one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide having a chimeric antigen receptor (including an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain), and a second engineered signaling polypeptide comprising at least one lymphoproliferative component; wherein expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is modulated by an in vivo control component; and (iii) an activating module on its surface, wherein the activating module is capable of binding to T cells and/or NK cells and is not encoded by the polynucleotide in the recombinant retroviral particle. In some embodiments, a promoter active in T cells and/or NK cells is not active in a packaging cell line, or is only active in an inducible manner in a packaging cell line. In any of the embodiments disclosed herein, one of the first and second engineered signaling polypeptides may have a chimeric antigen receptor and the other engineered signaling polypeptide may have at least one lymphoproliferative component.

Provided throughout the invention are various components and combinations of components, such as pseudotyping components, activating components, and membrane-bound interleukins, included in replication-defective recombinant retroviral particles, as well as nucleic acid sequences, such as but not limited to nucleic acids encoding CARs, included in the genome of the replication-defective recombinant retroviral particles; a nucleic acid encoding a lymphoproliferative component; nucleic acids encoding control components (such as riboswitches); promoters, particularly promoters that are constitutively active or inducible in T cells; and nucleic acids encoding inhibitory RNA molecules. In addition, the various aspects provided herein (such as methods of making recombinant retroviral particles, methods of performing adoptive cell therapy, and methods of transducing T cells) produce and/or include replication-defective recombinant retroviral particles. The replication deficient recombinant retroviruses produced and/or included in such methods themselves form, as an independent aspect of the invention, replication deficient recombinant retroviral particle compositions, which 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 known in the art for storage and use of the retroviral particles.

Thus, as a non-limiting example, in another aspect, provided herein is a replication-deficient recombinant retroviral particle having in its genome a polynucleotide having one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, in some cases the nucleic acid sequence comprising a first nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence encoding a chimeric antigen receptor or CAR, as described herein, in other embodiments there is a third nucleic acid sequence encoding at least one lymphoproliferative component that is not an inhibitory RNA molecule previously described herein.

Recombinant retroviral particle embodiments herein include those in which the retroviral particle comprises a genome comprising one or more nucleic acids encoding one or more inhibitory RNA molecules. Various alternative embodiments of such nucleic acids encoding inhibitory RNA molecules that can be included in the genome of a retroviral particle, including combinations of such nucleic acids with other nucleic acids encoding CARs or lymphoproliferative components other than inhibitory RNA molecules, are included in, for example, the inhibitory RNA portions provided herein, as well as in various other paragraphs that combine these embodiments. Furthermore, various alternatives to such replication-defective recombinant retroviruses can be identified by the exemplary nucleic acids disclosed within the packaging cell line aspects disclosed herein. The skilled artisan will recognize that the disclosure of this portion of the recombinant retroviral particle, which includes 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 inhibitory RNA molecules provided elsewhere herein. Furthermore, the skilled artisan will recognize that such nucleic acids encoding one or more inhibitory RNA molecules can be combined with various other functional nucleic acid components provided herein, as disclosed, for example, herein with emphasis on inhibitory RNA molecules and the nucleic acids encoding such molecules. Furthermore, the various embodiments of specific inhibitory RNA molecules provided elsewhere herein can be used in the recombinant retroviral particle aspects of the present invention.

For example, lentiviral particles typically include packaging components REV, GAG, and PO L, which may be delivered to the packaging cell line via one or more packaging plasmids, pseudotyping components, various examples provided herein, which may be delivered to the packaging cell line via pseudotyped plasmids, and genomes, which result from polynucleotides delivered to the host cell via transfer plasmids, such polynucleotides typically including viral L TR and psi packaging signal.5 'L TR ", may be chimeric 5' L TR fused to a heterologous promoter, which includes 5 'L TR. transfer plasmids that are self-inactivating, e.g., by removal of the U3 region of 3' L TR, in some non-limiting embodiments, for any composition or method provided herein including retroviral particles, such as the same and embodiments, the inclusion of Vpu-like and Vpu-packaging polypeptides in a non-limiting Vpu-like packaging (Vpu-Vpu-like) to facilitate the intracellular packaging of a non-Vpu-like polypeptide, such as Vpu-packaging polypeptides, and Vpu-like polypeptides provided herein, in some non-Vpu-packaging embodiments, including Vpu-packaging polypeptides, and Vpu-vp.

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

The skilled artisan will recognize that the functional modules discussed herein may be delivered to packaging cells and/or to T cells using different types of vectors such as expression vectors illustrative aspects of the invention utilize retroviral vectors, and in some particular illustrative embodiments lentiviral vectors other suitable expression vectors may be used to achieve certain embodiments herein such expression vectors include, but are not limited to, viral vectors (e.g., based on vaccinia Virus; poliovirus; adenovirus (see, e.g., L i et al; Invest Opthalmol Vis Sci 35: 25432549,1994; Borras et al, Gene Therr 6: 515524,1999; L i and Davidson, PNAS 92: 77007704,1995; Kamoto et al, H Gene Ther 5: 10881097,1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 35356 and WO 95/00655); adeno-associated Virus (see, e.g., Ali et al, the SaemGen Hur 9: WO 27, the Sar Alrol Virus; the Sarshiy Virus; the human Sarcoma Virus; WO 28; bovine Sarcoma Virus; such as 19810: 468; bovine leukemia Virus; bovine leukemia; 2; see, et al; 35; 3695; 35; P35; P35; P35; P35; P35; P35; P.

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, a DNA-containing viral particle is used in place of a recombinant retroviral particle. Such viral particles may be adenovirus, adeno-associated virus, herpes virus, cytomegalovirus, poxvirus, vaccinia virus, influenza virus, Vesicular Stomatitis Virus (VSV), or Sindbis virus (Sindbis virus). The skilled artisan will understand how to modify the methods disclosed herein for different viruses and retroviruses or retroviral particles. When using viral particles comprising a DNA genome, the skilled artisan 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 a T cell transduced with such a virus.

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

In one aspect, the invention provides a container (such as a commercial container or package) or a kit comprising the same, comprising an isolated replication-defective recombinant retroviral particle according to any one of the replication-defective recombinant retroviral particle aspects provided herein. Moreover, in another aspect, provided herein is a container (such as a commercial container or package) or a kit comprising the same, comprising an isolated packaging cell according to any of the packaging cell and/or packaging cell line aspects provided herein, in illustrative embodiments, 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 deficient recombinant retroviral particles provided herein in any aspect, in the manufacture of a kit for genetically modifying T cells or NK according to any aspect provided herein. Furthermore, in certain aspects, provided herein is the use of any packaging cell and/or packaging cell line provided herein in any aspect, in the manufacture of a recombinant retroviral particle for use in the production of a replication deficient recombinant retroviral particle according to any aspect provided herein.

In one aspect, provided herein are commercial containers comprising a replication-defective recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, and instructions for use in treating tumor growth in an individual. In some embodiments, a nucleic acid sequence of the one or more nucleic acid sequences can encode a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, a nucleic acid sequence of the one or more nucleic acid sequences can encode one or more inhibitory RNA molecules directed against one or more RNA targets.

The container containing the recombinant retroviral particles can be a tube, vial, well plate, or other container for storing the recombinant retroviral particles. The kit may comprise two or more containers, wherein a second or other container may comprise, for example, a solution or medium for transducing T cells and/or NK cells, and/or the second or other container may comprise a pH-adjusting pharmaceutical agent. Any of these containers may be of industrial strength and grade. The replication-defective recombinant retroviral particles in such aspects, including kits and nucleic acids encoding inhibitory RNA molecules, can be any of the embodiments of such replication-defective recombinant retroviral particles provided herein, including any of the embodiments of inhibitory RNA provided herein.

In another aspect, provided herein is a use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying T cells and/or NK cells, wherein the use of the kit comprises: contacting a T cell or NK cell ex vivo with a replication-defective recombinant retroviral particle, wherein the replication-defective recombinant retroviral particle comprises a pseudotyped component on the surface and a T cell activation component on the surface, wherein the contacting facilitates transduction of the T cell or NK cell by the replication-defective recombinant retroviral particle, thereby producing a genetically modified T cell or NK cell. In some embodiments, the T cell or NK cell may be from an individual. In some embodiments, the T cell activation module may be membrane bound. In some embodiments, the contacting may be performed at the lower end of the range for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours and at the upper end of the range for between 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, and 24 hours, such as between 1 hour and 12 hours. The replication-defective recombinant retroviral particles for use in manufacturing the kit may comprise any one of the aspects, embodiments, or sub-embodiments discussed elsewhere herein.

In another aspect, provided herein is a pharmaceutical composition for treating or preventing cancer or tumor growth comprising a replication-defective recombinant retroviral particle as an active ingredient. In another aspect, provided herein is an infusion composition for use in treating or preventing cancer or tumor growth comprising a replication-defective recombinant retroviral particle. The replication-defective recombinant retroviral particles of a pharmaceutical composition or an infusion composition may comprise any of the aspects, embodiments or sub-embodiments discussed above or elsewhere herein.

Genetically modified T cells and NK cells

In the embodiments of the methods and compositions herein, genetically modified lymphocytes that are themselves a separate aspect of the invention are generated. Such genetically modified lymphocytes may be transduced lymphocytes. In one aspect, provided herein are genetically modified T cells or NK cells, wherein the T cells or NK cells are genetically modified to express a first engineered signaling polypeptide. In some embodiments, the first engineered signaling polypeptide may be a lymphoproliferative component or a CAR comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the T cell or NK cell can further comprise a second engineered signaling polypeptide that can be a CAR or lymphoproliferative component. In some embodiments, the lymphoproliferative component can be a chimeric lymphoproliferative component. In some embodiments, the T cell or NK cell may further comprise a pseudotyped component on the surface. In some embodiments, the T cell or NK cell may further comprise an activating component on the surface. The genetically modified T cell or NK cell CAR, lymphoproliferative component, pseudotyping component and activating component may comprise any of the aspects, embodiments or sub-embodiments disclosed herein. In an illustrative embodiment, the activating component may be anti-CD 3 scfvffc.

Some aspects provided herein include genetically modified T cells or NK cells made by transducing resting T cells and/or resting NK cells according to a method comprising contacting resting T cells and/or resting NK cells ex vivo with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises a pseudotyping component on its surface and a membrane bound T cell activating component on its surface, wherein the contacting facilitates transduction of resting T cells and/or NK cells by the replication deficient recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells, and wherein the contacting is performed at a low end of a range of 1 hour, 2 hours, 3 hours, 4 hours, or 6 hours versus a high end of a range of 4 hours, 6 hours, 8 hours, a, Between 10 hours, 12 hours, 18 hours, 20 hours or 24 hours, for example between 1 hour and 12 hours.

In some embodiments, the genetically modified lymphocytes are lymphocytes, such as T cells or NK cells, that have been genetically modified to express a first engineered signaling polypeptide comprising at least one lymphoproliferative component and/or a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain in some embodiments of any of the aspects herein, the NK cells are NKT cells, the NKT cells are a subset that express CD3 and typically co-express αβ T cell receptors and also express a plurality of molecular markers (NK1.1 or CD56) typically associated with NK cells.

The genetically modified lymphocytes of the invention have a heterologous nucleic acid sequence that has been introduced into the lymphocytes by recombinant DNA methods. For example, the heterologous sequences in the illustrative embodiments are inserted into lymphocytes during the methods for transducing lymphocytes provided herein. The heterologous nucleic acid is found within the lymphocyte, and in some embodiments is integrated or not integrated into the genome of the genetically modified lymphocyte.

In illustrative embodiments, the heterologous nucleic acid is integrated into the genome of the genetically modified lymphocyte. In illustrative embodiments, such lymphocytes are produced using the methods provided herein for transducing lymphocytes using recombinant retroviral particles. Such recombinant retroviral particles may comprise a polynucleotide encoding a chimeric antigen receptor that typically comprises at least one Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In other parts of the invention, provided herein are various embodiments of replication-defective recombinant retroviral particles that can be used to generate genetically modified lymphocytes that themselves form another aspect of the invention, and polynucleotides encoded in the genome of the replication-defective retroviral particles.

The genetically modified lymphocytes of the invention can be isolated in vitro. For example, such lymphocytes can be found in the media and other solutions for ex vivo transduction as provided herein. Lymphocytes can be present in the blood collected from an individual in the methods provided herein in a form that has not been genetically modified, followed by genetic modification during the transduction method. The genetically modified lymphocytes can be found inside an individual after they have been genetically modified after they have been introduced or reintroduced into the individual. The genetically modified lymphocytes can be resting T cells or resting NK cells, or the genetically modified T cells or NK cells can actively divide, particularly after their expression is followed by some of the functional components provided in the nucleic acid inserted into the T cells or NK cells following transduction as disclosed herein.

In one aspect, provided herein are transduced and/or genetically modified T cells or NK cells comprising a recombinant polynucleotide comprising in its genome one or more transcription units operably linked to a promoter active in the T cells and/or NK cells, said transcription units expressing one or more of the functional components provided in any of the aspects and embodiments of the invention, for example, one or more transcription units may express a CAR, which may include any of the CAR components provided herein, such as an ASTR (as a non-limiting example MBR-ASTR), a transmembrane domain, and an intracellular signaling domain, and may further include a regulatory domain as a non-limiting example, further provided herein are one or more of the constitutive components, lymphoproliferative components, e.g., the constitutive active I L-7 receptor mutant, or other lymphoproliferative components, recognition domains, and/or abrogative domains that are not inhibitory RNA molecules (e.g., miRNA or shRNA).

In one aspect, provided herein is a genetically modified T cell or NK cell comprising:

a. one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets; and

b. a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain,

and/or nucleic acids encoding inhibitory RNA molecules and CARs directed against one or more RNA targets, wherein the one (e.g., two) or more inhibitory RNA molecules and CARs or nucleic acids encoding the same encode or are encoded by or are genetically modified nucleic acid sequences of T cells and/or NK cells.

The genetically modified T cells or NK cells can be a population of genetically modified T cells and/or NK cells that include one (e.g., two) or more inhibitory RNA molecules directed against one or more RNA targets; and a CAR.

In some embodiments of the aspects immediately above where the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and a CAR or nucleic acid encoding the same, components that may be included as part of the CAR or may be expressed with or used to control a lymphoproliferative component may include any of the embodiments provided herein.

In some embodiments immediately above the aspect in which the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and a CAR or nucleic acid encoding the same, the CAR is a microenvironment-restricted biological (MRB) -CAR and/or the genetically modified T cell or NK cell can further comprise at least one lymphoproliferative component that is not an inhibitory RNA molecule and/or a nucleic acid encoding a lymphoproliferative component.

In some embodiments of aspects immediately above where the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and the CAR or nucleic acid encoding the same, the inhibitory RNA molecule is a precursor of a miRNA or shRNA. In some embodiments of this aspect, the one (e.g., two) or more inhibitory RNA molecules are polycistronic. In some embodiments of this aspect, the one (e.g., two) or more inhibitory RNA molecules are directed against the same or (in illustrative embodiments) different RNA targets. In some embodiments of this aspect, most or all of the one (e.g., two) or more inhibitory RNA molecules reduce expression of endogenous TCRs.

In some embodiments of the immediately above aspect in which the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and the CAR or nucleic acid encoding the same, the RNA target is an mRNA transcribed from a gene selected from the group consisting of PD-1, CT L a4, TCR α, TCR β, CD3 ζ, SOCS, SMAD2, miR-155 target, IFN γ, cCB L, TRAI L2, PP2A, and abcg 1.

In some embodiments of the aspect immediately above where the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and the CAR or nucleic acid encoding the same, the ASTR of the CAR is the MRB ASTR and/or the ASTR of the CAR binds to an antigen associated with the tumor. Furthermore, in some embodiments of the above aspects, the first nucleic acid sequence is operably linked to a riboswitch, which is, for example, capable of binding a nucleoside analog, and in illustrative embodiments, an antiviral drug, such as 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 a nucleoside analog and, when present, expresses one or both of the engineered signaling polypeptides.

The genetically modified lymphocytes disclosed herein can also have on their surface a polypeptide that is the residue of the fusion of replication-defective recombinant retroviral particles during the transduction methods provided herein. Such polypeptides may include an activating module, a pseudotyping module, and/or one or more fusion polypeptides that include an interleukin.

In one aspect, provided herein are genetically modified T cells and/or NK cells that express one or more (e.g., two or more) inhibitory RNA molecules and chimeric antigen receptors or CARs against one or more RNA targets, as disclosed herein. In some embodiments, the genetically modified T cell and/or NK cell further expresses at least one lymphoproliferative component disclosed herein that is not an inhibitory RNA molecule. In certain embodiments, the genetically modified T cell and/or NK cell also expresses one or more riboswitches that control the expression of one or more inhibitory RNA molecules, a CAR, and/or at least one lymphoproliferative component that is not an inhibitory RNA molecule. In some embodiments, the genetically modified T cells and/or NK cells express two to 10 inhibitory RNA molecules. In other embodiments, the genetically modified T cells and/or NK cells express two to six inhibitory RNA molecules. In illustrative examples, genetically modified T cells and/or NK cells express four inhibitory RNA molecules.

Nucleic acids

The present invention provides nucleic acids encoding the polypeptides of the invention. In some embodiments, the nucleic acid will be DNA, including, for example, a recombinant expression vector. In some embodiments, the nucleic acid will be RNA, e.g., RNA synthesized in vivo.

In some cases, the nucleic acid provides for production of a polypeptide of the invention, e.g., in a mammalian cell. In other cases, the individual nucleic acid provides amplification of a nucleic acid encoding a polypeptide of the invention.

The nucleotide sequence encoding the polypeptide of the present invention is operably linked to transcriptional control elements such as promoters and enhancers, etc.

Suitable promoter and enhancer components are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, λ P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoters and enhancer components; cytomegalovirus immediate early promoter; a herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; a promoter present in the long terminal repeat of a retrovirus; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from a number of organisms, such as eukaryotes and prokaryotes. Modifications to reversible promoters derived from a first organism (e.g., first and second prokaryotes, etc.) for use in a second organism 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 regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoters, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal regulated promoters (e.g., metallothionein promoter system, etc.), related pathogen regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), (ii) promoter systems, Thermoregulation is at promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, etc.)), light regulated promoters, synthetic inducible promoters, and the like.

In some cases, loci or constructs or transgenes containing suitable promoters are irreversibly switched via induction of an inducible system. Suitable systems for inducing irreversible transformation are well known in the art, e.g., the induction of irreversible transformation can utilize Cre-lox mediated recombination (see, e.g., Fuhrmann-Benzakein et al, PNAS (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 generate the irreversibly switched promoter. Methods, mechanisms, and requirements for performing site-specific recombination as described elsewhere herein and well known in the art for generating promoters for irreversible transformation are described, for example, in Grindeley et al (2006) Annual Review of Biochemistry, 567-friendly 605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the disclosure of which is incorporated herein by reference.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter can be used, see, e.g., Salmonon et al (1993) Proc. Natl. Acad. Sci. USA 90:7739 and Marodon et al (2003) Blood 101: 3416. As another example, the CD8 gene promoter may be used. NK cell-specific expression can be achieved by using the Neri (page 46) promoter; see, e.g., Eckelhart et al (2011) Blood 117: 1565.

In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, and the like, or regulatable promoters, such as the GA L I promoter, the GA L10 promoter, the ADH2 promoter, the PH05 promoter, the CUP1 promoter, the GA L7 promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the L EU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia pastoris).

Suitable promoters for use In prokaryotic host cells include, but are not limited to, phage T7RNA polymerase promoter, trp promoter, lac operator promoter, hybrid promoters (e.g., lac/Tac hybrid promoter, Tac/Trc hybrid promoter, trp/lac promoter, T7/lac promoter), Trc promoter, Tac promoter and the like, araBAD promoter, promoters regulated In vivo such as ssaG promoter or related promoters (see, for example, U.S. Pat. No. 20040131637), pagC promoter (Pulken and Miller, J.Bacterial, 1991:173(1): 86-93; Alpuche-Aranda et al, PNAS, 1992; 89(21):10079-83), nirB promoter (Harborne et al (1992) Mall.6: 2805-2813), and the like (see, for example, see, Protein Nos. 7, WO 25, WO 27; for example, Protein map, Protein.

The nucleotide sequence encoding the polypeptide of the present invention may be present in an expression vector and/or a cloning vector. The nucleotide sequences encoding the two separate polypeptides may be cloned in the same or different vectors. The expression vector may include a selectable marker, a source of replication, and other components that provide for replication and/or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

A large number of suitable vectors and promoters are known to the skilled worker, many of which are commercially available for the production of individual recombinant constructs bacterial vectors are provided by way of example pBs, phagescript, PsiXl74, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, L a Jolla, CA, USA), pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5(Pharmacia, Uppsala, Sweden). eukaryotic vectors are provided by way of example pW L neo, pSV2cat, pOG44, PXRl, pSG (Stratagene) pSVK3, pBPV, pMSG, and PXV L (Pharmacia).

Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for insertion of a nucleic acid sequence encoding a heterologous protein. There may be a selectable marker that is operable in the expression host.

As noted above, in some embodiments, the nucleic acid encoding a polypeptide of the invention will be RNA in some embodiments, e.g., RNA synthesized in vitro. Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA that includes a nucleotide sequence encoding a polypeptide of the invention. Methods of introducing RNA into a host cell are known in the art. See, e.g., Zhao et al (2010) Cancer Res.15: 9053. Introduction of RNA comprising a nucleotide sequence encoding a polypeptide of the invention 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 invention.

In certain embodiments, the polynucleotide, nucleic acid sequence, and/or transcription unit and/or vector comprising the same further comprises one or more of a Kozak-type sequence (also referred to herein as a Kozak-related sequence), a woodchuck hepatitis virus post-transcriptional regulator component (WPRE), and a double or triple stop codon, wherein the one or more stop codons of the double or triple stop codon define a read stop from at least one of the one or more transcription 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 5' nucleotides within 10 nucleotides upstream of the start codon of at least one of the one or more transcription units, in certain embodiments, the polynucleotide, nucleic acid sequence, and/or transcription unit(s) of a Kozak consensus sequence (GCC) GCCRCCATG (SEQ ID NO:530), wherein R is a purine (a or a nucleotide sequence) in 1987, 15) or nucleotide sequence, in certain embodiments, the nucleotide sequence, or nucleotide sequence of a triple stop codon, wherein the nucleotide sequence, or codon, in certain embodiments, includes a codon, or nucleotide sequence, wherein the first nucleotide, the nucleotide, or nucleotide, in certain nucleotide, the nucleotide sequence, the nucleotide, or nucleotide, in certain embodiments, is represented by a codon for example, the same or nucleotide sequence of a codon, the codon for example, the codon of a codon, the same or nucleotide sequence of a codon for example, the same as a codon, the nucleotide, the codon of a codon, the nucleotide.

Triple stop codons herein include three stop codons, one in each reading frame, within 10 nucleotides of each other, and preferably having overlapping sequences, or three stop codons in the same reading frame, preferably at consecutive codons. By a double stop codon is meant two stop codons, each in a different reading frame, within 10 nucleotides of each other, and preferably having an overlapping sequence, 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 genome of the host cell, is performed using methods that do not utilize replication-defective recombinant retroviral particles. For example, other viral vectors may be utilized, such as those derived from adenovirus, adeno-associated virus, or herpes simplex virus type 1, as non-limiting examples.

In one of the embodiments disclosed herein that utilizes a non-viral vector to transfect a target cell, a non-viral vector, including naked DNA, can be introduced into a target cell, such as PBMC, B cell, T cell, and/or NK cell, using methods that include electroporation, nuclear transfection, liposome formulations, lipids, dendrimers, cationic polymers, such as poly (ethylenimine) (PEI) and poly (l-lysine) (P LL), nanoparticles, cell penetrating peptides, microinjection, and/or non-integrated lentiviral vectors.

In some embodiments of the methods provided herein, the Integration of DNA into the genome using a transposon-based vector system may be accomplished by co-transfecting, co-nucleofecting, or co-electroporating the target DNA into a plasmid containing transposon ITR fragments at the 5 'and 3' ends of the gene of interest or into a transposase vector system into DNA or mRNA or protein or Site-Specific serine recombinases, such as phiC31 that integrates the gene of interest into a pseudo-attP Site in the human genome, in which case the DNA vector may contain from 34bp to 40bp attB sites, which are recognition sequences for the recombinases (Bhastar Thyagara jan et al

Integrase, Mol Cell biol.2001, 6 months; 3926-3934) and co-transfected with a recombinase. For T cells and/or NK cells, transposon-based systems useful in certain methods provided herein utilize Sleeping Beauty DNA vectors in the form of DNA, mRNA, or proteinThe systemic system (see, e.g., U.S. Pat. No. 6,489,458 and U.S. patent application No. 15/434,595, which are incorporated herein by reference in their entirety), the PiggyBac DNA vector system (see, e.g., Manuri et al, Hum Gene Ther.2010, 4; 21(4):427-37, which is incorporated herein by reference in its entirety) or the Tol2 transposon system (see, e.g., Tsukahara et al, Gene Ther.2015, 2; 22(2): 209-215, which is incorporated herein by reference in its entirety). in some embodiments, the transposon and/or transposase of the transposon-based vector system can be produced as a microintegrated DNA vector prior to introduction into T cells and/or NK cells (see, e.g., Hudeck et al, Recessels Cancer Res.2016; 209:37-50 and Monjezi et al, ReceelEzi et al, Receeljun Reservs Rescer Res Res.2016; 209:37-50 and 11, 31, 11, 31, and 31, which are specifically joined to the CRISPR 14, 120, via the homologous DNA targeting sequences of the homologous DNA sequences of the DNA sequences incorporated herein incorporated by the homologous DNA sequences of the homologous DNA sequences (see, such as described by the homologous DNA sequences of the DNA sequences of SEQ ID No. 35, 120, 35.

Packaging cells

The present invention provides packaging cells and mammalian cell lines that are packaging cell lines that produce replication-defective recombinant retroviral particles that genetically modify target mammalian cells and mammalian cell lines thereof. In illustrative embodiments, the packaging cell comprises a nucleic acid sequence encoding the packagable RNA genome of the replication defective recombinant retroviral particle, the REV protein, the gag polypeptide, the pol polypeptide, and the pseudotyped component.

Suitable mammalian cell lines include, but are not limited to, He L a cells (e.g., American Type Culture Collection (ATCC) No. CC L-2), CHO cells (e.g., ATCC No. CR L09618, CC L, CR L29096), HEK293 cells (e.g., ATCC No. CR L-1573), suspension adapted HEK293 cells, Vero cells, NIH 3T3 cells (e.g., ATCC No. CR L-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CC 2 5965), PC12 cells (ATCC No. CR L), COS cells, COS-7 cells (ATCC No. CR 6861), mouse TtL cells, RANK 6I cells (ATCC No. CR 1655), CR1725 cells (ATCC No. CR 6342), and kidney cells (e.g., pG 4642-L), kidney cells (e.g., kidney) WO L, kidney 4642-29), and the like.

In some embodiments, the allogeneic cells may be immortalized cells, in some embodiments, the T cells are genetically engineered such that at least one of their T cell receptor chains is inoperative or at least partially absent, in some embodiments, the allogeneic cells may be modified such that they lack all or a portion of B2, in some embodiments, the autologous cells may be used to produce lymphocytes useful in the methods of the invention, e.g., in the form of the cells, e.g., in the form of the cells.

Method for activating immune cells

The present invention provides methods for activating immune cells (in illustrative embodiments, lymphocytes) in vitro, in vivo, or ex vivo. The methods generally involve contacting an immune cell (in vitro, in vivo, or ex vivo) with an activating component or with one or more target antigens, wherein the immune cell is genetically modified to produce a CAR of the invention (which in some embodiments is a MRB-CAR). In the presence of one or more target antigens, the CAR activates the immune cell, thereby generating an activated immune cell. Immune cells include, for example, cytotoxic T lymphocytes, NK cells, CD4⁺T cells, T regulatory (Treg) cells, gamma T cells, NK-T cells, neutrophils, etc. In other embodiments, the T cell is contacted with a T cell activator to activate the T cell. These methods are provided herein and discussed in the activation component section herein.

Contacting a genetically modified immune cell (e.g., T lymphocyte, NK cell) with one or more target antigens can increase the production of the cytokine by the immune 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 10-fold as compared to the amount of the cytokine produced by the immune cell in the absence of the one or more target antigens.

Contacting a genetically modified immune cell (e.g., a cytotoxic T lymphocyte) with an AAR can increase the cytotoxic activity of the cytotoxic 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 10-fold as compared to the cytotoxic activity of the cytotoxic cell in the absence of the one or more target antigens.

Contacting a genetically modified immune cell (e.g., a cytotoxic T lymphocyte) with one or more target antigens can increase the cytotoxic activity of the cytotoxic 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 10-fold as compared to the cytotoxic activity of the cytotoxic cell in the absence of the one or more target antigens.

In other embodiments, for example, contacting a genetically modified host cell with an antigen can increase or decrease cell proliferation, cell survival, cell death, and the like, depending on the host immune cell.

Inhibitory RNA molecules

In certain embodiments, the methods of the invention provided herein comprise inhibiting the expression of one or more endogenous genes expressed in T cells and/or NK cells. The methods provided herein illustrate the ability to make recombinant retroviral particles that express one or more (and in illustrative embodiments, two or more) inhibitory RNA molecules, such as miRNA or shRNA, useful in such methods. Indeed, the methods provided herein illustrate that such inhibitory RNA molecules can be encoded within introns including, for example, the Ef1a intron. This utilizes the present methods teaching to maximize functional components that can be included in the packaged retroviral genome, to overcome the disadvantages of the previous teachings, and to maximize the effectiveness of such recombinant retroviral particles in adoptive T cell therapy.

In some embodiments, the inhibitory RNA molecule comprises a5 '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 18 to 25 nucleotide RNA duplex within a 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 'and 3' strands may be the same or different lengths, and the RNA duplex may include one or more mismatches. Alternatively, the RNA duplex has no mismatches.

The inhibitory RNA molecules included in the compositions and methods provided herein are, in certain illustrative embodiments, not present in and/or not naturally expressed in the T cell into which they are inserted into their genome. In some embodiments, the inhibitory RNA molecule is a miRNA or shRNA. In some embodiments, when reference is made herein or in the priority application to a nucleic acid encoding an siRNA, particularly in the context of a nucleic acid that is part of the genome, it will be understood that such nucleic acid is capable of forming an siRNA precursor, such as an miRNA or shRNA, in a cell treated by DICER to form a double stranded RNA that typically interacts with or is part of the RISK complex. In some embodiments, the inhibitory molecule in an embodiment of the invention may be a precursor of a miRNA (such as Pri-miRNA or Pre-miRNA), or a precursor of a 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 processed into siRNA (either transcribed or artificially introduced) or 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 disposed in a serial or multiplex arrangement in the first nucleic acid molecule such that multiple miRNA sequences are simultaneously expressed from a single polycistronic miRNA transcript. In some embodiments, inhibitory RNA molecules can be linked to each other directly or indirectly using a non-functional linker sequence. In some embodiments, the linker sequence may be between 5 and 120 nucleotides in length, and in some embodiments may be between 10 and 40 nucleotides, as non-limiting examples. In illustrative embodiments, a first nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNAs and a second nucleic acid sequence encoding a CAR (e.g., MRB-CAR) are operably linked to a promoter that is constitutively active or inducible in T cells or NK cells. Thus, the inhibitory RNA molecule (e.g., miRNA) and CAR are expressed as polycistrons. In addition, functional sequences may be expressed from the same transcript. For example, any of the lymphoproliferative components provided herein that are not inhibitory RNA molecules can be expressed from the same transcript as the CAR and one or more (e.g., two or more) inhibitory RNA molecules.

In some embodiments, the inhibitory RNA molecule is a naturally occurring miRNA, such as but not limited to miR-155. Alternatively, artificial mirnas may be generated in which sequences capable of forming hybrid/complementary stem structures and directed against the target RNA are placed in a miRNA framework comprising microrna flanking sequences and loops for microrna processing, and optionally derived from naturally occurring mirnas that are identical to the flanking sequences between the stem sequences. Thus, in some embodiments, the 5 'to 3' orientation of the inhibitory RNA molecule comprises: a5 ' microRNA flanking sequence, a5 ' stem, a loop, a3 ' stem partially or fully complementary to the 5 ' stem, and a3 ' microRNA flanking sequence. In some embodiments, the 5 'stem (also referred to herein as the 5' arm) can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3 'stem (also referred to herein as the 3' arm) can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop is3 to 40, 10 to 40, 20 to 40, or 20 to 30 nucleotides in length, and in illustrative embodiments, the loop can be 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, one stem is two nucleotides longer than the other stem. The longer stem may be a5 'stem or a 3' stem.

In some embodiments, the 5 'microRNA flanking sequence, the 3' microRNA flanking sequence, or both are derived from a naturally occurring miRNA, 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 mus musculus or homo sapiens. The insertion of synthetic miRNA stem loops into the miR-155 framework (i.e., the 5 'microRNA flanking sequence, the 3' microRNA flanking sequence, and the loop between the miRNA5 'stem and 3' stem) is known to those of ordinary skill in the art (Chung, K. et al 2006.Nucleic Acids research.34(7): e 53; US 7,387,896). The SIBR (synthetic inhibitory BIC-derived RNA) sequence (Chung et al. 2006, supra) has, for example, a5 'microRNA flanking sequence consisting of nucleotides 134 to 161(SEQ ID NO:256) of a mouse BIC non-coding mRNA (Genbank IDAY096003.1) and a 3' microRNA flanking sequence consisting of nucleotides 223 to 283 of a mouse BIC non-coding mRNA (Genbank ID AY 096003.1). In one study, the SIBR sequence was modified (eSIBR) to enhance expression of mirnas (Fowler, d.k. et al. 2015.Nucleic acids Research 44(5): e 48). In some embodiments of the invention, the miRNA can be placed in the SIBR or eSIBR miR-155 framework. In the illustrative examples herein, the miRNAs are placed in a miR-155 framework, which includes the 5 'microRNA flanking sequence of miR-155 displayed by SEQ ID NO:256, the 3' microRNA flanking sequence displayed by SEQ ID NO:260 (nucleotides 221 to 265 of the mouse BIC non-coding mRNA); and a modified miR-155 loop (SEQ ID NO: 258). Thus, in some embodiments, the 5' microrna flanking sequence of miR-155 is SEQ ID NO:256 or a functional variant thereof, such as the same length as SEQ ID NO:256, or is 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID NO:256 or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less in length; and a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 256. In some embodiments, the 3' microrna flanking sequence of miR-155 is SEQ ID NO:260 or a functional variant thereof, e.g., the same length as SEQ ID NO:260, or is 95%, 90%, 85%, 80%, 75%, or 50% or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less of the length of SEQ ID NO: 260; and a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 260. However, any known microrna framework that functions to provide appropriate processing within the 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 invention.

In some embodiments, at least one, at least two, at least three, or at least four of the inhibitory RNA molecules encoded by the nucleic acid sequences in the polynucleotide of the replication-defective recombinant retroviral particle have the following arrangement in a5 'to 3' orientation: a5 ' microRNA flanking sequence, a5 ' stem, a loop, a3 ' stem partially or fully complementary to the 5 ' stem, and a3 ' microRNA flanking sequence. In some embodiments, all inhibitory RNA molecules have the following arrangement in a5 'to 3' orientation: a5 ' microRNA flanking sequence, a5 ' stem, a loop, a3 ' stem partially or fully complementary to the 5 ' stem, and a3 ' microRNA flanking sequence. As disclosed herein, inhibitory RNA molecules may be separated by one or more connector sequences that, in some embodiments, do not function other than as spacers between the inhibitory RNA molecules.

In some embodiments, when two or more inhibitory RNA molecules are included (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 (such as mRNA transcribed from an associated gene). In illustrative embodiments, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 3 and 5, or between 3 and 6 inhibitory RNA molecules are included in the first nucleic acid sequence. In an illustrative embodiment, four inhibitory RNA molecules are included in the first nucleic acid sequence.

In some embodiments, the RNA target is mRNA transcribed from a gene expressed by a T cell, such as, but not limited to, PD-1 (to prevent inactivation), CT L a4 (to prevent inactivation), TCRa (to safely prevent autoimmunity), TCRb (to safely prevent autoimmunity), CD3Z (to safely prevent autoimmunity), SOCS1 (to prevent inactivation), SMAD2 (to prevent inactivation), miR-155 target (to promote activation), IFN γ (to reduce CRS), cCB L (to prolong signaling), TRAI L2 (to prevent death), PP2A (to prolong signaling), ABCG1 (to increase cholesterol micro-content by limiting cholesterol clearance.) in illustrative examples, mirnas inserted into the T cell into the genome are directed against the target in the methods provided herein such that proliferation of the T cell is induced and/or enhanced and/or apoptosis is inhibited.

In some embodiments, the RNA target comprises mRNA encoding a component of a T Cell Receptor (TCR) complex such a component may comprise a component for generating and/or forming a T cell receptor complex and/or a component for proper function of a T cell receptor complex, thus, in one embodiment, at least one of two or more inhibitory RNA molecules causes a reduction in the formation and/or function of a TCR complex (in illustrative embodiments, one or more endogenous TCR complexes of a T cell) the T cell receptor complex comprises TCRa, TCRb, CD3d, CD3e, CD3g, and CD3z, these components are known to have a complex interdependency such that a reduction in expression of any one subunit will result in a reduction in expression and function of the complex, thus, in one embodiment, the RNA target is mRNA expressed from one or more of TCRa, TCRb, CD3d, CD3 364, CD3g, and CD z endogenous TCRa, which is transduced T cells, in certain embodiments, the RNA target RNA sequence is transcribed from a TCR RNA α or mRNA sequence encoding a T cell miRNA, CD3 CD 585, which has similar utility for inhibiting transcription of one or more target genes from IFN transcription from a target gene from a TCR mRNA from a target gene, which may be expressed from TCR mRNA, in certain embodiments, TCR CD3, TCR molecules which inhibit transcription of a target gene from RNA transcribed from a target gene from a mRNA from mRNA sequence comprising one or more of a target gene from a target gene, which may be transcribed from a target gene from IFN CD3, which is expected in certain target gene mRNA from TCR CD3, which is transcribed from a target gene.

In some embodiments provided herein, two or more inhibitory RNA molecules may be delivered in a single intron, such as but not limited to EF1- α a intron a. an intron sequence that may be used to carry a miRNA of the present invention includes any intron that is processed within a T cell.

In some embodiments, inhibitory RNA molecules may be provided on multiple nucleic acid sequences, which may be included on the same or different transcription 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, the two or more inhibitory RNA molecules are located on a first nucleic acid sequence expressed from a single promoter. Promoters for expressing such mirnas are typically inactive promoters in the packaging cells used to express the retroviral particle that delivers the miRNA in its genome to the target T cell, 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.

Characterization and commercial production method

The present invention provides methods and compositions useful as research reagents in scientific experiments and for commercial production. This scientific experiment can include methods for characterizing lymphocytes, such as NK cells, and in illustrative embodiments, T cells, using methods for genetically modifying (e.g., transducing) lymphocytes provided herein. These methods are useful, for example, for studying the activation of lymphocytes and the detailed molecular mechanisms by which these cells are rendered transducible. In addition, genetically modified lymphocytes that will be useful, for example, as research tools to better understand the factors that influence T cell proliferation and survival are provided herein. These genetically modified lymphocytes (such as NK cells, and in illustrative embodiments, T cells) can additionally be used in commercial production, e.g., for the production of certain factors, such as growth factors and immunomodulators, that can be harvested or tested or used to produce commercial products.

Scientific experiments and/or characteristics 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 replication defective recombinant retroviral particles provided herein that include a polynucleotide. In some embodiments, the transduced T cells and/or NK cells can comprise a polynucleotide comprising a polynucleotide encoding a polypeptide of the invention, e.g., a CAR, a lymphoproliferative component, and/or an activating component. In some embodiments, the polynucleotide may comprise an inhibitory RNA molecule as discussed elsewhere herein. In some embodiments, the lymphoproliferative component can be a chimeric lymphoproliferative component.

Method of treatment

The present invention provides various methods of treatment using the CAR. The CARs of the invention can mediate cytotoxicity to a target cell in the presence of a T lymphocyte or NK cell. The CAR of the invention binds to an antigen present on a target cell, thereby mediating killing of the target cell by a T lymphocyte or NK cell that is genetically modified to produce the CAR. The ASTR of the CAR binds to an antigen present on the surface of the target cell.

The invention provides methods of killing or inhibiting the growth of a target cell, the method comprising contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell or NK cell) that is genetically modified to generate an individual CAR, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of the target cell, and mediates killing of the target cell.

The present invention provides a method of treating a disease or disorder in a subject having the disease or disorder, the method comprising: a. introducing an expression vector comprising a polynucleotide sequence encoding a CAR into peripheral blood cells obtained from an individual to generate genetically engineered cytotoxic cells; administering the genetically engineered cytotoxic cell to a subject.

In the methods provided herein that include administering genetically modified T cells and/or NK cells to a subject (particularly a subject having or suspected of having cancer), the method can further comprise delivering an effective dose of an immune checkpoint inhibitor to the subject. This checkpoint inhibitor delivery can occur before, after, or simultaneously with the administration of the genetically modified T cells and/or NK cells. Immunodetection point inhibitors are known and various compounds are approved and in clinical studies. Where reference is made to some immune checkpoint inhibitors, checkpoint molecules (many of which are targets of checkpoint inhibitor compounds) include the following:

this molecule supports antigen-specific expansion of naive T cells and is essential for generating T cell memory. Celldex Therapeutics is studying CDX-1127, which is a monoclonal antibody that agonizes anti-CD 27.

Cd28. this molecule is constitutively expressed on almost all human CD4+ T cells and on about half of all CD 8T cells. CD28 is the target for TGN1412 "superagonists".

CD40. this molecule (found on various immune system cells including antigen presenting cells) has CD 40L, otherwise known as CD154, and is transiently expressed as its ligand on the surface of activated CD4+ T cells.

Cd122. this molecule, which is a subunit of the interleukin-2 receptor β, is known to increase proliferation of CD8+ effector T cells Nektar Therapeutics has developed NKTR-214, which is a CD122 biased immunostimulatory interleukin.

When this molecule (also called 4-1BB) is bound by a CD137 ligand, the result is T-cell proliferation. Pieris Pharmaceuticals has developed engineered lipocalins with dual specificity for CD137 and HER 2.

OX40. this molecule (also known as CD134) has OX 40L or CD252 as its ligand, like CD27, OX40 promotes the expansion of effector and memory T cells, however, it is also known to inhibit the differentiation and activity of T regulatory cells, and also its ability to modulate the production of interleukins AstraZeneca has three developers that target OX40, MEDI0562 is a human OX40 agonist, MEDI6469, a murine OX4 agonist, and MEDI6383(OX40 agonist).

GITR is an abbreviation for glucocorticoid-induced TNFR family-related genes, which promotes T cell expansion, including Treg expansion. TG Therapeutics is studying anti-GITR antibodies.

This molecule is an abbreviation for inducible T cell costimulatory factor and is also known as CD278, which is expressed on activated T cells. Jounce Therapeutics is developing ICOS agonists.

A2ar the adenosine A2A receptor is considered an important checkpoint in cancer therapy because adenosine in the immune microenvironment causing activation of the A2a receptor is a negative immune feedback loop and the tumor microenvironment has a relatively high concentration of adenosine.

B7-H3 (also known as CD276) was originally understood as a costimulatory molecule but is now considered to be co-inhibitory. MacroGenics is studying MGA271, an Fc optimized naive antibody that targets B7-H3.

B7-H4 (also known as VTCN1) is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape.

BT L a, this molecule is an abbreviation for B and T lymphocyte attenuator and is also known as CD272, which has HVEM (herpes virus entry mediator) as its ligand.

CT L A-4 is an abbreviation for interleukin T lymphocyte-associated protein 4 and is also known as CD152, which is the target of the compound ipilimumab (Yervoy) of Bristol-Myers Squibb.

IDO is an abbreviation for indoleamine 2, 3-dioxygenase, a tryptophanase enzyme with immunosuppressive properties. Another important molecule is TDO, tryptophan 2, 3-dioxygenase. IDO. newlink Genetics and Incyte have identified inhibitors of the IDO pathway.

Bristol-Myers Squibb has developed Liriluzumab (L iriumab), a simple line antibody to KIR.

L AG3, which is a contraction of lymphocyte activation gene-3, acts to suppress immune responses by acting on Tregs, and CD8+ T cells have a direct impact Bristol-Myers Squibb has developed an anti-L AG3 simple line antibody called BMS-986016.

PD-1 is an abbreviation for programmed death 1(PD-1) receptor having two ligands PD-L1 and PD-L2, the checkpoint is the target of the melanoma drug mersarong (Keytrud) by Merck & co.

TIM-3 is an abbreviation for T cell immunoglobulin domain and leukocyte domain 3, which is expressed on activated human CD4+ T cells and modulates Th1 and Th17 interleukins.

VISTA (protein), an abbreviation for V-domain Ig suppressor of T cell activation, is initially expressed on hematopoietic cells.

Is suitable for the treated individual

A variety of subjects are amenable to treatment by the methods and compositions of the present invention. Suitable subjects include any subject, e.g., a human or non-human animal, that has a disease or disorder, has been diagnosed with a disease or disorder, is at risk of having a disease or disorder, has had a disease or disorder and is at risk of recurrence of a disease or disorder, has been treated with a drug for the disease or disorder and failed to respond to such treatment, or has been treated with a drug for the disease or disorder but has had a recurrence following a preliminary response to such treatment.

Individuals suitable for treatment by immunomodulating methods include individuals with autoimmune diseases; individuals that are recipients of organ or tissue transplants and the like; (ii) an immunodeficient individual; and individuals infected with pathogens.

Illustrative embodiments

This exemplary embodiments section provides exemplary aspects and embodiments provided herein and discussed further throughout this specification. For the sake of brevity and convenience, all disclosed aspects and embodiments, and all possible combinations of disclosed aspects and embodiments, are not listed in this section. It should be understood that the embodiments provided are specific embodiments to numerous aspects, as discussed throughout this disclosure. It is intended that in view of the complete disclosure herein, any individual embodiment described below or in this complete disclosure can be combined with any aspect described below or in this complete disclosure, where it is an additional element that can be added to an aspect or because it is a narrower element than what is presented in an aspect. This combination is specifically discussed in other sections of this detailed description.

In one aspect, provided herein is a replication-defective recombinant retroviral particle comprising a polynucleotide comprising:

A. one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a polypeptide comprising a Chimeric Antigen Receptor (CAR); and

B. a pseudotyping module and an activating module (e.g., an NK cell activating module, or in an illustrative embodiment, a T cell activating module) on its surface, wherein the activating module is not encoded by a polynucleotide in a replication defective recombinant retroviral particle.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR) and a second polypeptide comprising a lymphoproliferative component. In this section and in this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activating components, pseudotyped components, control components, CARs, and other components are provided herein.

In another aspect, provided herein are replication-defective recombinant retroviral particles comprising a polynucleotide that includes 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 a first polypeptide comprising a Chimeric Antigen Receptor (CAR) and a second polypeptide comprising a chimeric lymphoproliferative component (e.g., a constitutively active chimeric lymphoproliferative component). In illustrative embodiments, the chimeric lymphoproliferative component does not comprise an interleukin linked to its cognate receptor or to a fragment of its cognate receptor. In this section and in this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activating components, pseudotyped components, control components, CARs, and other components are provided herein.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising:

A. a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a Chimeric Antigen Receptor (CAR); and

B. a pseudotyping module and a T cell activation module on its surface, wherein the T cell activation module is not encoded by the polynucleotide in the replication deficient recombinant retroviral particle, and wherein the T cell activation module is an anti-CD 3 scfvffc antibody. In this section and in this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activating components, pseudotyped components, control components, CARs, and other components are provided herein.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising:

A. one or more pseudotyped components capable of binding to T cells and/or NK cells and facilitating membrane fusion of replication-defective recombinant retroviral particles thereto;

B. 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 a first engineered signaling polypeptide comprising a chimeric antigen receptor (comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain) and a second engineered signaling polypeptide comprising at least one lymphoproliferative component; wherein expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is modulated by a control component; and

C. an activating module on its surface, wherein the activating module is capable of binding to T cells and/or NK cells and is not encoded by a polynucleotide in a replication defective recombinant retroviral particle. In this section and in this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activating components, pseudotyped components, control components, CARs, and other components are provided herein.

In some aspects, provided herein are genetically modified lymphocytes (such as T cells or NK cells) made by ex vivo transduction of resting T cells and/or resting NK cells with any of the replication deficient recombinant retroviral particles provided herein, wherein the contacting facilitates transduction of resting T cells and/or NK cells by the replication deficient recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. In some embodiments, the replication-defective recombinant retroviral particle comprises a pseudotyped component on its surface and a membrane-bound T cell activation component on its surface. In this section and in this disclosure, various embodiments of such cells are provided herein, such as, but not limited to, contact time, lymphoproliferative elements, activating elements, pseudotyping elements, controlling elements, and other elements.

In another aspect, provided herein is the use of any replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying T cells and/or NK cells, wherein use of the kit comprises the steps of any one of the method aspects and embodiments provided herein. For example, in another aspect, provided herein is a use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying T cells or NK cells, wherein the use of the kit comprises: contacting T cells or NK cells ex vivo with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises a pseudotyped component on a surface and a T cell activation component, typically on a surface, wherein the contacting facilitates transduction of T cells or NK cells by the replication deficient recombinant retroviral particle, thereby producing genetically modified T cells or NK cells. In some embodiments, the T cell activation module may be membrane bound. In illustrative embodiments, the T cell activation component activates T cells via a T cell receptor associated receptor complex. In some embodiments, the contacting may be performed at the lower end of the range for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours and at the upper end of the range for between 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, and 24 hours, such as between 1 hour and 12 hours or between 1 hour and 5 hours. In this and other aspects of use herein, the replication-defective recombinant retroviral particles for use in the manufacture of a kit may comprise any one of the replication-defective recombinant retroviral particle aspects and embodiments provided herein. Various other embodiments of these methods are provided herein, such as (but not limited to) other contact times, lymphoproliferative components, activating components, pseudotyped components, transfected/genetically modified cell percentages, control components, and other components.

In another aspect, provided herein is a method of producing a genetically modified T cell and/or NK cell, comprising contacting a lymphocyte, typically a T cell and/or NK cell, with a replication-deficient recombinant retroviral particle, wherein the replication-deficient recombinant retroviral particle typically comprises a pseudotyped component on its surface, wherein the contacting (which may also be considered to be incubated under contacting conditions) facilitates transduction of the resting T cell and/or NK cell by the replication-deficient recombinant retroviral particle, thereby producing the genetically modified T cell and/or NK cell. The pseudotyped module is generally capable of binding resting T cells and/or NK cells and generally facilitates membrane fusion of itself or binding to other proteins of replication-defective recombinant retroviral particles. In some embodiments of the methods, the replication-defective recombinant retroviral particle comprises an activation module, such as a T cell activation module that activates T cells via a T cell receptor associated complex. This activating module may be an anti-CD 3 antibody, such as anti-CD 3scFv or anti-CD 3 scfvffc. Various exemplary and illustrative contact times are provided herein. Various other embodiments of these methods such as (but not limited to) contact time, lymphoproliferative components, activating components, pseudotyped components, percentage of transfected/genetically modified cells, control components, Kozak-type sequences, WPRE components, triple termination sequences, and other components are provided herein.

In another aspect, provided herein is a method for genetically modifying and/or transducing a lymphocyte, the method comprising contacting the lymphocyte ex vivo with a replication-defective recombinant retroviral particle, wherein the replication-defective recombinant retroviral particle comprises a pseudotyping component on its surface and a membrane-bound T cell activation component on its surface, wherein the contacting facilitates transduction of the lymphocyte by the replication-defective recombinant retroviral particle, thereby producing a genetically modified lymphocyte. In some embodiments, the membrane-bound T cell activation module is an anti-CD 3 antibody, e.g., anti-CD 3scFv or anti-CD 3 scfvffc. Various other embodiments of these methods such as (but not limited to) contact time, lymphoproliferative components, activating components, pseudotyped components, percentage of transfected/genetically modified cells, control components, Kozak-like sequences, WPRE, triple termination, and other components are provided herein.

In another aspect, provided herein is a method of transducing a resting lymphocyte of an individual, the method comprising contacting a resting T cell and/or a resting NK cell ex vivo with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises on its surface a pseudotyping component capable of binding to the resting T cell and/or the resting NK cell and facilitating fusion of the replication deficient recombinant retroviral particle to its membrane, wherein the contacting facilitates transduction of the resting T cell and/or NK cell by the replication deficient recombinant retroviral particle, thereby producing a genetically modified T cell and/or NK cell. In illustrative embodiments of this aspect, at least 10%, 20%, or 25% of resting T cells and/or NK cells, or between 10% and 70%, between 10% and 50%, or between 20% and 50% of T cells and/or NK cells, are transduced as a result of the method. Various other embodiments of these methods such as (but not limited to) contact time, lymphoproliferative components, activating components, pseudotyping components, transfected/genetically modified cell percentages, control components, and other components are provided herein.

In another aspect, provided herein is a method for transducing resting T cells and/or resting NK cells from an isolated college, comprising:

A. collecting blood from a subject;

B. isolating Peripheral Blood Mononuclear Cells (PBMCs) comprising resting T cells and/or resting NK cells;

C. contacting ex vivo a resting T cell and/or a resting NK cell of an individual with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises on its surface a pseudotyping component capable of binding to the resting T cell and/or the resting NK cell and facilitating binding of the replication deficient recombinant retroviral particle to its membrane, wherein the contacting facilitates transduction of at least 5% of the resting T cell and/or NK cell by the replication deficient recombinant retroviral particle, thereby producing a genetically modified T cell and/or NK cell, thereby transducing the resting T cell and/or NK cell. Various other embodiments of these methods such as (but not limited to) contact time, lymphoproliferative components, activating components, pseudotyping components, transfected/genetically modified cell percentages, control components, and other components are provided herein.

Typically, packaging cell lines are used to produce the replication-defective recombinant retroviral particles discussed elsewhere herein. In some embodiments, the packaging cell line can be a suspension cell line. In an illustrative example, a packaging cell line can be grown in serum-free media. In some embodiments, the lymphocytes can be from an individual. In an illustrative embodiment, the lymphocytes can be from the blood of an individual. Various other embodiments of these methods such as (but not limited to) contact time, lymphoproliferative components, activating components, pseudotyping components, transfected/genetically modified cell percentages, control components, and other components are provided herein.

In another aspect, provided herein is a method of transducing a lymphocyte with a replication-defective recombinant retroviral particle comprising:

A. transfecting a packaging cell with a set of vectors comprising components of a replication-defective recombinant retroviral particle, wherein the packaging cell is grown in a serum-free medium comprising a suspension;

B. collecting the replication-defective recombinant retroviral particles from a serum-free medium; and

C. the lymphocytes are contacted with the replication-defective recombinant retroviral particle. Various embodiments of these methods such as (but not limited to) contact time, lymphoproliferative components, activating components, pseudotyping components, transfected/genetically modified cell percentages, control components, and other components are provided herein.

In another aspect, provided herein is a method of transducing lymphocytes with a replication-defective recombinant retroviral particle comprising:

A. culturing a packaging cell in suspension in a serum-free medium, wherein the packaging cell comprises a nucleic acid sequence encoding the packagable RNA genome of the replication-defective retroviral particle, the REV protein, the gag polypeptide, the pol polypeptide, and the pseudotyping component;

B. collecting the replication-defective recombinant retroviral particles from a serum-free medium; and

C. contacting the lymphocyte with the replication-defective recombinant retroviral particle, wherein the contacting is performed for less than 12 hours, thereby transducing the lymphocyte. Various other embodiments of these methods are provided herein, such as (but not limited to) other contact times, lymphoproliferative components, activating components, pseudotyped components, transfected/genetically modified cell percentages, control components, and other components.

Other embodiments of METHODS FOR genetically MODIFYING AND/OR TRANSDUCING PBMCs, lymphocytes, NK cells, AND/OR T cells in illustrative embodiments, OR related METHODS, uses, OR products of the METHODS aspects herein, are provided in other sections of the disclosure within AND outside this illustrative embodiments section, such as in the section entitled "METHODS FOR TRANSDUCING AND/OR genetically MODIFYING lymphocytes (METHODS FOR transporting AND/OR genenetica LL Y modified genes L YMPHOCYTES". other embodiments of any of the related uses OR products of the above METHODS OR method aspects FOR genetically MODIFYING AND/OR TRANSDUCING may include replication-defective recombinant retroviral particles, lymphoproliferative components, CARs, pseudotyped components, riboswitches, activated components, membrane-bound interleukin, mirnas, Kozak-like sequences, WPREs, stop codons, AND/OR other components disclosed in this illustrative embodiments of the invention.

Unless not compatible with or stated to the aspects, in illustrative embodiments, the packaging cell is an immortalized cell comprising a DNA nucleic acid encoding a packagable RNA genome stably integrated therein. In some embodiments, the gag and pol polypeptides are expressed from one or more inducible promoters, wherein the method further comprises: during culturing, trans-activating factors are added to induce expression of the gag and pol polypeptides from one or more inducible promoters. Various embodiments of these methods, such as (but not limited to) contact time, lymphoproliferative components, activation components, control components, and other components are provided herein.

Unless otherwise incompatible with or stated in the state, in the use for genetically modifying and/or transducing lymphocytes (T lymphocytes), in an illustrative embodiment of any one of the method aspects for genetically modifying and expanding lymphocytes, such as T lymphocytes, or performing cell therapy or similar methods herein, the lower end of the range in which the contact can be effected is between 5 seconds, 10 seconds, 15 seconds, 30 seconds or 45 seconds or1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes or 45 minutes or1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours, and the upper end of the range is between 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours and 72 hours. For example, in illustrative embodiments, the contacting is carried out between 2 hours and 24 hours, between 2 hours and 20 hours, between 2 hours and 6 hours, between 1 hour and 20 hours, between 1 hour and 12 hours, between 1 hour and 4 hours, between 4 hours and 12 hours, or between 4 hours and 8 hours. In some embodiments, after addition of the cells to be genetically modified and/or transduced, the replication-defective recombinant retroviral particle may be washed immediately such that the contact time is carried out for the length of time it takes to wash the replication-defective recombinant retroviral particle. Thus, typically, contacting comprises at least an initial contacting step in which the retroviral particles are contacted with the cells in suspension in a transduction reaction mixture. In some embodiments, unless stated, or otherwise stated in the broadest aspects provided herein, in any of the methods, methods to produce products, or uses thereof, the contacting of PBMCs, NK cells, and in illustrative embodiments T cells with replication-defective recombinant retroviral particles can be performed for between 1 minute and 12 hours, between 5 minutes and 12 hours, between 10 minutes and 12 hours, between 15 minutes and 12 hours, between 30 minutes and 12 hours, or between 1 hour and 24 hours, such as between 1 hour and 12 hours or between 1 hour and 6 hours. In some embodiments, the contacting can be performed for less than 24 hours, e.g., less than 12 hours, less than 8 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. In illustrative embodiments, the contacting is performed for between 1 minute and 12 hours, between 5 minutes and 12 hours, between 15 minutes and 12 hours, between 30 minutes and 12 hours, between 1 hour and 14 hours, between 1 hour and 12 hours, between 1 hour and 6 hours, between 1 hour and 4 hours, or between 2 hours and 14 hours. Such methods may be performed without prior activation.

Unless incompatible with, OR stated in, a state, in an illustrative embodiment of any one of, OR a similar method FOR, genetically modifying AND/OR TRANSDUCING lymphocytes (e.g., T lymphocytes), FOR genetically modifying AND expanding lymphocytes (e.g., T lymphocytes), OR a method state FOR performing cell therapy herein, other embodiments of the time ranges of METHODS envisioned FOR genetically modifying AND/OR TRANSDUCING, OR related uses OR products of the method states herein are provided within other sections of the disclosure within AND outside this illustrative embodiment section, FOR example in the section entitled "(method FOR TRANSDUCING AND/OR genetically modifying lymphocytes) METHODS FOR transporting AND/OR gene modifying lymphocytes LL YMODIFYING ying L ymphotes".

Unless incompatible with, or stated in, a state, in an illustrative embodiment of any of the method states for genetically modifying and/or transducing lymphocytes (e.g., T lymphocytes), for genetically modifying and expanding lymphocytes (e.g., T lymphocytes), or for performing cell therapy herein, or a similar method, the cells are activated during the contacting, and are either not activated at all or are not activated for more than 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours prior to the contacting. In certain illustrative embodiments, activation by components not present on the surface of a retroviral particle does not require genetically modifying and/or transducing the cell. Thus, no such activating or stimulating means are required, other than on the retroviral particle, before, during or after contact.

Unless incompatible with, or stated in, a state, in an illustrative embodiment of any one of, or a similar method for genetically modifying and/or transducing lymphocytes (e.g., T lymphocytes), for genetically modifying and amplifying lymphocytes, or for performing a method state of a cell therapy herein, wherein the replication-defective recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), and in an illustrative embodiment encodes a second polypeptide comprising a chimeric lymphoproliferative component, the genetically modified T cells or NK cells are capable of, suitable for, possess the property that, and/or are modified for survival in the absence of added I L-2 or in the presence of an added cytokine (such as I L-2, I L-15, or I L-7) and survival in culture on at least day 14, day 21, day 28, day 42, day 60, day 28, day 42, day 60, day 42, or day 60 from day 14, day 42, day 60, day, or day 60, day from day.

Unless incompatible with or already stated in a state, in the methods, kits, uses or compositions provided herein (e.g., polynucleotides, packaging cells or replication deficient recombinant retroviral particles), the replication deficient recombinant retroviral particles comprise or further comprise on their surface an activating module capable of activating T cells and/or NK cells. Typically, an activation module, such as a T cell activation module, is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle.

Unless otherwise incompatible with or stated in a state, in the methods, kits, uses or compositions (e.g., polynucleotides, packaging cells or replication-defective recombinant retroviral particles) provided herein that recite a T cell and/or an NK cell or a resting T cell and/or a resting NK cell, in certain illustrative embodiments, the cell is a T cell.

Unless not compatible with or stated to be within the context, generally, a recombinant retroviral particle in any one of the methods, kits, uses, or compositions provided herein (e.g., a polynucleotide, a packaging cell, or a replication-defective recombinant retroviral particle) is replication-defective, i.e., non-replicable. In an illustrative embodiment, the retrovirus is a lentivirus, such as a replication or replication-defective HIV lentivirus. In an illustrative embodiment, the retrovirus is a lentivirus, such as a replication-defective or replication-defective HIV lentivirus particle. Thus, in illustrative embodiments in any of the methods, kits, uses, or compositions provided herein (e.g., a polynucleotide, a packaging cell, or a replication-defective recombinant retroviral particle), if not described or otherwise stated, the replication-defective recombinant retroviral particle is a lentiviral particle and/or the genetically modified cell is a genetically modified T cell.

Unless incompatible with or already stated in a state, in an illustrative embodiment of any of the states provided herein, the lymphocyte can be a T cell and/or an NK cell. In further embodiments, the T cells and/or NK cells may be resting T cells and/or resting NK cells.

Unless not compatible with the state or already stated in the state, in illustrative embodiments of any of the method states for transducing, genetically modifying and expanding lymphocytes or for performing adaptive cell therapy or similar therapies herein, between 10% and 75%, or between 10% and 70%, or between 10% and 60%, or between 10% and 50%, or between 10% and 25%, or between 20% and 75%, or between 20% and 50%, or at least 10%, 20% or 25% of the quiescent T cells are transduced or between 0% and 75% of the NK cells are transduced. In other aspects, resting NK cells between 5% and 80%, or between 10% and 70%, or between 10% and 60%, or between 10% and 50%, or between 10% and 25%, or between 10% and 20%, or between 20% and 50% are transduced.

Unless not compatible with or stated in the aspect, in an illustrative embodiment of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adaptive cell therapy or similar therapy herein or any composition provided herein, the expression of the second engineered signaling polypeptide is regulated by a control component.

Unless otherwise incompatible with the state or stated in the state, in any of the methods, uses, process products or compositions herein comprising a genetically modified T cell or NK cell or replication deficient recombinant retroviral particle, such a genetically modified T cell or NK cell or replication deficient recombinant retroviral particle may comprise a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR) and/or a second polypeptide comprising a T cell lymphoproliferative component. In some embodiments, the expression of the CAR and/or lymphoproliferative component is controlled by the control component or a different control component for each. In some embodiments, the CAR and/or lymphoproliferative component may be expressed on the same polypeptide, and in illustrative embodiments, the CAR and/or lymphoproliferative component are expressed as separate polypeptides. In some embodiments, the CAR is a MRB CAR. In some embodiments, the chimeric lymphoproliferative component is constitutively active, e.g., a constitutively active chimeric interleukin receptor. In this embodiment, the constitutively active chimeric interleukin receptor may be controlled by the control component.

Unless incompatible with or stated in a state, or in a state including polynucleotides, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR) and/or a second polypeptide comprising a T-cell lymphoproliferative component, the polynucleotides may further comprise one or more of a Kozak-related sequence, a WPRE component, and a multiple termination sequence, such as a dual termination codon or a triple termination codon, wherein the one or more termination codons of the dual termination codon or the triple termination codon define a stop reading from at least one of the one or more transcription units, unless incompatible with or stated in a state including polynucleotides, wherein the polynucleotides comprise a polynucleotide comprising a nucleotide sequence of the type ccacak/ug (SEQ ID NO:515), a gcgcg/ug g) (SEQ ID NO:516 (g) (SEQ ID NO:517) or SEQ ID No.: GCCGCCGCCAT/ug (g) or wpg 32/ug g) in which the polynucleotide comprises a combination of the one or more of the aforementioned polynucleotide comprising a polynucleotide, wherein the polynucleotide comprises a polynucleotide comprising a nucleotide sequence of the type of Kozak + 3, WPRE, wherein the polynucleotide comprises a combination of the polynucleotide comprising a polynucleotide of the type of the aforementioned, and/or the polynucleotide, wherein the polynucleotide comprises a polynucleotide, in each of the sequence of the type of the polynucleotide, or the polynucleotide, in one or the case, wherein the polynucleotide comprises a combination of the polynucleotide, wherein the polynucleotide, comprises a polynucleotide, in one or the case, the polynucleotide, comprises a polynucleotide, the type of the polynucleotide, comprises a WPRE-5, wherein the type of the polynucleotide, comprises a.

In any of the embodiments herein comprising a chimeric lymphoproliferative component, the chimeric lymphoproliferative component may be a chimeric interleukin receptor in any of the embodiments herein comprising a chimeric lymphoproliferative component, the chimeric lymphoproliferative component may be an I α -7 linked to I L-7 receptor α, or an I α -7 linked to I α -7 receptor α in any of the embodiments herein comprising a chimeric lymphoproliferative component, the chimeric interleukin receptor may comprise an I L-7 linked to I α -7 receptor α, or a fragment thereof that retains the ability to promote proliferation of T cells and/or NK cells, and wherein the chimeric interleukin receptor is constitutively active, in some embodiments, the chimeric interleukin receptor comprises a transmembrane signaling domain linked to an I3625-7 receptor covalently linked to an I3625-7 receptor capable of binding I L-7, a fragment of an I3625-7 receptor, a transmembrane signaling domain of I L-I637 receptor 5392-7 domain I-7 receptor.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the chimeric lymphoproliferative component can be a chimeric interleukin receptor, the chimeric interleukin receptor comprises an intracellular signaling domain of the I L-7 receptor, an intracellular signaling domain of the I L-12 receptor, an intracellular signaling domain of the I L2-15/I L-2 receptor (such as the I L-15/I L-2L receptor), an intracellular signaling domain of the I L-21 receptor, an intracellular signaling domain of the I L-23 receptor, an intracellular signaling domain of the I L-27 receptor, a TGF 9-7 receptor (TGF β) receptor, an intracellular signaling domain of the TGF 9-9 receptor, or an intracellular signaling domain of the TGF 9-9 receptor comprising the chimeric interleukin receptor fused to the cd7 receptor in illustrative embodiments provided herein that include a chimeric interleukin receptor.

In addition to being incompatible with or stated otherwise in one aspect, illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component that may include a T cell survival motif, which may include all or a functional fragment of the I L-7 receptor, the I L-15 receptor, or CD28, may include, in other embodiments, an interleukin or an interleukin receptor polypeptide, or a fragment thereof that includes a signaling domain, for example, the lymphoproliferative component may include an interleukin polypeptide and the lymphoproliferative component may further include a portion of its cognate interleukin receptor polypeptide covalently linked via a linker, in general, this portion of a cognate interleukin receptor includes a functional portion capable of binding an extracellular domain of an interleukin and a transmembrane domain, in some embodiments, the intracellular domain is an intracellular portion of a cognate interleukin receptor, in some embodiments, the intracellular domain is an intracellular portion of a different interleukin receptor that is capable of promoting proliferation of an intracellular portion of a lymphoblastic portion of a modified cell, in the absence of exposure to a CAR cell, such as an extracellular portion of an interleukin receptor, and/or an extracellular portion of a cell surface of a cell that is not capable of expressing a human antigen, such as an antigen, and/or cell, and is expressed in vitro upon a cell culture in a cell culture under conditions that indicate that no in vivo, or cell activation by a cell growth on a cell culture, such as an in a cell culture, or on a cell culture medium, such as a cell type 367, or on a cell, or cell culture, and/or on a cell culture medium.

Alternatively, the lymphoproliferative component can be the intracellular signaling domain of the I L-7 receptor, the intracellular signaling domain of the I L-12 receptor, the intracellular signaling domain of the I L-23 receptor, the intracellular signaling domain of the I L-27 receptor, the intracellular signaling domain of the I L-15 receptor, the intracellular signaling domain of the I L-21 receptor, or the intracellular signaling domain of the transforming growth factor β (TGF β) decoy receptor.

In some illustrative embodiments, the lymphoproliferative component can comprise an I L InsPPC L mutant, unless incompatible with or stated in one aspect, the lymphoproliferative component can be any of the C L E listed in the lymphoproliferative component section herein in any of the aspects that include a lymphoproliferative component, for example, in illustrative embodiments, C L E comprises a domain from a gene specifically referred to as being of particular interest and/or an optimal construct (Top Constructs) relative to the examples herein, unless incompatible with or stated in one aspect.

Unless incompatible with or stated to be in one state, in any of the states and embodiments disclosed herein that include lymphoproliferative components, such as products of methods, uses, compositions and process states, in illustrative embodiments, genetically modified PBMCs, lymphocytes or genetically modified T cells and/or NK cells are capable of being implanted in vivo in the absence of exposing the cells to interleukins (such as I L-15, I L-7, and in illustrative embodiments, I L-2) and optionally to targets of ASTR expressed by the cells, or in certain illustrative embodiments, in the presence of antigens recognized by the CARs (particularly for embodiments in which the method comprises genetically modifying and/or transducing retroviral particles having a pseudotyped component on their surface and optionally an isolated or fused activation domain and generally not requiring pre-activation) in culture for the presence and/or ex vivo expansion of the live cells and/or the proliferation/or proliferation/proliferation of the CAR/or proliferation/proliferation domain in vitro for at least 6 days, 7 days, 21 days, or longer in vivo in the culture of the cells that include lymphoproliferative cells that are capable of being implanted for any of being exposed to the target cells in vivo for 35 days.

In illustrative embodiments of any of the methods and compositions provided herein that include a replication-defective recombinant retroviral particle, the replication-defective recombinant retroviral particle may comprise an activating module on its surface, unless incompatible with or stated in one aspect. In illustrative embodiments, the activation component activates T cells via a T cell receptor associated complex. In some cases, the activation component may activate only T cells. In other cases, the activation component requires activation via the TCR receptor complex to further activate the T cell. Thus, in some embodiments, the activation assembly comprises:

A. a membrane-bound polypeptide capable of binding to CD 3; and/or

B. A membrane-bound polypeptide capable of binding to CD28.

Furthermore, the membrane-bound polypeptide capable of binding to CD3 is a polypeptide capable of binding to CD3 fused to a heterologous GPI-anchor linkage sequence, and the membrane-bound polypeptide capable of binding to CD28 may be a polypeptide capable of binding to CD28 fused to a heterologous GPI-anchor linkage sequence. In some embodiments, the membrane-bound polypeptide capable of binding to CD28 is CD80, CD86, or a functional fragment thereof capable of inducing CD 28-mediated activation of Akt (such as the extracellular domain of CD 80).

In an illustrative embodiment of any of the methods and compositions provided herein that are replication-deficient recombinant retroviral particles or that comprise replication-deficient recombinant retroviral particles, unless incompatible with or stated in a state, the activation component may be a membrane-binding polypeptide capable of binding to CD3, such as anti-CD 3scFv or anti-CD 3 scffc in an illustrative embodiment of any of the methods and compositions provided herein that comprise replication-deficient recombinant retroviral particles and anti-CD 3, anti-CD 3 may be anti-CD 3 scffc fused to a heterologous GPI-anchor linker sequence in an illustrative embodiment of any of the methods and compositions provided herein that comprise replication-deficient recombinant retroviral particles, a membrane-binding polypeptide capable of binding to CD3 may be anti-CD 3 bound to a CD14 GPI-anchor linker sequence, and a membrane-binding polypeptide capable of binding to CD28 may be a CD 45 bound to a CD B GPI-anchor linker sequence or a cell-anchor linker sequence thereof in an illustrative embodiment of a cell-binding polypeptide comprising a CD 963 scFv-binding to a CD16 GPI-anchor linker sequence, or a cell-scFv-8, a cell-binding polypeptide comprising a heterologous GPI-anchor linker sequence, a heterologous GPI-binding to a heterologous GPI-anchor linker sequence, a heterologous GPI-binding domain, a cell-binding polypeptide, or a cell-scFv-binding polypeptide, and a cell-binding domain comprising a cell-binding domain, a heterologous GPI-binding polypeptide, a replication-binding domain, a replication-binding polypeptide, a replication-binding to a replication-binding domain, a replication-binding domain of any of a replication-binding to a replication-binding polypeptide, a replication-binding to a replication-binding domain of any of a replication-binding domain of a replication-binding to a replication-binding domain of a replication.

Unless otherwise stated, or stated in a broad aspect, in an illustrative embodiment of any of the methods and compositions provided herein that include a replication-defective recombinant retroviral particle, the replication-defective recombinant retroviral particle may comprise a membrane-bound interleukin on its surface the membrane-bound interleukin may be I L-7, I L-15, or an activated fragment thereof in other embodiments, the fusion polypeptide in which the membrane-bound interleukin is I L-7, or an activated fragment thereof, and DAF, for example, the fusion polypeptide may comprise a DAF signal sequence (nucleotides 1 to 34 of SEQ ID NO: 286), I L-7 without its signal sequence (nucleotides 35 to 186 of SEQ ID NO: 286), and a DAF fragment comprising its GPI anchor linker sequence (nucleotides 187 to 532 of SEQ ID NO: 286).

Unless incompatible with or stated in one aspect, in illustrative embodiments of any of the method and composition aspects provided herein, the pseudotyped component may comprise one or more heterologous envelope proteins. In other examples, the pseudotyping module may comprise one or more viral polypeptides recognized by T cells. The one or more pseudotyped components may comprise a measles virus F polypeptide, a measles virus H polypeptide, and/or a fragment thereof. The one or more pseudotyped components may be cytoplasmic domain deleted variants of a measles virus F polypeptide and/or a measles virus H polypeptide.

In an illustrative embodiment of any of the methods and compositions provided herein that include a control element, unless incompatible with or stated in one aspect, the control element is a control element that modulates a lymphoproliferative element, wherein the lymphoproliferative element is inactive or less active in promoting proliferation of T cells and/or NK cells in the absence of a compound, and wherein the compound is a chaperone that binds to and induces activity of the lymphoproliferative element.

Unless incompatible with or stated in one aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a control component, the control component can be a polynucleotide comprising a riboswitch. The riboswitch may be capable of binding a nucleoside analog and the compound that binds the control component is a nucleoside analog. The nucleoside analog can be an antiviral agent. The antiviral agent may be acyclovir or penciclovir.

Unless incompatible with or stated in one aspect, in illustrative embodiments of any of the methods and compositions provided herein that include any of the engineered signaling polypeptides (which include ASTRs), the ASTRs of one or both of the engineered signaling polypeptides can bind to a tumor-associated antigen. In some illustrative embodiments, the antigen-specific targeting region of the second engineered polypeptide is a microenvironment-restricted antigen-specific targeting region (MRB-ASTR). In some embodiments, the microenvironment may be an in vivo microenvironment, such as a tumor, tissue, non-tumor tissue, normal tissue, or tissue that has undergone a transient change in pH. For example, tissues that typically undergo a transient change in pH include muscle tissue under anaerobic conditions or muscle tissue that undergoes exercise or inflamed tissue or tissue that is undergoing inflammation. In some embodiments including a target mammalian cell, the target mammalian cell can be a tumor cell or a non-tumor cell or a normal cell.

In illustrative embodiments of any of the methods and compositions provided herein that include one or more replication-defective recombinant retroviral particles, unless incompatible with or stated in one aspect, the replication-defective recombinant retroviral particle may encode a recognition domain of a single, biologically-validated antibody, if not specifically recited in the broadest aspect. In some embodiments, the recognition domain is expressed on the same transcript as the chimeric antigen receptor, and wherein the recognition domain is separated from the chimeric antigen receptor by ribosome skipping and/or cleavage signals. The ribosome skipping and/or cleavage signal can be 2A-1. The recognition domain may comprise a polypeptide or epitope thereof that is recognized by an antibody that recognizes EGFR. The recognition domain may be an EGFR mutant that is recognized by EGFR antibodies and expressed on the surface of transduced T cells and/or NK cells as another control mechanism provided herein. In related embodiments, the recognition domain may comprise a polypeptide or epitope thereof recognized by an antibody that recognizes EGFR.

Unless incompatible with any of the aspects, methods, uses, process products or stated in an aspect, the method may further comprise collecting blood from the subject. The method can further comprise reintroducing the genetically modified T cells and/or NK cells into the subject after ex vivo contact with resting T cells and/or resting NK cells of the subject. In certain embodiments, the contacted resting T cells and/or resting NK cells are from the blood of the individual, and wherein expansion of the genetically modified T cells and/or NK cells occurs in the individual. In certain embodiments, the step between collecting blood and reintroducing the genetically modified T cells and/or NK cells is performed in no more than 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, or 4 hours. Other times are provided herein. In certain embodiments, the subject is not exposed to the lymphodepleting agent within 7 days of the contacting, during the contacting, and/or within 7 days after reintroduction of the genetically modified T cells and/or NK cells into the subject.

In one aspect, provided herein is a method of transducing and/or genetically modifying lymphocytes (e.g., T cells and/or NK cells), in illustrative embodiments resting lymphocytes (resting T cells and/or NK cells), the method comprises contacting ex vivo resting T cells and/or resting NK cells of an individual with replication defective recombinant retroviral particles, wherein the replication deficient recombinant retroviral particle comprises on its surface a pseudotyped component and on its surface a membrane bound anti-CD 3scFvFc antibody capable of binding to resting T cells and/or resting NK cells and facilitating fusion of the replication deficient recombinant retroviral particle therewith, wherein the contacting facilitates transduction of resting T cells and/or NK cells with replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells.

In one aspect, provided herein is a method of transducing and/or genetically modifying resting T cells and/or resting NK cells from isolated blood, the method comprising: collecting blood from a subject; isolating Peripheral Blood Mononuclear Cells (PBMCs) comprising resting T cells and/or resting NK cells; and contacting ex vivo a resting T cell and/or resting NK cell of the individual with a replication deficient recombinant retroviral particle for an effective time, wherein the replication deficient recombinant retroviral particle comprises a pseudotyped component on its surface and a membrane bound anti-CD 3 scfvffc antibody on its surface, thereby producing a genetically modified T cell and/or NK cell, thereby transducing the resting T cell and/or NK cell.

In aspects of the transduction and/or genetic modification of T lymphocytes (e.g., T cells and/or NK cells) herein that include a membrane-bound anti-CD 3 scfvffc antibody, the pseudotyped component is in certain embodiments a herpetic oral virus envelope protein (VSV-G). In some embodiments, the replication-defective recombinant retroviral particle further comprises a membrane-bound polypeptide capable of binding to CD28, which may include, for example, the extracellular domain of CD80, CD86, or a functional fragment thereof that retains the ability to bind to CD28. In some embodiments, the anti-CD 3 scffc antibody is fused to a heterologous GPI anchor linking sequence. In some embodiments, the anti-CD 3 scffc antibody is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle.

In aspects of the transduction and/or genetic modification of T lymphocytes (e.g., T cells and/or NK cells) that include membrane-bound anti-CD 3 scfvffc antibodies herein, the recombinant retroviral particle may further comprise a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor. In some embodiments, the membrane-bound polypeptide capable of binding to CD3 is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle. In some embodiments, the anti-CD 3 scfvffc antibody is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle.

In another aspect, provided herein is a method of transducing and/or genetically modifying a resting lymphocyte of an individual, the method comprising contacting a resting T cell and/or a resting NK cell of the individual ex vivo with a replication-deficient recombinant retroviral particle, wherein the replication-deficient recombinant retroviral particle comprises on its surface a pseudotyped component and on its surface a membrane-bound polypeptide capable of binding to CD3, and not on its surface a membrane-bound polypeptide capable of binding to CD28 and activating CD28, wherein the contacting facilitates transduction of the resting T cell and/or NK cell with the replication-deficient recombinant retroviral particle, thereby producing the genetically modified T cell and/or NK cell.

In another aspect, provided herein is a method of transducing and/or genetically modifying resting T cells and/or resting NK cells from isolated blood, the method comprising: collecting blood from a subject; isolating Peripheral Blood Mononuclear Cells (PBMCs) comprising resting T cells and/or resting NK cells; and contacting ex vivo resting T cells and/or resting NK cells of the individual with replication deficient recombinant retroviral particles for an effective time, wherein the replication deficient recombinant retroviral particles comprise on their surface a pseudotyped component and on their surface a membrane bound polypeptide capable of binding to CD3, but not a membrane bound polypeptide capable of binding to CD28 and activating CD28, thereby producing genetically modified T cells and/or NK cells, thereby transducing the resting T cells and/or NK cells.

In the aspect of transducing and/or genetically modifying resting T lymphocytes herein, the resting T lymphocytes include on their surface a pseudotyped component, which can be, for example, a herpetic oral virus envelope protein (VSV-G), and on their surface a membrane-bound polypeptide capable of binding to CD3, rather than a membrane-bound polypeptide capable of binding to CD28 and activating CD28. In illustrative embodiments, the membrane-binding polypeptide capable of binding to CD3 is an anti-CD 3 scffc antibody, which in some embodiments is fused to a heterologous GPI anchor linking sequence. In some embodiments of this aspect, the contacting is performed for at least 2 hours, or between 2 hours and 24 hours, or between 2 hours and 6 hours. In some embodiments, the detectable marker is encoded by the genome of the replication-defective recombinant retroviral particle and is detected in T cells and/or NK cells following transduction. In some embodiments, the membrane-bound polypeptide capable of binding to CD3 is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle. In some embodiments, the detectable marker is encoded by the genome of the replication-defective recombinant retroviral particle and is detected in T cells and/or NK cells following transduction.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising: one or more pseudotyped elements; a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor; and a pseudotyped component on its surface and an activating component on its surface, wherein the activating component is capable of binding to T cells and/or NK cells and is not encoded by the polynucleotide in the replication defective recombinant retroviral particle, and wherein the activating component is an anti-CD 3 scffc antibody.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising: one or more pseudotyped components capable of binding to T cells and/or NK cells and facilitating fusion with a membrane to which replication defective recombinant retroviral particles are attached;

a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor; and a pseudotyped component on its surface and an activating component on its surface, wherein the activating component is capable of binding to T cells and/or NK cells and is not encoded by the polynucleotide in the replication deficient recombinant retroviral particle, and wherein the activating component is a membrane bound polypeptide capable of binding to CD3 on its surface, but not to CD28 and activates CD28 on its surface.

In some embodiments of the replication-defective recombinant retroviral particle aspects herein, the recombinant retroviral particle further comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor. In some embodiments of these aspects, the membrane-bound polypeptide capable of binding CD3 is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle. In some embodiments of these aspects, the anti-CD 3 scffc antibody is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle.

In any of the aspects and embodiments herein, those such as those comprising a polypeptide or nucleic acid encoding the same include, for example, an activation module present on the surface of a replication-defective recombinant retroviral particle, which activation module may further comprise, for example, a fusion polypeptide having a polypeptide capable of binding to CD28 or, in an illustrative embodiment, CD3, a dimerization motif that may be active in the absence of a dimerization agent. In some embodiments, the activating component may be an anti-CD 3 single chain antibody and the dimerization motif may be selected from the group consisting of: CD69, CD71, CD72, CD96, CD105, CD161, CD162, CD249, CD271, and CD324, and mutants and/or active fragments thereof that retain dimerization capacity.

In any of the aspects and embodiments herein, those such as those comprising a polypeptide or nucleic acid encoding the same include, for example, an activation module present on the surface of a replication-defective recombinant retroviral particle, which activation module may further comprise, for example, a fusion polypeptide having a polypeptide capable of binding to CD28 or, in an illustrative embodiment, CD3, a dimerization motif that may be active in the absence of a dimerization agent. In some embodiments, the activating component may be an anti-CD 3 single chain antibody and the dimerization motif may be selected from the group consisting of: FKBP and rapamycin or analogues thereof, GyrB and coumaromycin or analogues thereof, DHFR and methotrexate or analogues thereof, or DmrB and AP20187 or analogues thereof, as well as mutants and/or active fragments of said dimeric protein retaining the ability to dimerize.

In any of the aspects and embodiments herein, those such as those comprising a polypeptide or nucleic acid encoding the same include, for example, an activation module present on the surface of a replication-defective recombinant retroviral particle, which activation module may further comprise an anti-CD 3-scfvffc-GPI moiety (UCHT1) and optionally VSV-G.

In one embodiment of the above aspects, the polynucleotide further comprises a Kozak-type sequence, a WPRE component, and one or more of a double or triple stop codon, wherein one or more of the double or triple stop codons define a stop read from at least one of the one or more transcription units in certain embodiments, the polynucleotide comprises a Kozak-type sequence selected from CCACCAT/ug (g) (SEQ ID NO:515), CCGCCAT/ug (g) (SEQ ID NO:516), GCCGCCGCCAT/ug (g) (SEQ ID NO:517) or GCCGCCACCAT/ug (g) (SEQ ID NO:532) and in certain embodiments, nucleotides-3 and +4 relative to the start codon of the first nucleic acid sequence both comprise g in another embodiment that can be combined with the foregoing embodiments comprising a Kozak-type sequence and/or the following embodiments including a triple stop codon, the triple polynucleotide comprises a WPRE component in certain embodiments, the WPRE component is located at the 3 'of the stop codon of the one or more of the transcription unit, i.e., in embodiments, the polynucleotide comprises one or more of the double or triple stop codon, wherein the embodiment comprises a double or triple stop codon, the WPRE component, in certain embodiments, the WPRE component, is located at the 3' 3, in certain embodiments, or the other embodiments, wherein the polynucleotide comprises a double or triple stop codon, comprises a double stop codon, or triple stop codon, and/or triple stop codon, comprises a sequence of the embodiment, wherein the polynucleotide comprises a Kozak-type sequence, wherein the polynucleotide.

In some embodiments of any of the aspects provided herein that include ASTR, the ASTR can be anti-tagASTR. The method can further include, for example, tag-bound antibodies that bind to a target molecule, such as a target protein on a target cell.

In any of the embodiments and aspects provided herein that include B cells, T cells, or NK cells, the cells may be allogeneic cells, or they may not be allogeneic cells.

In any of the aspects and embodiments disclosed herein, transduction of DNA into PBMC, B cells, T cells, and/or NK cells, and optionally incorporation of the DNA into the host cell genome, can be performed using methods that do not utilize replication-defective recombinant retroviral particles, for example, other viral vectors, such as those derived from adenovirus, adenovirus-associated virus, or herpes simplex virus type 1, can be utilized as non-limiting examples in other embodiments, these aspects and embodiments can include transfection and/or transduction of target cells with non-viral vectors in any of the embodiments disclosed herein that utilize non-viral vectors to transfect target cells, non-viral vectors including naked DNA can be introduced into target cells (such as PBMC, B cells, T cells, and/or NK cells) using electroporation, nuclear transfection, liposome formulations, lipids, dendrimers, cationic polymers such as poly (ethyleneimine) (PEI) and poly (l-ionone) (P LL), nanoparticles, cell penetrating peptides, microinjection, and/or non-integrated lentiviral vectors provided herein, and transposons can be used as DNA co-transfection vectors for example, transposing DNA into target cells, or gene-transposons containing fragments of interest, e DNA, e-gene-transfecting systems, e-3' or transposons in certain embodiments provided herein, e-transfecting vectors, and methods, e-like DNA-transposing vectors, DNA-like DNA-transposing DNA-like DNA-transposing vectors, DNA-like DNA-transposing systems, DNA-like DNA-transposing systems, DNA.

In another aspect, herein is provided a replication-defective recombinant retroviral particle, each comprising:

A. a pseudotyped component on its surface, which is capable of binding to T cells and/or NK cells and which facilitates fusion of a replication deficient recombinant retroviral particle to its membrane, wherein the pseudotyped component comprises a cytoplasmic domain deleted variant of a measles virus F polypeptide and/or a measles virus H polypeptide;

B. a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen specific targeting region, a transmembrane domain and an intracellular activation domain and a second engineered signaling polypeptide comprising a constitutively active I L-7 receptor mutant, wherein expression of the I L-7 receptor mutant is regulated by a riboswitch binding a nucleoside-like antiviral drug, and

C. a polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD28, wherein the polypeptide is expressed on the surface of a replication-defective recombinant retroviral particle, is capable of binding to a T cell and/or an NK cell, and is not encoded by a polynucleotide in the replication-defective recombinant retroviral particle.

In one aspect, provided herein is a method of genetically modifying and amplifying lymphocytes of an individual, the method comprising:

A. contacting ex vivo, without prior ex vivo stimulation, resting T cells and/or NK cells of an individual with a replication-defective recombinant retroviral particle comprising:

i. a pseudotyped module on its surface, which is capable of binding to T cells and/or NK cells and facilitates membrane fusion of replication-defective recombinant retroviral particles thereto; and

a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component,

wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with replication-defective recombinant retroviral particles, thereby producing T cells and/or NK cells that can be genetically modified;

B. introducing genetically modified T cells and/or NK cells into an individual; and

C. genetically modified T cells and/or NK cells are exposed in vivo to a compound that binds a control component to affect expression of the first engineered signaling polypeptide and facilitate and/or enhance expansion, transplantation, and/or persistence of lymphocytes in vivo to genetically modify and expand lymphocytes of the individual. In illustrative embodiments, transduction is performed without the need for in vitro stimulation.

In any of the above aspects and method aspects for genetically modifying and amplifying lymphocytes or for performing cell therapy herein, if not recited in the broadest aspects, in certain embodiments the polynucleotide further comprises a transcription unit encoding a second engineered signaling polypeptide comprising a first chimeric antigen receptor comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another aspect, provided herein is a method for performing adoptive cell therapy on an individual, the method comprising:

A. collecting blood from a subject;

B. contacting ex vivo resting T cells and/or NK cells from blood of an individual with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises

i. A pseudotyped module on its surface, which is capable of binding to T cells and/or NK cells and facilitates membrane fusion of replication-defective recombinant retroviral particles thereto; and

a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide comprising at least one lymphoproliferative component, the expression of which is regulated by a control component, and a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain,

wherein the contacting produces at least some of the resting T cells and/or NK cells that become genetically modified; and

C. reintroducing the genetically modified T cells and/or NK cells into the individual, wherein expansion, transplantation and/or persistence of the genetically modified T cells and/or NK cells occurs in the individual, and wherein the method between collecting blood and reintroducing the genetically modified T cells and/or NK cells is performed in no more than 24 hours, thereby performing adoptive cell therapy to the individual.

Another aspect herein provides a method for performing adoptive cell therapy for an individual, the method comprising:

A. collecting blood from a subject;

B. isolating Peripheral Blood Mononuclear Cells (PBMCs) comprising resting T cells and/or resting NK cells;

C. contacting ex vivo resting T cells and/or resting NK cells of an individual with replication deficient recombinant retroviral particles, wherein the replication deficient recombinant retroviral particles comprise on their surface a pseudotyping module capable of binding to the resting T cells and/or NK cells and facilitating membrane fusion of the replication deficient recombinant retroviral particles thereto, wherein the contacting facilitates transduction of the resting T cells and/or NK cells with the replication deficient recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells; and

D. within 24 hours of collecting blood from the individual, the genetically modified cells are reintroduced into the individual, thereby performing adoptive cell therapy in the individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the method can further comprise exposing the genetically modified T cells and/or NK cells in vivo to a compound that binds a control component to affect expression of the first engineered signaling polypeptide and, optionally, the second engineered signaling polypeptide, and to promote expansion, transplantation, and/or persistence of lymphocytes in vivo.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the genetically modified T cells and/or NK cells undergo 8, 7, 6, 5, 4, 3, or fewer cell divisions ex vivo prior to being introduced or reintroduced into an individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the expansion, transplantation and/or persistence in vivo of the genetically modified T cells and/or NK cells depends on the presence or absence, and in illustrative embodiments on the presence, of a compound that binds to a control component.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the subject is not exposed to a lymphodepleting agent within 7 days, 14 days, or 21 days of performing the contacting, or during the contacting, and/or within 7 days, 14 days, or 21 days after introducing the modified T cells and/or NK cells into the subject. In other embodiments, the subject is not exposed to the lymphodepleting agent during the contacting.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the T cell and/or NK cell conditions for the contacting step can be any of the conditions, such as the contact time, provided in other paragraphs in this illustrative embodiments section and other sections herein for methods for genetically modifying and/or transducing, including those that do not include an explicit in vivo step.

In an illustrative embodiment of any one of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the method further comprises the step of isolating the replication-defective recombinant retroviral particle from the T cell and/or NK cell after the contacting. In methods involving introduction or reintroduction, the separation is performed prior to introduction. In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the exposing step comprises administering to the subject a dose of the compound prior to or during the contacting, and/or after the genetically modified T cells and/or NK cells have been introduced into the subject.

In an illustrative embodiment of any one of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the method comprises collecting blood comprising T cells and/or NK cells from an individual prior to contacting the T cells and/or NK cells with replication deficient recombinant retroviral particles ex vivo, and wherein the introducing is reintroducing. For example, between 20ml and 250ml of blood is drawn from the subject.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, no more than 8 hours, 12 hours, 24 hours, or 48 hours between the time blood is collected from the individual and the time modified T cells and/or NK cells are reintroduced into the individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, the range between the time of blood collection from the individual and the time of reintroduction of the modified T cells and/or NK cells into the individual is between 4 or 8 hours at the low end and 12, 24, 36 or 48 hours at the high end.

In aspects and embodiments provided herein that include administering to a subject (particularly where the subject has or is suspected of having cancer) a genetically modified T cell and/or NK cell, the method can further comprise delivering to the subject an effective amount of an immune checkpoint inhibitor.

In an illustrative embodiment of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly recited in the broadest aspects, all steps after blood collection and before reintroduction are performed in a closed system in which one person monitors the closed system throughout the process. In another embodiment, after collection of the blood and before reintroduction of the blood, it is performed in a closed system kept in the same room as the individual.

In illustrative embodiments of any of the methods and compositions provided herein that include one or more transcriptional units or one or more engineered signaling polypeptides, if not specifically recited in the broadest aspects, one of the transcriptional units or one of the engineered signaling polypeptides may encode or comprise or further comprise an antigen-specific targeting region (ASTR) and a transmembrane domain that links the ASTR to a lymphoproliferative component, the ASTR of such engineered signaling polypeptide is capable of binding to a first tumor antigen, and, when present, the ASTR of a second engineered signaling polypeptide is capable of binding to a second tumor antigen.

In any of the methods or compositions provided herein that include a lymphoproliferative component, the method or composition may further include an inhibitory RNA molecule (such as miRNA or shRNA) that stimulates STAT5 pathway or inhibits SOCS pathway, or the lymphoproliferative component may be replaced by the inhibitory RNA molecule, for example, the inhibitory RNA molecule may bind a nucleic acid encoding a protein selected from the group consisting of ABCG1, SOCS, TGFbR2, SMAD2, cCB L, and pd1 in illustrative embodiments of any of the replication-deficient recombinant retroviral particles or transduced cells provided herein or methods including the same, such replication-deficient recombinant retroviral particles or zeta cells may encode two or more inhibitory RNA molecules, such as miRNA or shRNA in an intron, in some embodiments 1, 2,3, or 4 inhibitory RNA molecules, that bind a nucleic acid encoding one or more of the genes expressed by a target T cell, PD-1, CT 46 4, TCR α, TCR β, 852, CD 892, CD 8926, CD 9626, TCR-CD 2, TCR 9626, TCR-CD 2, miRNA, TCR 9626, and miR, CD3, and miR 3, CD 38, and CD 38 in another illustrative embodiment, CD3, CD 38, and CD 38.

In illustrative embodiments of any of the methods and compositions provided herein, the replication-defective recombinant retroviral particles, mammalian cells, and/or packaging cells can comprise a Vpu polypeptide. The Vpu polypeptide can be, for example, a fusion polypeptide, and in some examples, particularly in packaging cells, a membrane-bound Vpu polypeptide. In illustrative embodiments of any of the methods and compositions provided herein, the replication-defective recombinant retroviral particles, mammalian cells, and/or packaging cells can comprise both a Vpu polypeptide and a Vpx polypeptide.

In illustrative embodiments of any of the methods and compositions provided herein, the replication-defective recombinant retroviral particles, mammalian cells, and/or packaging cells can comprise a Vpx polypeptide. The Vpx polypeptide can be, for example, a fusion polypeptide, and in some examples, particularly in packaging cells, a membrane-bound Vpx polypeptide.

In any of the methods or compositions provided herein, the one or more pseudotyped components can include a vesicular oral virus envelope protein (VSV-G), a feline endogenous virus (RD114) envelope protein, an oncogenic retroviral amphotropic envelope protein, or a functional fragment thereof.

In one aspect, provided herein are genetically modified T cells and/or NK cells, wherein the T cells and/or NK cells have been genetically modified to express a first engineered signaling polypeptide comprising a lymphoproliferative component and a second engineered signaling polypeptide comprising a CAR comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another aspect, provided herein are genetically modified T cells and/or NK cells comprising: a pseudotyped component on the surface of the T cell or NK cell and an activating component on the surface of the T cell or NK cell, wherein the T cell or NK cell has been genetically modified to express a first engineered signaling polypeptide comprising a lymphoproliferative component and a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activating domain.

In another aspect, provided herein are genetically modified T cells and/or NK cells comprising:

A. a first engineered signaling polypeptide comprising at least one lymphoproliferative component; and

B. a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain.

In some embodiments, the genetically modified T cells and/or NK cells are capable of living and/or proliferating and/or expanding ex vivo or in vitro in culture for a period of 6 days, 7 days, 14 days, 21 days or 35 days, 42 days, or more, during culture, in the absence of exposing the cells to an interleukin (such as I L-15, I L-7, and in illustrative embodiments, I L-2), and optionally a target of an ASTR directed against a CAR expressed by the cells, or in certain illustrative embodiments, in the presence of an antigen recognized by the CAR (particularly where the genetically modified T cells or NK cells are formed by genetically modifying and/or transforming retroviral particles having a pseudotyped component and optionally an isolated or fused activation domain on their surface and generally do not require pre-activation).

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for making replication-defective recombinant retroviral particles), for example, a packageable RNA genome is encoded by a polynucleotide operably linked to a promoter, wherein the promoter is constitutively active or inducible by a first transactivator or a second transactivator.

In addition, the packagable RNA genome comprises, from 5 'to 3':

1.) a 5' long terminal repeat or an activated fragment thereof;

2.) a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

3.) a nucleic acid sequence encoding a first target polypeptide and/or a nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNA molecules;

4.) a promoter active in the target cell; and

5.) a 3' long terminal repeat or an activated fragment thereof.

In some embodiments, the nucleic acid sequence encoding the first target polypeptide is in an opposite orientation to the RNA encoding the retroviral component for packaging and assembly and the 5' L TR.

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for making replication-defective recombinant retroviral particles), for example, the first target polypeptide comprises a first engineered signaling polypeptide and wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component. The packagable RNA genome can further comprise a nucleic acid sequence encoding a second target polypeptide. The second target polypeptide may comprise a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising:

1.) a first antigen-specific targeting region;

2.) a first transmembrane domain; and

3.) a first intracellular activation domain.

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for making replication-defective recombinant retroviral particles (e.g., mammalian cells)), for example, the packaging cells can include a nucleic acid sequence encoding a Vpu polypeptide, e.g., on the second transcription unit or an optional third transcription unit, or on other transcription units operably linked to a first inducible promoter. In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for making replication-defective recombinant retroviral particles (e.g., mammalian cells)), for example, the packaging cells can include a nucleic acid sequence encoding both a Vpx polypeptide and a Vpu polypeptide, e.g., on the second transcription unit or an optional third transcription unit, or on other transcription units operably linked to a first inducible promoter. The mammalian cell, which may be a packaging cell, may be a 293 cell.

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for making replication-defective recombinant retroviral particles (e.g., mammalian cells)), for example, the packaging cells can include a nucleic acid sequence encoding Vpx, e.g., on the second transcription unit or an optional third transcription unit, or on other transcription units operably linked to the first inducible promoter. The mammalian cell, which may be a packaging cell, may be a 293 cell.

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for making replication-defective recombinant retroviral particles), the first ligand can be rapamycin and the second ligand can be tetracycline or doxorubicin, or the first ligand can be tetracycline or doxorubicin and the second ligand can be rapamycin.

In some aspects, provided herein are cells that have been transduced by any one of the replication defective recombinant retroviral particles provided herein. The cell may be, for example, a lymphocyte, such as a T cell or NK cell. In an illustrative embodiment, the cell is a human cell.

In one aspect, provided herein is a method for expanding modified T cells and/or NK cells in an individual, the method comprising:

A. contacting isolated resting T cells and/or resting NK cells obtained from the individual with replication-defective recombinant retroviral particles of any one of the embodiments disclosed herein;

B. introducing genetically modified T cells and/or NK cells into an individual; and

C. providing to the subject an effective amount of acyclovir, a prodrug of acyclovir, penciclovir or a prodrug of penciclovir, wherein the modified T cells and/or NK cells proliferate in the subject following administration of acyclovir, a prodrug of acyclovir, penciclovir or a prodrug of penciclovir, thereby expanding the modified T cells and/or NK cells in the subject.

In another aspect, provided herein is a method for stopping the expansion, transplantation, and/or persistence of modified T cells and/or NK cells in an individual, the method comprising:

A. contacting isolated resting T cells and/or NK cells obtained from the individual with a replication-defective recombinant retroviral particle of any one of the embodiments disclosed herein;

B. introducing the modified T cells and/or NK cells into an individual;

C. administering to the subject an effective amount of acyclovir, a prodrug of acyclovir, penciclovir or a prodrug of penciclovir to expand modified T cells and/or NK cells in the subject, wherein the modified T cells and/or NK cells proliferate in the subject following administration of acyclovir, a prodrug of acyclovir, penciclovir or a prodrug of penciclovir to expand modified PB L in the subject, and

D. stopping administration of acyclovir, an acyclovir prodrug, penciclovir, or a penciclovir prodrug, wherein the modified T cells and/or NK cells stop proliferating in the subject after stopping administration of acyclovir, an acyclovir prodrug, penciclovir, or a penciclovir prodrug, thereby controlling the expansion, transplantation, and/or persistence of the modified T cells and/or NK cells in the subject.

In another aspect, provided herein is a method of treating cancer in an individual, the method comprising:

A. contacting isolated resting T cells and/or NK cells obtained from the individual with replication deficient recombinant retroviral particles according to any one of the embodiments disclosed herein;

B. introducing genetically modified T cells and/or NK cells into an individual; and

C. administering to the subject an effective amount of acyclovir, a prodrug of acyclovir, penciclovir, or a prodrug of penciclovir to expand modified T cells and/or NK cells in the subject, wherein the modified T cells and/or NK cells proliferate in the subject following administration of acyclovir, a prodrug of acyclovir, penciclovir, a prodrug of penciclovir, and wherein chimeric antigen receptors in the modified T cells and/or NK cells bind to cancer cells in the subject, thereby treating cancer in the subject.

In another aspect, provided herein is a transduced T cell and/or NK cell comprising a recombinant 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 a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises a constitutively active I L-7 receptor mutant, and wherein the control component is capable of binding to a compound in vivo, and/or is designed and/or configured to bind to a compound.

In another aspect, provided herein is a retroviral packaging system comprising:

a mammalian cell comprising:

A. a first trans-activator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of a contrasting deletion of the first ligand;

B. a second trans-activator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence of the second ligand in the absence of the second ligand; and

C. against the packagable RNA genome of the retroviral particle,

wherein the first transactivator modulates expression of the second transactivator and retroviral REV protein, wherein the second transactivator modulates expression of a gag polypeptide, a pol polypeptide, and one or more pseudotyped components capable of binding to a target cell and facilitating membrane fusion thereto, and wherein the retroviral protein is derived from a retrovirus. Embodiments of this aspect may include any of the embodiments provided herein for that embodiment in other aspects.

In another aspect, provided herein is a method for making a replication-defective recombinant retroviral particle comprising:

A. culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed from a first constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the first ligand as compared to the absence, and wherein expression of the second transactivator and retroviral REV protein is regulated by the first transactivator;

B. incubating a population of packaging cells comprising the accumulated first transactivator in the presence of a first ligand to accumulate a second transactivator and retroviral REV protein, wherein the second transactivator is capable of binding to the second ligand and a second inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the second ligand compared to the absence; and

C. incubating a population of packaging cells comprising the accumulated second transactivator and retroviral REV protein in the presence of a second ligand, thereby inducing expression of a gag polypeptide, a pol polypeptide, and one or more pseudotyped components, thereby producing replication-defective recombinant retroviral particles,

wherein the packagable RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is constitutively active or inducible by the first transactivator or the second transactivator, and wherein the one or more pseudotyped components are capable of binding to a target cell and/or facilitating fusion of a membrane with a replication-defective recombinant retroviral particle.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particles, the mammalian cell further comprises an activation module capable of binding to and activating a target cell (typically a lymphocyte, such as an NK cell or in illustrative embodiments a T cell), and the first transactivator regulates expression of the activation module. The activating module is on the surface of the replication-defective recombinant retroviral particle and wherein the activating module may comprise: a membrane-bound polypeptide capable of binding to CD 3; and/or a membrane-bound polypeptide capable of binding to CD28. A membrane-bound polypeptide capable of binding CD3 is a polypeptide capable of binding to CD3 fused to a heterologous GPI anchor linker sequence, and a membrane-bound polypeptide capable of binding CD28 is a polypeptide capable of binding to CD28 fused to a heterologous GPI anchor linker sequence. A membrane-binding polypeptide capable of binding to CD28 in some embodiments comprises CD80, CD86, or a functional fragment thereof (such as the extracellular domain of CD 80) capable of inducing CD 28-mediated activation of Akt. In other embodiments, the membrane-binding polypeptide capable of binding CD3 is anti-CD 3scFv or anti-CD 3 scfvcfc that binds to a CD14 GPI-anchored linker sequence, and wherein the membrane-binding polypeptide capable of binding CD28 is CD80, or an extracellular fragment thereof that binds to a CD16B GPI-anchored linker sequence.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle aspects, the mammalian cell further comprises a membrane-bound interleukin, and the first transactivator regulates expression of the membrane-bound interleukin can be, for example, I L-7, I L-15, or an activating fragment thereof the membrane-bound interleukin in embodiments can be a fusion polypeptide of I L-7 or an activating fragment thereof and DAF, for example, the fusion polypeptide can comprise a DAF signal sequence and I L-7 without its signal sequence, followed by residues 36 to 525 of DAF.

In some embodiments of the retroviral packaging systems provided herein and methods for making a replication-defective recombinant retroviral particle state, a mammalian cell comprises, associated with its membrane, an activation component comprising anti-CD 3scFv or anti-CD 3scFvFc that binds to a CD14 GPI-anchor linker, or CD80 or an extracellular fragment thereof that binds to a CD16B GPI-anchor linker, and a membrane-bound interleukin comprising a fusion polypeptide of I L-7 or an activation fragment thereof and DAF comprising a GPI-anchor linker, and wherein the first transactivator modulates the expression of each of the activation component and the membrane-bound interleukin, in some embodiments, I L-7 or an activation fragment thereof and the DAF fusion, anti-CD 3scFv or anti-CD 3scFvFc, and CD80 or an extracellular fragment thereof each comprise a DAF signal sequence.

In some embodiments of the retroviral packaging systems provided herein and methods for making a replication-defective recombinant retroviral particle form, the mammalian cell further comprises a Vpu polypeptide. In some embodiments of the retroviral packaging systems provided herein and methods for making a replication-defective recombinant retroviral particle state, the mammalian cell further comprises a Vpu polypeptide and a Vpx polypeptide. In some embodiments of the retroviral packaging systems provided herein and methods for making a replication-defective recombinant retroviral particle form, the mammalian cell further comprises a Vpx polypeptide. In these or other embodiments, the one or more pseudotyped components comprise one or more viral polypeptides recognized by T cells. The one or more pseudotyped components may comprise a measles virus F polypeptide, a measles virus H polypeptide, and/or a fragment thereof. In certain illustrative embodiments, the one or more pseudotyped components are cytoplasmic domain deleted variants of a measles virus F polypeptide and/or a measles virus H polypeptide.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle aspects, the packagable RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is constitutively active or inducible by the first transactivator or the second transactivator. In illustrative embodiments, the packagable RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is inducible by a second transactivator.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle forms, the packagable RNA genome further comprises, from 5 'to 3':

a) a 5' long terminal repeat or an activated fragment thereof;

b) a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c) a nucleic acid sequence encoding a first target polypeptide and optionally a second target polypeptide;

d) a fourth promoter operably linked to the first target polypeptide and optionally the second target polypeptide, wherein the fourth promoter is active in the target cell but inactive in the packaging cell line; and

e) 3' long terminal repeats or activated fragments thereof.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle patterns comprising the immediately above constructs, the third promoter promotes transcription or expression in the opposite direction to the transcription and expression promoted by the fourth promoter.

In some embodiments of the retroviral packaging systems provided herein and methods for making a replication-defective recombinant retroviral particle state, the packageable RNA genome encodes a replication-defective recombinant retroviral particle of any embodiment disclosed herein, wherein the first and second target polypeptides are first and second engineered signaling polypeptides, respectively. In some embodiments, for example, the packagable RNA genome further comprises a control component operably linked to a nucleic acid encoding the first engineered signaling polypeptide or the second engineered signaling polypeptide. The control component is a riboswitch in the illustrative embodiment. The riboswitch in the illustrative embodiment is capable of binding a compound and the compound that binds the control component is a nucleoside analog, and the nucleoside analog can be an antiviral drug, such as acyclovir or penciclovir.

In some embodiments of the retroviral packaging systems and methods for making replication-defective recombinant retroviral particle aspects provided herein, the packagable RNA genome further comprises an intron comprising a polynucleotide encoding an inhibitory RNA molecule (such as miRNA or shRNA). The intron can be adjacent to or downstream of the fourth promoter.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle patterns, the target cells can be T cells and/or NK cells.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle aspects, the one or more pseudotyped components comprise a vesicular stomatitis virus envelope protein (VSV-G), a feline endogenous virus (RD114) envelope protein, an oncogenic retroviral amphotropic envelope protein, or a functional fragment thereof.

In some embodiments of the retroviral packaging systems provided herein and methods for making replication-defective recombinant retroviral particle forms, the packageable RNA genome size is 11,000KB or less, or 10,000KB or less. In some embodiments of the retroviral packaging systems provided herein and methods for making a replication-defective recombinant retroviral particle state, a first target polypeptide comprises a first engineered signaling polypeptide and wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component and a second target polypeptide comprises a second engineered signaling polypeptide comprising a CAR.

In illustrative embodiments of any of the methods and compositions provided herein that include a control component can be a polynucleotide comprising a riboswitch. The riboswitch may be capable of binding a nucleoside analog and the compound that binds the control component is a nucleoside analog. The nucleoside analog can be an antiviral agent. The antiviral agent may be acyclovir or penciclovir. The riboswitch may preferably be coupled to acyclovir via penciclovir or preferably to penciclovir via acyclovir. Riboswitches can reduce binding to nucleoside analog antiviral drugs at temperatures above 37 ℃, 37.5 ℃,38 ℃, 38.5 ℃, or 39 ℃ (e.g., above 39 ℃). Riboswitches can be between the low end of the range of 35, 40, 45, and 50 nucleotides in length and the high end of the range of 60, 65, 70, 75, 80, 85, 90, 95, and 100 nucleotides in length, such as between 45 and 80 nucleotides in length. In illustrative embodiments of any of the methods and compositions provided herein that include a riboswitch, the polynucleotide target polynucleotide modulated by the riboswitch may include a region encoding a miRNA, shRNA, and/or polypeptide. The polynucleotide target polynucleotide may encode a lymphoproliferative component. The polynucleotide target polynucleotide may be operably linked to a promoter. The polynucleotide target polynucleotide may include a region encoding a polypeptide, and the polypeptide may include a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain. In illustrative embodiments of any of the methods and compositions provided herein that include a riboswitch, the functional switching domain can modulate an internal ribosome entry site, pre-mRNA splice donor accessibility in a viral gene construct, translation, transcription termination, transcription degradation, miRNA expression, or shRNA expression, thereby modulating expression of a polynucleotide target polynucleotide. The riboswitch can include a ribonuclease. In illustrative embodiments of any of the methods and compositions provided herein that include a riboswitch, the isolated polynucleotide can be a molecular cloning vector or an expression vector. In illustrative embodiments of any of the methods and compositions provided herein that include a riboswitch, the isolated polynucleotide can be integrated into a retroviral genome or into a mammalian chromosome or fragment thereof. In illustrative embodiments of any of the aspects and embodiments herein comprising a riboswitch, the riboswitch modulates pre-mRNA splicing. For example, a riboswitch can comprise a branch point sequence between two exons as is common on polynucleotides and/or transcription units herein. Thus, the binding of nucleoside-like antiviral compounds or drugs to riboswitches modulates exon splicing. Such embodiments may include a polynucleotide comprising a first exon, a second exon, and a riboswitch between the first exon and the second exon, wherein the riboswitch comprises a branch point sequence, and wherein the binding of a nucleoside-like antiviral compound or drug (e.g., acyclovir) to the riboswitch modulates exon splicing of the first and second exons. In illustrative embodiments of any of the aspects and embodiments herein comprising riboswitches, ribose disclosure may control gene expression by modulating pre-mRNA trans-splicing. In such embodiments, the riboswitch is located within the trans-splicing intron. For example, one aspect can include a polynucleotide comprising a first exon, a second exon, and a riboswitch between the first exon and the second exon, wherein the riboswitch comprises a branch point sequence, and wherein the binding of acyclovir to the riboswitch modulates exon splicing of the first and second exons.

In another illustrative embodiment, the replication-defective recombinant retroviral particle further comprises on its surface a fusion polypeptide comprising an interleukin covalently linked to DAF, in some cases the interleukin may be I L-7 or I L-15, and the fusion polypeptide may comprise a DAF signal sequence, I L-7 without its signal sequence, and a fragment of DAF comprising a GPI-anchor linker sequence.

In another illustrative embodiment of any of the replication-defective recombinant retroviral particle aspects herein, the riboswitch further controls expression of the chimeric antigen receptor in a manner regulated by the riboswitch in combination with a nucleoside-like antiviral drug, in some cases acyclovir and/or penciclovir in another embodiment, the constitutively active I L-7 can be replaced by a miRNA or shRNA or a nucleic acid encoding a miRNA or shRNA, and there can be I L-7 the miRNA or shRNA can be encoded by a nucleic acid within an intron.

Another aspect provided herein is a method for making a replication-defective recombinant retroviral particle, comprising:

A. culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed from a constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the first ligand compared to the absence, and wherein expression of the second transactivator and retroviral REV protein is regulated by the first transactivator;

B. incubating a population of packaging cells comprising the accumulated first transactivator in the presence of a first ligand to accumulate a second transactivator and retroviral REV protein and, typically on its surface, an activation component comprising a polypeptide capable of binding to CD3 and comprising a polypeptide capable of binding to CD28, wherein the second transactivator is capable of binding to the second ligand and a second inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the second ligand as compared to in the absence; and

C. incubating a population of packaging cells comprising the accumulated second transactivator and retroviral REV protein in the presence of a second ligand, thereby inducing expression of a gag polypeptide, a pol polypeptide and a pseudotyped component capable of binding to T cells and/or NK cells and facilitating fusion with the replication deficient recombinant retroviral particle membrane, wherein the pseudotyped component comprises a cytoplasmic domain deleted variant of a measles virus F polypeptide and/or a measles virus H polypeptide,

wherein the packagable RNA genome is encoded by a polynucleotide operably linked to a third promoter, and wherein the promoter is inducible by the second transactivator,

wherein the packagable RNA genome comprises, from 5 'to 3':

i.5' long terminal repeat or an activated fragment thereof;

a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

a nucleic acid sequence encoding a first engineered signaling polypeptide comprising a chimeric antigen receptor and a second engineered signaling polypeptide comprising a constitutively active I L-7 receptor mutant, separated by a cleavage signal;

a fourth promoter active in T cells and/or NK cells; and

v.3' long terminal repeats or activated fragments thereof,

thereby producing a replication-defective recombinant retroviral particle.

In an illustrative embodiment of the method, the packagable RNA genome further comprises a riboswitch that binds to a nucleoside analog antiviral drug, wherein binding of the riboswitch to the nucleoside analog antiviral drug increases expression of the I L-7 receptor mutant, in some embodiments, the riboswitch further controls expression of the chimeric antigen receptor by binding to the riboswitch to the nucleoside analog antiviral drug, in another illustrative embodiment, the nucleoside analog antiviral drug is acyclovir and/or penciclovir, in another illustrative embodiment, the packagable RNA genome further comprises a recognition domain, wherein the recognition domain comprises a polypeptide recognized by an antibody that recognizes EGFR or an epitope thereof, in another illustrative embodiment, the first ligand is rapamycin and the second ligand is tetracycline or doxorubicin, or the first ligand is tetracycline or doxorubicin, and the second ligand is rapamycin, in another illustrative embodiment, the packaging cell further comprises a fusion polypeptide encoding a miRNA, or other transcription-inducible promoter operably linked to a third transcription unit, or other transcription unit encoding a miRNA, or a miRNA encoding a miRNA, and optionally a CD-shRNA polypeptide capable of binding to a CD-homing molecule encoding a miRNA, wherein the miRNA, or homing molecule is capable of binding to a miRNA, and optionally, wherein the miRNA is capable of binding to a miRNA, and the miRNA, or homing molecule, and optionally, and optionally, the miRNA is capable of being linked to a further capable of binding to a cell, wherein the cell, comprises a chimeric antigen receptor, wherein the chimeric antigen encoding a chimeric antigen receptor is capable of binding to a chimeric antigen encoding a miRNA, wherein the chimeric antigen encoding a chimeric antigen binding to a chimeric antigen encoding a miRNA, wherein the miRNA, the chimeric antigen is capable of expressing a chimeric antigen encoding a chimeric antigen is capable of the chimeric antigen encoding a chimeric antigen binding to a miRNA, wherein the chimeric antigen encoding a miRNA, wherein the miRNA, the chimeric antigen encoding a miRNA, the miRNA, wherein the miRNA, the chimeric antigen is capable of expressing a chimeric antigen, wherein the chimeric antigen encoding a chimeric antigen, wherein the chimeric antigen, the chimeric antigen is capable of expressing a chimeric antigen encoding a chimeric antigen is capable of expressing a chimeric antigen is capable of the chimeric antigen is capable of expressing a chimeric antigen, wherein the chimeric antigen is capable of the chimeric antigen, the chimeric antigen encoding a chimeric antigen-2 is capable of expressing a chimeric antigen, the chimeric antigen or chimeric antigen, the chimeric antigen is capable of expressing a chimeric antigen, the chimeric antigen.

In another aspect herein is provided a genetically modified lymphocyte comprising:

A. a first engineered signaling polypeptide comprising a constitutively active I L-7 receptor mutant, and

B. a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another illustrative embodiment, the expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is regulated by a riboswitch that binds a nucleoside analog antiviral drug, wherein binding of the nucleoside analog antiviral drug to the riboswitch increases the expression of the I L-7 receptor mutant.

Another aspect herein provides a genetically modified T cell and/or NK cell comprising:

a. a first engineered signaling polypeptide comprising at least one lymphoproliferative component; and

b. a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain.

In an illustrative embodiment of the genetically modified T cell and/or NK cell-like, the lymphoproliferative component is constitutively active, and in some cases constitutively active mutated I L-7 receptor or fragment thereof in another illustrative embodiment, expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is regulated by a control component in some cases the control component is a polynucleotide comprising a riboswitch in some cases the riboswitch is capable of binding a nucleoside analog and, when present, expresses the first engineered signaling polypeptide and/or the second engineered signaling polypeptide in other illustrative embodiments, the genetically modified T cell and/or NK cell has on its surface an activating component, a pseudotyped component and/or a membrane-bound cytokine, in some cases the activating component comprises a membrane-bound polypeptide capable of binding to CD3, and/or a membrane-bound polypeptide capable of binding to CD28.

In one aspect, provided herein is a method for genetically modifying and amplifying lymphocytes of a subject, comprising:

A. contacting ex vivo resting T cells and/or NK cells of an individual with a replication-defective recombinant retroviral particle, which replication-defective recombinant retroviral particle comprises:

i. a pseudotyped module on its surface, which is capable of binding to T cells and/or NK cells and facilitates membrane fusion with replication-defective recombinant retroviral particles; and

a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units: encoding a first engineered signaling polypeptide that is modulated by a control component, wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component; and optionally encoding a second engineered signaling polypeptide that is optionally regulated by the control component, wherein the second engineered signaling polypeptide comprises an intracellular activation domain and optionally other components of the CAR, wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells;

B. introducing genetically modified T cells and/or NK cells into an individual; and

genetically modified T cells and/or NK cells are exposed in vivo to a compound that acts as a control component to affect expression of the first engineered signaling polypeptide and promote expansion, transplantation, and/or persistence of lymphocytes in vivo, thereby genetically modifying and expanding the lymphocytes of the individual.

In illustrative embodiments, transduction is performed without the need for in vitro stimulation. In illustrative embodiments, the compound is a chaperone, such as a small chaperone. In illustrative embodiments, binding of a chaperone to a lymphoproliferative component increases the proliferative activity of the lymphoproliferative component. The chaperone can be administered to the subject prior to collection of blood, during contact, and/or after introduction of T cells and/or NK cells into the subject. It will be appreciated by this aspect that the compound is a control component, the compound typically being capable of binding to a lymphoproliferative component and/or a component of the CAR, and binding to such lymphoproliferative component or CAR component during performance of the method. Other embodiments and teachings related to the methods provided herein that include transfecting T cells and/or NK cells with replication-defective recombinant retroviral particles apply to this aspect, including chaperone embodiments as well.

In another aspect, provided herein is a chimeric antigen receptor for binding a target antigen, the chimeric antigen receptor comprising:

a) a microenvironment-restricted antigen-specific targeting region that exhibits increased binding to a target antigen under aberrant conditions compared to normal physiological conditions, wherein the antigen-specific targeting region binds to the target;

b) a transmembrane domain; and

c) an intracellular activation domain.

In illustrative embodiments of any of the methods and compositions provided herein that include an antigen-specific targeting domain (ASTR) that is restricted by a microenvironment, binding affinity of the ASTR can be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% for a target antigen in an assay under abnormal conditions as compared to normal conditions, abnormal conditions can be hypoxia, acidic pH, higher concentration of lactic acid, higher concentration of hyaluronic acid, higher concentration of albumin, higher concentration of adenosine, higher concentration of R-2-hydroxyglutarate, higher concentration of PAD enzyme, higher pressure, higher oxidation reaction and lower nutrient availability as compared to ASTR that is restricted by a microenvironment at a pH of 7.4 can exhibit an increase in antigen binding at a pH of 6.7. ASTR that is restricted by a microenvironment can exhibit an increase in antigen binding in a tumor environment associated with the corresponding physiological conditions and/or in vivo tumor environment assay conditions as well as 4-1, ST, BB, AA, MAX, CD-125, CD-CD receptor, CD-receptor CD-kinase, CD-RANK L, RON, ROR1, ROR2 SCH900105, SDC1, S L AMF7, TAG-72, tenascin C, TGF β 2, TGF-P, TRAI L-R1, TRAI L-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin ASTR can be an antibody, antigen, ligand, receptor binding domain of a ligand, receptor, ligand binding domain of a receptor, or an affibody ASTR can be a full-length antibody, a single-chain antibody, a Fab fragment, a Fab 'fragment, (Fab')₂Fragments, Fv fragments, and bivalent single chain or diabodies. The ASTR may include heavy and light chains from an antibody. The antibody may be a single chain variable fragment. In some embodiments, the heavy and light chains may be separated by a linker, wherein the linker is between 6 and 100 amino acids in length. In some embodiments, the heavy chain may be positioned N-terminal to the light chain on the chimeric antigen receptor, and in some embodiments the light chain may be positioned N-terminal to the heavy chain on the chimeric antigen receptor.

In illustrative embodiments of any of the methods provided herein that include a polypeptide display library, the polypeptide display library can be a phage display library or a yeast display library. The polypeptide display library may be an antibody display library. The antibody display library may be a human or humanized antibody display library. The antibody display library may be a native library. The method may comprise infecting bacterial cells with the collected phage to produce a refined phage display library, and repeating the contacting, incubating, and collecting for 1 to 1000 cycles, using the refined phage display library produced by the previous cycle.

In another aspect, provided herein is a transduced T cell and/or NK cell comprising a recombinant polynucleotide comprising one or more transcriptional units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcriptional units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises a constitutively active I L-7 receptor mutant, and wherein the control component is capable of binding, or is configured to bind, a compound in vitro or in vivo.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising a recombinant 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 a first engineered signaling polypeptide that is regulated by a control component (which may be an in vivo control component), wherein the first engineered signaling polypeptide comprises a constitutive I L-7 receptor mutant, and wherein the control component is capable of binding to a compound in vivo, or is configured to bind a compound in vivo.

In another aspect, provided herein is a method of transducing a T cell and/or NK cell, the method comprising contacting the T cell and/or NK cell with a replication deficient recombinant retroviral particle comprising a recombinant polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises a constitutive I L-7 receptor mutant, and wherein an in vivo control component is capable of binding to a compound in vivo or in vitro under transduction conditions, thereby transducing the T cell and/or NK cell.

In illustrative embodiments of the transduced T cell and/or NK cell forms, replication-deficient recombinant retroviral particle forms, and method forms provided in the preceding paragraphs, the recombinant polynucleotide further comprises a transcription unit encoding a second engineered signaling polypeptide comprising a first chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain in other illustrative embodiments, the lymphoproliferative component comprises a mutated I L-7 receptor, or a fragment thereof in other illustrative embodiments, the control component is a polynucleotide comprising a riboswitch in some cases, the riboswitch is capable of binding a nucleoside analog and the compound that binds the control component is a nucleoside analog in some cases, the nucleoside analog is an antiviral agent such as acyclovir or penciclovir in some embodiments, the antiviral agent is acyclovir in other illustrative embodiments, the constitutive active I L-7 receptor mutant is fused to EGFR or its epitope in other illustrative embodiments, the constitutive active I L-7 receptor mutant comprises a wild type T cell insert activity insert in other illustrative embodiments, the constitutive active I L-7 g mutant comprises a wild type T receptor insert at position 399-598 in other illustrative embodiments, the constitutive active I-9-7-9-c insert is inserted in other illustrative embodiments, the wild type T cell insert activity of the mutated T-9-c insert.

In one aspect, provided herein is a replication-defective recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein:

a. a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, an

b. A second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another aspect of this document there is provided a mammalian packaging cell line comprising a packageable RNA genome of a replication defective recombinant retroviral particle, wherein the packageable RNA genome comprises:

a 5' long terminal repeat or an activated fragment thereof;

b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c. a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acids encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain; and

a 3' long terminal repeat or an activated fragment thereof.

In some embodiments of the mammalian packaging cell line aspects, the polynucleotide of (c) may be in an opposite orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging component (b), the 5 'long terminal repeat (a), and/or the 3' long terminal repeat (d).

In some embodiments of the mammalian packaging cell line aspects, expression of the packagable RNA genome is driven by an inducible promoter active in the mammalian packaging cell line.

In some embodiments of the mammalian packaging cell line aspects, the retroviral cis-acting RNA packaging component can comprise a central polypurine tract (cPPT)/central termination sequence, HIV Psi, or a combination thereof.

Another aspect herein provides a retroviral vector comprising a packageable RNA genome for a replication-defective recombinant retroviral particle, wherein the packageable RNA genome comprises:

a 5' long terminal repeat or an activated fragment thereof;

b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c. a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acids encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain; and

a 3' long terminal repeat or an activated fragment thereof.

In some embodiments of the retroviral vector aspects, the polynucleotide of (c) may be in an opposite orientation to the nucleic acid sequence (b), the 5 'long terminal repeat (a), and/or the 3' long terminal repeat (d) encoding the retroviral cis-acting RNA packaging component.

In some embodiments of the retroviral vector aspects, expression of the packagable RNA genome is driven by an inducible promoter active in a mammalian packaging cell line.

In some embodiments of the retroviral vector aspects, the retroviral cis-acting RNA packaging component can comprise a central polypurine tract (cPPT)/central termination sequence, HIV Psi, or a combination thereof. The retroviral vector may optionally include an antibiotic resistance gene and/or a detectable marker.

In another aspect, provided herein is a method for genetically modifying or transducing a lymphocyte (e.g., T cell and/or NK cell) or a population thereof of an individual, the method comprising contacting the lymphocyte (e.g., T cell and/or NK cell) or population thereof of the individual in vitro with a replication-deficient recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in the lymphocyte (e.g., T cell and/or NK cell), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR), the chimeric antigen receptor comprises an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contacting facilitates genetic modification and/or transduction of a lymphocyte (e.g., T cell and/or NK cell) or at least some of a lymphocyte (e.g., T cell and/or NK cell) with a replication-defective recombinant retroviral particle, thereby generating a genetically modified and/or transduced lymphocyte (e.g., T cell and/or NK cell).

In some embodiments of the methods provided immediately above, the genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or populations thereof are introduced into an individual. In some embodiments, the genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or populations thereof undergo 4 or fewer cell divisions ex vivo prior to being introduced or reintroduced into an individual. In some embodiments, the lymphocyte is a resting T cell and/or resting NK cell that is contacted with the replication-defective recombinant retroviral particle for between 1 hour and 12 hours. In some embodiments, the time between the time blood is collected from the individual and the time the genetically modified T cells and/or NK cells are reintroduced into the individual is no more than 8 hours. In some embodiments, all steps after collection of blood and before reintroduction of blood are performed in a closed system that is monitored by a person throughout the treatment.

In another aspect, provided herein is a genetically modified T cell and/or NK cell comprising:

a. one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets; and

b. a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the one or more (e.g., two or more) inhibitory RNA molecules and the CAR are encoded by a nucleic acid sequence that is genetically modified by a T cell and/or NK cell.

In some embodiments of the genetically modified T cell and/or NK cell population, the genetically modified T cell and/or NK cell also comprises at least one lymphoproliferative component that is not an inhibitory RNA molecule, wherein the lymphoproliferative component is encoded by a nucleic acid that is genetically modified by the T cell and/or NK cell. In some embodiments, the inhibitory RNA molecule, CAR, and/or at least one lymphoproliferative component are expressed as polycistronic material. In illustrative embodiments, the inhibitory RNA molecule is expressed from a single polycistronic transcript.

In another aspect, provided herein is a replication-defective recombinant retroviral particle for use in a method for genetically modifying lymphocytes of an individual for treating tumor growth, wherein the replication-defective recombinant retroviral particle comprises in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and a second nucleic acid sequence encodes a lymphoproliferative component, wherein the method comprises contacting T cells and/or NK cells of the individual ex vivo, and the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells. In some embodiments, the CAR is a MRB-CAR. In any of the aspects provided herein that include MRB-CARs, the MRB-CAR can have reduced binding to its cognate antigen at one pH value than at a different pH value. In some embodiments, the MRB-CAR can have reduced binding to its cognate antigen at a pH above 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 than at a pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In other embodiments, the MRB-CAR can have reduced binding at higher pH than at lower pH. For example, the MRB-CAR can have reduced binding to its cognate antigen at a pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 than at a pH above 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In other embodiments, the MRB-CAR exhibits increased binding in the pH of the tumor compared to the pH of the blood. In some embodiments, the agent is used in a method further comprising introducing genetically engineered T cells and/or NK cells into the subject. In some embodiments, the pharmaceutical agent can be sodium bicarbonate, tris-hydroxymethylaminomethane, an isotonic solution of sodium bicarbonate and sodium carbonate, or a proton pump inhibitor such as esomeprazole (esomeprazole), esomeprazole and naproxen (naproxen), lansoprazole (lansoprazole), omeprazole (omeprazole), and rabeprazole (rabeprazole).

In another aspect, provided herein is a replication-defective recombinant retroviral particle for use in a method for genetically modifying T cells and/or NK cells of an individual for treating tumor growth, wherein the method comprises:

a) contacting T cells and/or NK cells of an individual ex vivo with replication deficient recombinant retroviral particles comprising a polynucleotide in their genome, the polynucleotide comprises one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR), the chimeric antigen receptor comprises an Antigen Specific Targeting Region (ASTR), a transmembrane domain and an intracellular activation domain, wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells; and

b) introducing the genetically modified T cells and/or NK cells into the individual, thereby genetically modifying the T cells and/or NK cells of the individual.

In the aspects immediately provided above, in some embodiments, the population of T cells and/or NK cells is contacted in the contacting step and introduced into the individual in the introducing step.

In another aspect, provided herein is the use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying T cells and/or NK cells of an individual, wherein the use of the kit comprises:

A. contacting T cells and/or NK cells of an individual ex vivo with replication deficient recombinant retroviral particles comprising a polynucleotide in their genome, the polynucleotide comprises one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR), the chimeric antigen receptor comprises an Antigen Specific Targeting Region (ASTR), a transmembrane domain and an intracellular activation domain, wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells; and

B. introducing the genetically modified T cells and/or NK cells into the individual, thereby genetically modifying the T cells and/or NK cells of the individual.

In another aspect, provided herein is the use of a replication-defective recombinant retroviral particle in the manufacture of a medicament for genetically modifying T cells and/or NK cells of an individual, wherein the use of the medicament comprises:

A) contacting T cells and/or NK cells of an individual ex vivo with replication deficient recombinant retroviral particles comprising a polynucleotide in their genome, the polynucleotide comprises one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR), the chimeric antigen receptor comprises an Antigen Specific Targeting Region (ASTR), a transmembrane domain and an intracellular activation domain, wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby producing genetically modified T cells and/or NK cells; and

B) introducing the genetically modified T cells and/or NK cells into the individual, thereby genetically modifying the T cells and/or NK cells of the individual.

In another aspect, provided herein is a commercial container comprising a replication-defective recombinant retroviral particle comprising in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and instructions for its use to treat tumor growth in an individual.

In some embodiments, in a aspect of the commercial container, the instructions direct the user to contact T cells and/or NK cells of the individual ex vivo to facilitate transduction of at least one resting T cell and/or NK cell of the individual with the replication-defective recombinant retroviral particle, thereby generating the genetically modified T cells and/or NK cells.

In any of the aspects provided herein that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, the polynucleotide may further comprise a third nucleic acid sequence encoding at least one lymphoproliferative component that is not an inhibitory RNA molecule.

In any of the aspects provided herein that include polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, the inhibitory RNA molecules may in some embodiments comprise a5 'strand and a 3' strand that are partially or fully complementary to each other, wherein the 5 'strand and the 3' strand are capable of forming an 18 to 25 nucleotide RNA duplex. In some embodiments, the 5 'strand length can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and the 3' strand length can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the 5 'and 3' strand lengths may be the same or different. In some embodiments, the RNA duplex may include one or more mismatches. In alternative embodiments, the RNA duplex has no mismatches.

In any of the aspects provided herein that include polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, which can be a miRNA or shRNA. In some embodiments, the inhibitory molecule may be a precursor of a miRNA, such as a Pri-miRNA or pre-miRNA, or a precursor of a shRNA. In some embodiments, the inhibitory molecule may be an artificially-derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule can be dsRNA processed into siRNA (transcribed or artificially introduced) or siRNA itself. In some embodiments, the inhibitory RNA molecule can be a miRNA or shRNA having a sequence not found in nature, or having at least one functional segment not found in nature, or having a combination of functional segments not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules is miR-155.

In any of the aspects provided herein that include polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, in some embodiments, the inhibitory RNA molecules can comprise a sequence from a5 'to a 3' orientation: a5 ' arm, a5 ' stem, a loop, a3 ' stem that is partially or fully complementary to the 5 ' stem, and a3 ' arm. In some embodiments, at least one of the two or more inhibitory RNA molecules has this arrangement. In other embodiments, all of the two or more inhibitory RNA molecules have this arrangement. In some embodiments, the 5' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop length can be 3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the 5 'arm, the 3' arm, or both are derived from a naturally occurring miRNA. In some embodiments, the 5 'arm, the 3' arm, or both are derived from a naturally occurring miRNA selected from the group consisting of: miR-155, miR-30, miR-17-92, miR-122 and miR-21. In illustrative embodiments, the 5 'arm, the 3' arm, or both are derived from miR-155. In some embodiments, the 5 'arm, the 3' arm, or both are derived from mouse (Musmusculus) miR-155 or Homo sapiens (Homo sapiens) miR-155. In some embodiments, the 5' arm has a sequence set forth in SEQ ID No. 256 or is a functional variant thereof, such as a sequence that is the same in length as SEQ ID No. 256, or 95%, 90%, 85%, 80%, 75% or 50% or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less or 25 nucleotides or less of the length of SEQ ID No. 256; and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 256. In some embodiments, the 3' arm has a sequence set forth in SEQ ID No. 260 or is a functional variant thereof, such as a sequence that is the same in length as SEQ ID No. 260, or 95%, 90%, 85%, 80%, 75% or 50% or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less or 25 nucleotides or less of the length of SEQ ID No. 260; and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO 260. In some embodiments, the 3' arm comprises nucleotides 221 to 283 of the mus musculus BIC.

In any of the aspects provided herein that include polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode two or more inhibitory RNA molecules against one or more RNA targets, and in some embodiments, the two or more inhibitory RNA molecules can be positioned consecutively in the first nucleic acid sequence. In some embodiments, inhibitory RNA molecules can be directly or indirectly contiguous with each other using a non-functional linker sequence. In some embodiments, the connector sequence may be between 5 and 120 nucleotides in length, or between 10 and 40 nucleotides in length.

In any of the aspects provided herein that include polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode two or more inhibitory RNA molecules against one or more RNA targets, in some embodiments, the first nucleic acid sequence encodes two to four inhibitory RNA molecules. In illustrative embodiments, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 5, or between 3 and 6 inhibitory RNA molecules are included in the first nucleic acid sequence. In an illustrative embodiment, four inhibitory RNA molecules are included in the first nucleic acid sequence.

In any of the aspects provided herein, including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, which one or more (e.g., two or more) inhibitory RNA molecules can be in an intron.

In any of the aspects provided herein that include polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets, in some embodiments the two or more inhibitory RNA molecules may be directed against different targets in an alternative embodiment, the two or more inhibitory RNA molecules are directed against the same target in some embodiments, the RNA targets are mRNA transcribed from genes expressed by T cells, such as, but not limited to, PD-1 (to prevent inactivation), CT L a4 (to prevent inactivation), TCRa (to prevent autoimmunity), TCRb (to prevent autoimmunity), CD3Z (to prevent autoimmunity), SOCS1 (to prevent inactivation), SMAD2 (to prevent inactivation), miR-155 targets (to promote activation), IFN γ (to decrease), CRS 2 (to prolong signal transduction), TRAI L (to prevent PP2 (to death), celiac 2 (to prolong signal transduction), mRNA 4835 (to increase the level of mRNA from cells expressing endogenous TCR receptor mRNA from cells, in some embodiments, the mRNA molecules encoding one or more RNA molecules that inhibit endogenous TCR receptor transcription of RNA receptors, TCR receptor mRNA from T cells, TCR receptor mRNA molecules, in some embodiments, the RNA target cells, RNA targeting RNA molecules encoding one or RNA targeting RNA molecules, which may be expressed by T cells.

In any of the aspects provided herein that include a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, in some embodiments, the CAR is a microenvironment-restricted organism (MRB) -CAR. In other embodiments, the ASTR of the CAR binds to a tumor-associated antigen. In other embodiments, the ASTR of the CAR is a microenvironment-restricted biological (MRB) -ASTR.

In any of the aspects provided herein, including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of the one or more nucleic acid sequences can encode one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a Chimeric Antigen Receptor (CAR), the chimeric antigen receptor comprises an Antigen Specific Targeting Region (ASTR), a transmembrane domain and an intracellular activation domain, and in some cases, the third nucleic acid sequence encoding one or more nucleic acid sequences of at least one lymphoproliferative component is not an inhibitory RNA molecule, in some embodiments, any or all of the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are 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 any of the aspects provided herein that include replication-defective recombinant retroviral particles, in some embodiments, the replication-defective recombinant retroviral particles comprise on their surface a pseudotyping component that is capable of binding to T cells and/or NK cells and facilitates membrane fusion with the replication-defective recombinant retroviral particles. In some embodiments, the pseudotyped component can be a measles virus F polypeptide, a measles virus H polypeptide, a VSV-G polypeptide, or any fragment thereof that retains the ability to bind to resting T cells and/or resting NK cells. In the illustrative embodiment, the pseudotyped component is VSV-G.

In any of the aspects provided herein that include replication-defective recombinant retroviral particles, in some embodiments, the replication-defective recombinant retroviral particle comprises on its surface an activation module comprising a membrane-bound polypeptide capable of binding to CD3, and/or a membrane-bound polypeptide capable of binding to CD 28. In some embodiments, the membrane-bound polypeptide capable of binding CD3 is fused to a heterologous GPI-anchor linker sequence and/or the membrane-bound polypeptide capable of binding CD28 is fused to a heterologous GPI-anchor linker sequence. In some embodiments, the membrane-binding polypeptide capable of binding to CD3 is anti-CD 3scFv or anti-CD 3 scfvffc. In an illustrative embodiment, the membrane-bound polypeptide capable of binding to CD3 is anti-CD 3 scfvffc. In illustrative embodiments, the membrane-bound polypeptide capable of binding to CD28 is CD80, or its extracellular domain that binds to the CD16B GPI-anchor linkage sequence.

In any of the aspects provided herein that include a replication-defective recombinant retroviral particle, in some embodiments, the replication-defective recombinant retroviral particle comprises on its surface a nucleic acid encoding a domain recognized by a biologically-validated single strain antibody.

In aspects provided below that include chimeric polypeptides that promote cell proliferation of PBMCs (e.g., B cells, T cells, and/or NK cells), the chimeric polypeptides may be referred to herein as chimeric lymphoproliferative components (C L E) embodiments provided below that include nucleic acid sequences encoding C L E and CARs are referred to herein as C L E CAR polynucleotide embodiments.

In embodiments and sub-embodiments provided herein that include any of the aspects and embodiments of a lymphoproliferative component, including those in the above paragraphs of this section, the lymphoproliferative component can be any of the chimeric lymphoproliferative components provided herein, including in the context of this section other embodiments and sub-embodiments of any of the aspects and embodiments of herein that include a CAR, the lymphoproliferative component in illustrative embodiments is any of the chimeric lymphoproliferative components encoded by the nucleic acids in the C L ECAR polynucleotide (and related polypeptide) embodiments provided herein, such as set forth below in this section.

Unless incompatible with, or stated in a state, any state or other embodiment provided herein that includes a lymphoproliferative component (L E) or a nucleic acid encoding a lymphoproliferative component may state that, in certain illustrative embodiments, the lymphoproliferative component satisfies, is suitable for satisfying, possesses the following properties, possesses the following ability or possesses the properties of any of the identified tests for identifying L E provided herein, or is capable of providing, is suitable for, possesses the following properties and/or is modified to achieve the results of any one or more of the following tests by a cell genetically modified and/or transduced with a retroviral particle (e.g., a lentiviral particle) having a genome encoding L E:

a) improving amplification of preactivated PBMCs transduced with retroviral particles comprising a nucleic acid encoding a lymphoproliferative component between days 7 and 21, 28, 35 and/or 42 of in vitro culture in the absence of exogenously added interleukins when transduced with a nucleic acid encoding an anti-CD 19 CAR comprising a CD3 ζ intracellular activation domain but no co-stimulatory domain compared to a control construct under identical conditions; and/or

b) Preactivated PBMCs transduced with retroviral particles comprising a nucleic acid encoding a lymphoproliferative component are amplified at least 2-fold, 3-fold, 4-fold, 6-fold, 7-fold, or 8-fold, or between 3-fold and 25-fold, 5-fold and 20-fold, 5-fold and 15-fold, 7-fold and 15-fold, or 7-fold and 12-fold, between day 7 and 21, day 28, day 35, and/or day 42 of in vitro culture, in the absence of exogenously added interleukins, when transduced with a nucleic acid encoding an anti-CD 19 CAR comprising a CD3 ζ intracellular activation domain but no co-stimulatory domain.

In some embodiments, a statistical test is used to show improved amplification according to element a) in the preceding paragraph the statistical test can use a cut-off value for a p-value, e.g., equal to or less than 0.10, 0.05, or 0.01.

In one aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) an extracellular domain and a transmembrane domain from an interleukin receptor or a hormone receptor, wherein at least one of the extracellular domain and the transmembrane domain comprises

i. Mutations found in constitutively active mutants of the interleukin receptor, and in which the extracellular sequence does not bind to a ligand of the interleukin receptor, or

The extracellular and transmembrane domains from L MP1, and

b) a first endodomain of a gene selected from the group consisting of genes having a first endodomain of selected polypeptides identified in tables 8 to 11, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells.

In one example of an aspect in the immediately preceding paragraph, the first nucleic acid sequence further encodes a second endodomain, wherein the second endodomain may be upstream (i.e., 5') of the first endodomain, or in an illustrative example the second endodomain is downstream of the first endodomain. In a sub-embodiment of this embodiment, the first and second combination of endodomains comprise the first and second combination of endodomains of the selected polypeptide. Thus, for clarity, in a non-limiting exemplary embodiment, the selected polypeptide is M008-S212-S075. In this embodiment, the first endodomain of the chimeric polypeptide comprises or is TNFRSF8 transcript variant 1(NM _001243_4) (S212) and the second polypeptide comprises or is FCGR2C (NM _201563_5) (S075).

Thus, for the sake of clarity, in one embodiment, the extracellular and transmembrane domains of the chimeric polypeptide comprise or are Myc L MP1(NC 007605_1) (M008), the first intracellular domain of the chimeric polypeptide comprises or is TNFRSF8 transcript 1(NM _001243_4) (S212), and the second intracellular domain of the chimeric polypeptide comprises or is FCGR2 gr2C (NM 201563_5) (S075). in a related embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains of the selected polypeptide, the first and second intracellular domains, in addition to the extracellular and transmembrane domains and the transmembrane domains, or comprises in a related embodiment, a second extracellular domain of the selected polypeptide, a transmembrane domain of a selected polypeptide, a first intracellular domain, and a second intracellular domain, in an embodiment, or a second extracellular domain of a selected polypeptide, or a transmembrane domain, or a second intracellular domain, in an embodiment, or a second intracellular domain comprising a transmembrane domain, a second intracellular domain, or a selectable polypeptide comprising a transmembrane domain, a second intracellular domain, a third intracellular domain, a fourth intracellular domain.

In another embodiment of this aspect, the polynucleotide further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first and second endodomain combinations are first and second endodomain combinations of the selected polypeptide, and the selected polypeptide is identified in tables 8-11. In another sub-embodiment of this embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in tables 8-11. In a related sub-embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of a selected polypeptide, optionally comprising a recognition or elimination domain other than the extracellular domain and the transmembrane domain, and wherein the recognition or elimination domain is a recognition or elimination domain of the extracellular domain of the selected polypeptide, or other recognition or elimination domain, and wherein the selected polypeptide is recognized in tables 8-11.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) an extracellular domain and a transmembrane domain from an interleukin receptor or hormone, wherein at least one of the extracellular domain and the transmembrane domain comprises

i. Mutations found in constitutively active mutants of the interleukin receptor, and in which the extracellular sequence does not bind to a ligand of the interleukin receptor, or

The extracellular and transmembrane domains from L MP1, and

b) a first endodomain selected from the group consisting of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, I L2 RA, I L3 RA, I L13 RA2, TNFRSF8, TNFRSF9, I L31 RA, or MyD88, wherein said chimeric polypeptide promotes cell proliferation of PBMCs, in illustrative embodiments B cells, T cells, and/or NK cells.

In one example of an aspect in the immediately preceding paragraph, the first nucleic acid sequence further encodes a second endodomain, wherein the second endodomain may be upstream (i.e., 5') of the first endodomain, or in an illustrative example the second endodomain is downstream of the first endodomain. In a sub-embodiment of this embodiment, the second endodomain is from a gene identified in any one of the tables of example 11 as producing an enrichment of greater than 0, 1, or 2 on day 35 versus day 7 of the selected polypeptide, wherein the first endodomain is from a gene of a first endodomain of the selected polypeptide. In this sub-embodiment or another sub-embodiment of this embodiment, the first endodomain and the second endodomain are combined to be a first endodomain and a second endodomain from a selected polypeptide identified in any one of the tables of example 11 as producing an enrichment at day 35 versus day 7 of greater than 0, 1, or 2. In another embodiment of the embodiments or sub-embodiments in this paragraph, the first endodomain is from a first gene of a selected first endodomain of a selected polypeptide in tables 8-11 and the second endodomain is from a second gene of a selected second endodomain of the selected polypeptide. In another embodiment of the embodiments or sub-embodiments in this paragraph, the first endodomain is from a selected polypeptide in tables 8-11 and the second endodomain is from a selected polypeptide in tables 8-11.

In another sub-embodiment of the immediately preceding embodiments, wherein the first nucleic acid sequence further encodes a second endodomain, the second endodomain is selected from the group consisting of those identified in any one of the tables in example 11 as producing an enrichment of greater than 0, 1, or 2 on day 35 versus day 7 for the corresponding first endodomain, in a sub-embodiment of these embodiments, the first endodomain is selected from the group consisting of endodomains of CD, or CD79, in other sub-embodiments of this embodiment, the first endodomains are selected from those identified in any one of the tables in example 11 as producing an enrichment of greater than 0, 1, or 2 on day 35 versus day 7 for CD3, CD8, CD79, IFNAR, I2 RA, I3 RA, I13 RA, TNFRSF, I31 RA, or MyD, in another embodiment of this paragraph, or sub-embodiment, the first endodomains from the tables 8, table 8, and the selected endodomains are derived from the chimeric endodomains of the first endodomains, a chimeric endodomains from the selected endodomains of this embodiment, a chimeric endodomains comprising a transmembrane recognition polypeptide from the first endodomains of the second endodomains, a chimeric endodomains from the table 8, a chimeric endodomains of example 11, a chimeric endodomains of this embodiment, a chimeric endodomains from the first endodomains of the second endodomains of example 11, a chimeric endodomains comprising a transmembrane recognition polypeptide, a transmembrane recognition polypeptide from the first endodomain, a transmembrane domain, a transmembrane recognition polypeptide, a transmembrane domain, a transmembrane recognition polypeptide, a transmembrane domain, a transmembrane recognition polypeptide, a transmembrane domain.

In another embodiment of this aspect, the first endodomain is selected from the endodomains of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, I L2 RA, I L3 RA, I L13 RA2, TNFRSF8, or TNFRSF9, the polynucleotide further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain, in a subset of this aspect, the first endodomains are from the gene of the first endodomains of the selected polypeptide and the second endodomains are from the gene of the second endodomains of the selected polypeptide, and the selected polypeptide is recognized in tables 8-11, in a subset of this aspect, the first endodomains and the second endodomains are combined as the first endodomains and the second endodomains of the selected polypeptide, and the selected endodomains are recognized in a subset of this aspect, and the first endodomains and the selected endodomains are either chimeric endodomains recognized in this embodiment, or eliminated from the selected polypeptide, and the selected endodomains are included in this aspect, and the chimeric endodomains recognized in one or one of the selected polypeptide, and wherein the selected endodomains, a chimeric endodomains, a polypeptide, a transmembrane domain, or a transmembrane domain, and a transmembrane domain, or a transmembrane domain, and a transmembrane domain, or a polypeptide, and a polypeptide, or a.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

(a) an extracellular domain and a transmembrane domain from an interleukin receptor or hormone, wherein at least one of the extracellular domain and the transmembrane domain comprises

i. Mutations found in constitutively active mutants of the interleukin receptor, and in which the extracellular sequence does not bind to a ligand of the interleukin receptor, or

The extracellular and transmembrane domains from L MP1, and

(b) a first endodomain selected from the group consisting of endodomains of CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B, CR L F2, FCGR2C, ICOS, I L2 RA, I L13 RA1, I L13 RA2, I L15 RA, TNFRSF9, and TNFRSF18, wherein said chimeric polypeptide promotes PBMC, and in illustrative embodiments cell proliferation of B cells, T cells, and/or NK cells.

In example 11, the endodomain described in the immediately preceding aspect was identified as the noteworthy second endodomain. However, it will be appreciated that in some embodiments, the endodomain identified as the noteworthy second endodomain is the first endodomain of the chimeric polypeptide of these embodiments.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) an extracellular domain and a transmembrane domain from an interleukin receptor or hormone, wherein at least one of the extracellular domain and the transmembrane domain comprises i.a mutation found on a constitutively active mutant of an interleukin receptor, and wherein the extracellular sequence does not bind a ligand of an interleukin receptor, or

The extracellular and transmembrane domains from L MP1, and

b) a first intracellular domain selected from the group consisting of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, I L2 RA, I L3 RA, I L13 RA2, TNFRSF8, TNFRSF9, I L31 RA, and MyD88,

wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments B cells, T cells, and/or NK cells, and wherein the extracellular domain and transmembrane domain are selected from the extracellular domain and transmembrane domain of I L7 RA Ins PPC L, L MP1, CR L F2F 232C, CSF2RB V449E, CSF3R T640N, EPOR L251C I252C, GHR E260C I270C, I L27 RA F523C, and MP L S505N.

In certain embodiments of any of the above aspects directed to polynucleotides encoding chimeric polypeptides, wherein the chimeric polypeptides promote cell proliferation of PBMCs, and in illustrative embodiments B cells, T cells, and/or NK cells, the ectodomain and transmembrane domain is selected from CSF3R T640N and MP L s505n in illustrative embodiments the ectodomain and transmembrane domain is from CSF3RT640N and in certain embodiments the isolated polynucleotide does not comprise a car in certain embodiments the ectodomain and transmembrane domain is CSF3R T640N provided in table 7, but the recognition domain and elimination domain are optional in certain embodiments the first ectodomain and the second ectodomain are from any of the polypeptides provided in examples 11 and tables herein comprising the ectodomain and the first ectodomain in other embodiments the ectodomain and ectodomain are from MP 7S 35505 and the elimination domain is other than MP L and optional N provided in table 7.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first endodomain of a gene selected from the group consisting of genes having a first endodomain of selected polypeptides identified in tables 12-17, wherein said chimeric polypeptide promotes cell proliferation and/or survival of B cells, T cells and/or NK cells.

As shown in additional example 12, such chimeric polypeptides are capable of promoting cell survival and/or proliferation of PBMCs in vivo or in vitro or ex vivo for 6 days, 7 days, 14 days, 21 days or 35 days, 42 days or more without exposure of PBMCs, T cells, B cells or NK cells to interleukins (such as I L-15, I L-7, and in illustrative embodiments, I L-2) and optionally in the absence of the target of ASTR of CAR expressed by the cells during culture.

In one embodiment of the aspect in the immediately preceding paragraph and in any embodiment of this aspect in the following text section, the first endodomain is an endodomain of a gene having a first endodomain in tables 12-17, and in other illustrative embodiments, one of the first 50, 25, or 10 constructs is listed in two, three, four, or five of the six tables 12-17, or all of the tables. In one embodiment, the first endodomain is an endodomain identified in table 18. Table 18 identifies constructs that promote proliferation of PBMCs between day 7 and day 21 or day 35, wherein the second endodomain is not present in the construct. In some of these embodiments, the chimeric polypeptide comprises a combination of a transmembrane domain and an intracellular domain, with or without an extracellular domain comprising a dimerization motif of table 18.

In any of the aspects or embodiments immediately following the aspects in the paragraphs above and in this aspect in the following section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments the extracellular domain comprises a dimerization motif, in any of the aspects or embodiments wherein the extracellular domain of C L E comprises a dimerization motif, the dimerization motif may be selected from the group consisting of polypeptides comprising leucine zipper motifs, CD69, CD71, CD72, CD96, CD105, CD161, CD162, 249, CD271, and CD324, and mutants and/or active fragments thereof that retain dimerization capacity, in any of the aspects or embodiments wherein the extracellular domain of C L E comprises a dimerization motif, the dimerization motif may require a dimerization motif, and the dimerization motif and associated dimerization motif may be selected from the group consisting of FKBP and rapamycin or analogs thereof, gbb and coumaromycin or analogs thereof, DHFR and pterin or analogs thereof, or dmr b and AP 87 or analogs thereof, and the mutant and/or active fragments of dimeric proteins that retain dimerization capacity.

In illustrative embodiments of any aspect or embodiment wherein the extracellular domain of C L E comprises a dimerization motif, the extracellular domain may comprise a leucine zipper motif in some embodiments, the leucine zipper motif is from a jun polypeptide, e.g., C-jun.

In one embodiment of this aspect, the first nucleic acid sequence further encodes a second endodomain, wherein the second endodomain may be upstream (i.e., 5') of the first endodomain, or in illustrative embodiments the second endodomain is downstream of the first endodomain. In a sub-embodiment of this embodiment, the first endodomain and second endodomain combination comprises the first endodomain of the selected polypeptide and any endodomain of any gene identified in the second endodomain of any candidate polypeptide in tables 12-17, and in illustrative embodiments in tables 12-17, and in other embodiments, one of the first 50, 25, or 10 constructs is listed in tables 12-17, and in other illustrative embodiments, one of the first 50, 25, or 10 constructs is listed in two, three, four, or five, or all of the tables 12-17. In a sub-embodiment of this embodiment, the combination of the first endodomain and the second endodomain comprises the first endodomain and the second endodomain of the selected polypeptide.

In one embodiment of this aspect, the chimeric polypeptide comprises a transmembrane domain, a first endodomain and a second endodomain of the selected polypeptide. In one embodiment of this aspect, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain and the second intracellular domain of the selected polypeptide, wherein the extracellular domain optionally comprises a recognition domain or an elimination domain.

In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain and the second intracellular domain of the selected polypeptide, with the exception that the extracellular and transmembrane domains optionally comprise a recognition domain or an elimination domain or do not comprise a recognition domain or an elimination domain. In a related sub-embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of the selected polypeptide, except that the extracellular domain and the transmembrane domain comprise a recognition domain or an elimination domain, or another recognition domain or elimination domain, of the selected polypeptide.

In another embodiment of this aspect, the polynucleotide further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first endodomain and second endodomain combination is a first endodomain and second endodomain combination of the selected polypeptide, and the selected polypeptide is identified in tables 12-17, and in other embodiments, one of the first 50, 25, or 10 constructs is listed in two, three, four, or five, or all of the tables 12-17. In another embodiment of this embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of a selected polypeptide, and the selected polypeptide is identified in tables 12-17, or in illustrative embodiments, one of the first 50, 25 or 10 constructs is listed in tables 12-17, in other embodiments, one of the first 50, 25 or 10 constructs is listed in two, three, four or five, or all of the tables 12-17. In a related sub-embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of the selected polypeptide, optionally comprising a recognition domain or an elimination domain other than the extracellular domain and the transmembrane domain, and wherein the recognition domain or the elimination domain is the recognition domain or the elimination domain, or another recognition domain or elimination domain, of the extracellular domain of the selected polypeptide, and wherein the selected polypeptide is identified in tables 12-17.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the intracellular domains of L EPR, MYD88, IFNAR2, MP L, I L18R 1, I L13 RA2, I L10 RB, I L23R, or CSF2RA,

wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells.

In one embodiment of the aspect in the immediately preceding paragraph and in any embodiment of this aspect in the following text section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In illustrative embodiments of this aspect, the ectodomain 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.

In another embodiment of this aspect, wherein the first endodomain is selected from the group consisting of L EPR, MYD88, IFNAR2, MP L, I L18R 1, I L13 RA2, I L10 RB, I L23R or CSF2RA, the first nucleic acid sequence further encodes a second endodomain, wherein the second endodomain may be upstream (i.e., 5') of the first endodomain, or in an illustrative embodiment, the second endodomain is downstream of the first endodomain in a sub-embodiment of this embodiment, the second endodomain is from a gene recognized in any one of the tables of example 12 as producing a second endodomain in the enriched selected polypeptide above 0, 1 or 2 at day 21 versus day 7, wherein the first endodomain is from a gene of the first endodomain of the selected polypeptide in this sub-embodiment or in another sub-embodiment, the first endodomain is from a gene of a first endodomain of the selected polypeptide, and the second endodomain is from a chimeric polypeptide of this selected polynucleotide of this example 12, or from a chimeric polypeptide of this embodiment, and the second endodomain is from a chimeric polypeptide of this selected polynucleotide encoding a chimeric polypeptide of this first endodomain in this example 12, or a chimeric polypeptide comprising a chimeric gene of this selected polynucleotide sequence encoding a chimeric polypeptide in this selected endodomain in this second endodomain in this example 12 or in this sub-embodiment, or in this chimeric polypeptide of this chimeric polypeptide, and a chimeric polypeptide of this example 2.

In one embodiment of this aspect or one sub-embodiment or other sub-embodiments of the immediately preceding paragraph comprising a second endodomain, the first endodomain is an endodomain of L EPR, MYD88, IFNAR2 and MP L in another embodiment, the first endodomain is an endodomain of L EPR, IFNAR2 and MP L in another embodiment, the first endodomain is not MYD88 in another embodiment, the first endodomain is not MYD88 and the second endodomain is not cd40 in a sub-embodiment of this embodiment in this paragraph, the polynucleotide further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain and an intracellular activation domain.

In one embodiment of this aspect of the invention, MP L is a first endodomain and a second endodomain is from a gene of a second endodomain in a selected polypeptide identified in tables 12-17. in one other sub-embodiment of this embodiment, the first endodomain and second endodomain in combination comprise a first endodomain and a second endodomain from a selected chimeric polypeptide identified in tables 12-17. in some sub-embodiments of these embodiments, the orientation of the first endodomain and the second endodomain is reversed such that the carboxy-terminal residue of the second domain binds to the first domain or to a spacer between the second domain and the first domain.

In one embodiment of this aspect of the invention L EPR is a first endodomain and a second endodomain is from a gene of a second endodomain in a selected polypeptide identified in tables 12 to 17 in other sub-embodiments of this embodiment the first and second endodomains in combination comprise first and second endodomains from a selected chimeric polypeptide identified in tables 12 to 17 in some sub-embodiments of these embodiments the orientation of the first and second endodomains is reversed such that the carboxy-terminal residue of the second domain binds to the first domain or to a spacer between the second domain and the first domain.

In one embodiment of this aspect of the invention, IFNAR2 is a first endodomain and the second endodomain is from a gene of a second endodomain in a selected polypeptide identified in tables 12 through 17. In other sub-embodiments of this embodiment, the first and second endodomain combinations comprise first and second endodomains from a selected chimeric polypeptide identified in tables 12-17. In some sub-embodiments of these embodiments, the orientation of the first and second intracellular domains is reversed such that the carboxy-terminal residue of the second domain binds to the first domain or a spacer between the second domain and the first domain.

In one embodiment of this aspect of the invention, MyD88 is a first endodomain and the second endodomain is from a gene of a second endodomain in a selected polypeptide identified in tables 12-17. In another sub-embodiment of this embodiment, the first endodomain and second endodomain combination comprise a first endodomain and a second endodomain from a selected chimeric polypeptide identified in tables 12-17. In some sub-embodiments of these embodiments, the orientation of the first intracellular domain and the second intracellular domain are reversed such that the carboxy-terminal residue of the second domain binds to the first domain or a spacer between the second domain and the first domain.

In some embodiments of the isolated polynucleotide aspects above in which the first endodomain is selected from the group consisting of the endodomains of L EPR, MYD88, IFNAR2, I L RD, I L017 RE, I L11 RAP, I L R, and MP L, the transmembrane domain is selected from the group consisting of the transmembrane domains of CD40, CD8B, CR L F2, CSF2RA, GCGR 2RA, ICOS, IFNAR RA, IFNGR RA, I RA RB, I RA R RA, I RA RAP, I RA RA, and PR RA R.

In some embodiments of the isolated polynucleotide aspect above wherein the first intracellular domain is selected from the group consisting of L EPR, MYD88, IFNAR2, I L RD, I L RE, I L RAP, I L R and the intracellular domains of MP L, the second intracellular domain is from CD40, CD79B or cd27. in some sub-embodiments, the first intracellular domain is from L EPR, MYD88, IFNAR2 and MP L. for example, in some embodiments, the first intracellular domain is MP L and the second intracellular domain is cd40. in all embodiments in this paragraph, the order of the first and second intracellular domains on the polypeptide may be reversed.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first endodomain of a gene selected from the group consisting of genes having a first endodomain of selected polypeptides identified in tables 12 to 17, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells

Wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells. In some embodiments, the NK cells are NKT cells.

In any of the aspects or embodiments in which the extracellular domain of C L E comprises a dimerization motif, the dimerization motif may be selected from the group consisting of polypeptides containing leucine zipper motifs, CD69, CD71, CD72, CD96, CD105, CD161, CD162, 249, CD271, and CD324, and mutants and/or active fragments thereof that retain dimerization capacity, in any of the aspects or embodiments in which the extracellular domain of C L E comprises a dimerization motif, the dimerization motif may require a dimerization agent, and the dimerization motif and related dimerization agent may be selected from the group consisting of dhbp and rapamycin or analogs thereof, GyrB and coumaromycin or analogs thereof, DHFR and pterin or analogs thereof, or DmrB and AP20187 or analogs thereof, and dimeric mutants and/or active fragments of the dimeric protein that retain dimerization capacity.

In illustrative embodiments of any aspect or embodiment herein in which the extracellular domain of C L E comprises a dimerization motif, the extracellular domain may comprise an leucine zipper motif in some embodiments, the leucine zipper motif is from a jun polypeptide, e.g., C-jun.

In one embodiment of this aspect, the first nucleic acid sequence further encodes a second endodomain, wherein the second endodomain may be upstream (i.e., 5') of the first endodomain, or in illustrative embodiments the second endodomain is downstream of the first endodomain. In a sub-embodiment of this embodiment, the first endodomain and second endodomain combination comprises a first endodomain of the selected polypeptide and any endodomain of any gene identified in a second endodomain of any candidate polypeptide in tables 12-17. In a sub-embodiment of this embodiment, the combination of the first endodomain and the second endodomain comprises the first endodomain and the second endodomain of the selected polypeptide.

In one embodiment of this aspect, the chimeric polypeptide comprises a transmembrane domain, a first endodomain and a second endodomain of the selected polypeptide. In one embodiment of this aspect, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain and the second intracellular domain of the selected polypeptide, wherein the extracellular domain optionally comprises a recognition domain or an elimination domain.

In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain and the second intracellular domain of the selected polypeptide, except that the extracellular and transmembrane domains optionally comprise a recognition domain or an elimination domain, or no recognition domain or elimination domain. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain and the second intracellular domain of the selected polypeptide, except that the extracellular and transmembrane domains optionally comprise the recognition domain or elimination domain, or another recognition domain or elimination domain, of the selected polypeptide.

In another embodiment of this aspect, the polynucleotide further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first and second endodomain combinations are first and second endodomain combinations of a selected polypeptide, and the selected polypeptide is identified in tables 12-17. In another embodiment of this embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of a selected polypeptide, and the selected polypeptide is identified in tables 12-17. In a related sub-embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain and a second intracellular domain of the selected polypeptide, optionally comprising a recognition domain or an elimination domain other than the extracellular domain and the transmembrane domain, and wherein the recognition domain or the elimination domain is the recognition domain or the elimination domain, or another recognition domain or elimination domain, of the extracellular domain of the selected polypeptide, and wherein the selected polypeptide is identified in tables 12-17.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

a first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising in amino to carboxy orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the group consisting of L EPR, MYD88, IFNAR2, I L17 RD, I L17 RE, I L1 RAP, I L23R, and MP L,

wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells.

In one embodiment of the aspect in the immediately preceding paragraph and in any embodiment of this aspect in the following text section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In illustrative embodiments of this aspect, the ectodomain 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.

In another embodiment of this aspect, wherein the first endodomain is selected from the group consisting of L EPR, MYD88, IFNAR2, I L17 RD, I L17 RE, I L1 RAP, I L23R and MP L endodomains, the first nucleic acid sequence further encodes a second endodomain, wherein the second endodomain may be upstream (i.e. 5') of the first endodomain, or in an illustrative embodiment, the second endodomain is downstream of the first endodomain in a first sub-embodiment of this embodiment, the second endodomain is from a gene that is recognized in any of the tables of example 12 as producing a second endodomain in the selected polypeptide enriched above 0, 1 or 2 at day 21 versus day 7in this sub-embodiment, wherein the first endodomain is from the first endodomain of the selected polypeptide in this sub-embodiment or another sub-embodiment, the first endodomain is from a first endodomain of the selected polypeptide in this sub-embodiment or embodiment, and the second endodomain is from a chimeric polypeptide of this selected endodomain in this sub-embodiment or embodiment, the first endodomain is from a chimeric endodomain in which the first endodomain is derived from the first endodomain of the selected polypeptide in this sub-embodiment, and the chimeric endodomain is derived from the selected endodomain in this chimeric polypeptide of this sub-embodiment, and the chimeric polypeptide comprises a chimeric endodomain from the first endodomain of the selected endodomain in the first endodomain in the chimeric polypeptide of example 7 of example 2 of the selected polypeptide of this sub-embodiment, and the chimeric endodomain of the chimeric polypeptide, and the chimeric polypeptide of example 7 of the chimeric polypeptide of this sub-embodiment, the chimeric polypeptide of the invention.

In certain embodiments of any of the methods herein that include a chimeric polypeptide or isolated polynucleotide embodiments, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells, the first and/or second endodomains (in illustrative embodiments the first endodomains) are endodomains from L EPR, MYD88, IFNAR2, MP L, I L18R 1, I L13 RA2, I L10 RB, I L23R, or CSF2RA in some embodiments, the first endodomains are from MP L in illustrative sub-embodiments, the chimeric polypeptide comprises an ectodomain comprising a dimerization motif as provided herein.

In certain embodiments of any of the methods herein that include a chimeric polypeptide or isolated polynucleotide embodiments, the transmembrane domain is from CD40, ICOS, FCGR2C, PR L R, I L RA, or I L ST, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells.

In certain embodiments of any of the methods herein that include a chimeric polypeptide or isolated polynucleotide embodiments, the first and/or second endodomain (in illustrative embodiments, the second endodomain) is an endodomain from CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells. In some illustrative embodiments, the second endodomain is from CD40, CD79B, or CD 27. In exemplary sub-embodiments, the chimeric polypeptide comprises an extracellular domain comprising a dimerization motif as provided herein.

In certain embodiments of any of the methods herein that include a chimeric polypeptide or isolated polynucleotide embodiments, the first and/or second endodomain is an endodomain (P3 or P4 component) of any one of the chimeric polypeptides in tables 12-17, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells. In some embodiments, the chimeric polypeptide comprises any P2, P3, and P4 combination of any one of the chimeric polypeptides in tables 12-17.

In an illustrative embodiment of any of these embodiments in this paragraph, the first intracellular domain is selected from the group consisting of L EPR, MYD88, IFNAR2, I L17 RD, I L17 RE, I L1 RAP, I L23R, and MP L.

In embodiments of any of the aspects and embodiments directed to polynucleotides comprising a nucleic acid sequence encoding a chimeric polypeptide, the chimeric polypeptide comprises an extracellular domain that may comprise a1, 2, 3, or 4 amino acid spacer within 20 amino acids of the carboxy-terminus of the extracellular domain (and in illustrative embodiments, at the carboxy-terminus of the extracellular domain) such that the spacer separates the extracellular domain and the transmembrane domain. In some embodiments, the 1,2, 3, or 4 amino acid spacers comprise alanine residues, and in some cases, only alanine residues. In an illustrative sub-embodiment of the embodiment comprising the spacer, the extracellular domain comprises a dimerization motif, such as a leucine zipper motif.

It will be appreciated that aspects and embodiments of the chimeric polypeptides provided herein comprising a cell proliferation-promoting component of PBMCs, B cells, T cells and/or NK cells may include mutants and/or fragments of the gene, provided that the chimeric polypeptide retains its ability to promote cell proliferation of PBMCs, B cells, T cells and/or NK cells. These mutants and/or fragments typically retain signaling activity such that the chimeric polypeptide retains its survival-promoting and proliferative activities. It is understood that the first endodomain and the second endodomain may be reversed so as to be used in any aspect and embodiment provided herein comprising a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells. Thus, in these embodiments, it is understood that the first endodomain is the second endodomain and vice versa.

In any of the aspects and embodiments provided herein comprising a polynucleotide, the polynucleotide comprises a nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, which nucleic acid sequence may further encode a recognition domain or an ablation domain. In some embodiments, the recognition domain or the elimination domain is a recognition domain of a biologically approved monoclonal antibody. In some embodiments, the recognition domain or the abrogation domain comprises a polypeptide, or epitope thereof, recognized by an antibody that recognizes EGFR. In some embodiments, the recognition domain or the elimination domain comprises a polypeptide, or epitope thereof, recognized by an antibody that recognizes MYC.

In any of the aspects and embodiments provided herein comprising a polynucleotide, the polynucleotide comprises a nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, the nucleic acid sequence and/or the second nucleic acid sequence being operably linked to a first promoter active in B cells, T cells, and/or NK cells. In some illustrative embodiments, the first promoter is active in T cells and/or NK cells.

In embodiments of any of the isolated polynucleotide aspects and embodiments provided herein having a nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, for polynucleotide embodiments comprising a second endodomain, a linker may be present between the transmembrane domain and the first endodomain and/or between the first endodomain and the second endodomain. In some embodiments, for embodiments comprising an extracellular domain, a linker between 1 and 4 alanine is present between the extracellular domain and the transmembrane domain.

In embodiments of any of the isolated polynucleotide or vector aspects and embodiments provided herein comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells and a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR), the chimeric antigen receptor comprises an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, the first and second nucleic acid sequences may be separated by a ribosome skip sequence. In some embodiments, the ribosome skipping sequence is F2A, E2A, P2A, or T2A.

In certain embodiments, the polynucleotide further comprises a Kozak-type sequence, a WPRE component, and one or more of a double or triple stop codon that defines a stop read from at least one of the one or more transcription units, in certain embodiments, the polynucleotide further comprises a Kozak-type sequence selected from the group consisting of ccacct/ug (g) (SEQ ID NO:515), CCGCCAT/ug (g) (SEQ ID NO:516), GCCGCCGCCAT/ug (g) (SEQ ID NO:517), and GCCGCCGCCAT/UG. in certain embodiments, the nucleotides-3 and +4 relative to the start codon of the first nucleic acid sequence both comprise g in certain embodiments, in another embodiment, the polynucleotide comprises one or more of the aforementioned polynucleotide in combination with the aforementioned embodiment comprising a Kozak-type sequence and/or the following embodiment comprising a triple stop codon, in certain embodiments, the WPRE 3 and/or the polynucleotide comprises one or more of the aforementioned stop codon in combination with the WPRE 3 '5, in embodiments, wherein the WPRE and WPRE' are located in certain embodiments.

In one aspect, provided herein are nucleic acid vectors comprising

Embodiments of any of the isolated polynucleotide aspects and embodiments provided herein that encode a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells,

a. a copy source;

b. an antibiotic resistance gene; and/or

c. One or more viral and/or retroviral sequences selected from the group consisting of a retroviral long terminal repeat sequence (L TR) or an active fragment thereof, a retroviral cis-acting RNA packaging component, and/or a retroviral central polypurine tract/Central Termination Sequence (CTS) component.

In some embodiments, the isolated polynucleotide is in reverse orientation with respect to a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component, a 5 'long-acting repeat sequence, and/or a 3' long-acting repeat sequence.

In some aspects, provided herein are replication-defective recombinant retroviral particles comprising a retroviral genome comprising any of the aspects and embodiments herein comprising an isolated polynucleotide comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, and further comprising an envelope and a capsid. The retroviral particle may additionally comprise any of the retroviral components described in this disclosure. In one embodiment, the retroviral genome further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence may be separated by a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence is F2A, E2A, P2A, or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are typically on the same transcriptional unit.

In another embodiment, provided herein is a mammalian cell, wherein the mammalian cell (e.g., a human cell) is a B cell, T cell, or NK cell comprising an isolated polynucleotide, which, according to any of the aspects and embodiments herein comprising an isolated polynucleotide, optionally can be integrated into the genome of the cell, the isolated polynucleotide comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells. In one embodiment, the isolated polynucleotide in the mammalian cell further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence may be separated by a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence is F2A, E2A, P2A, or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are typically on the same transcriptional unit. In some embodiments, the cell is a T cell or NK cell, and in certain illustrative embodiments, the cell is a human T cell.

In another aspect, provided herein is a packaging component comprising an isolated polynucleotide, which can be integrated into the genome of a cell, comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, according to any of the aspects and embodiments herein comprising the isolated polynucleotide. In some embodiments, the isolated polynucleotide in the cell further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an Antigen Specific Targeting Region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence may be separated by a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence is F2A, E2A, P2A, or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are typically on the same transcriptional unit. In some embodiments, the cell is a cell provided herein for use in the production of a retroviral particle.

In one aspect, provided herein is a method of promoting cell proliferation of PBMCs (e.g., B cells, T cells, and/or NK cells), the method comprising transducing and/or genetically modifying cells with a vector encoding a lymphoproliferative component (and in illustrative embodiments, a chimeric polypeptide according to any of the embodiments provided herein), wherein the chimeric polypeptide promotes PBMC proliferation.

In one aspect, provided herein is a method of transfecting, transducing, and/or genetically modifying a B cell, T cell, and/or NK cell (which in some non-limiting embodiments may be an allogeneic cell, an autologous cell, or a non-allogeneic cell), the method comprising contacting the cell with an isolated polynucleotide according to any one of the aspects and embodiments herein comprising an isolated polynucleotide comprising a first nucleic acid sequence (C L E aspects and embodiments) encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, in some embodiments of this method the cell is a T cell and the isolated polynucleotide further comprises a second nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular skipping domain, in other words, a method for transfecting, transducing, and/or genetically modifying a B cell, T cell, and/or NK cell, which method may be performed according to any one of the embodiments, the first polynucleotide sequence is contacted with a T cell, the isolated polynucleotide, and/or the isolated polynucleotide is a ribosomal polynucleotide according to any one of the embodiments herein, the first polynucleotide sequence is a polynucleotide sequence specified in some embodiments as a T A, in which embodiments, the method for transfecting a cell, and/or a cell is a ribosome-transfected, and/or a cell.

In some embodiments of any one of the aspects herein, the NK cell is an NKT cell.

The following non-limiting examples are provided merely by way of illustrating exemplary embodiments and in no way limit the scope and spirit of the present invention. Moreover, it is to be understood that any invention disclosed or claimed herein encompasses all variations, combinations, and permutations of any one or more features described herein. Any one or more features may be explicitly excluded from the claims even if a specific exclusion is not explicitly recited herein. It should also be understood that the disclosure of an agent for use in a method is intended to be synonymous with (and provide support for) a method involving the use of the agent, in accordance with the particular methods disclosed herein or other methods known in the art, unless one of ordinary skill in the art would understand. Additionally, unless the ordinarily skilled artisan will understand that the specification and/or claims disclose a method for which any one or more of the agents disclosed herein may be used.

Examples of the invention

Example 1 retroviral packaging and transduction systems were engineered to target resting T cells for selective T cell integration and expression by PBMCs.

Although it is possible to generate high titer lentiviral vectors using transient transfection, this approach carries the risk of generating replication-defective recombinant retroviruses (RCR) and is not amplifiable for clinical use. Here, stable retroviral packaging cell lines were generated by the simultaneous introduction of multiple constructs encoding inducible promoters and their regulators into HEK293 suspension-adapted cells (HEK293S) to stably produce viral components, CAR genes and their regulatory components. Two different inducible systems can be used to temporarily control gene expression. One system is rapamycin-or rapamycin analogue-induced dimerization based on two transcription factors. One transcription factor consists of three copies of the FKPB protein fused to the ZFHD1 DNA binding domain and the other transcription factor consists of the FRB protein fused to the p65 activation domain. Rapamycin or rapamycin analogs dimerize transcription factors to form ZFHD1/p65AD and can activate gene transcription at the 12xZFHD1 binding site.

The series of vectors as shown in figures 3A-3E are generated by flanking transposon sequences for integration into the HEK293S genome once integrated into the genome of the cell, these sequences act as regulatory components and lox sites and/or FRT sites (herein referred to as landing sites) for subsequent integration using Cre and/or flp recombinase once the initial 5 constructs contain an extracellular MYC marker-encoding polynucleotide sequence encoding puromycin resistance, GFP, RFP and targeting the cell membrane via N-terminal P L ss (bovine prolactin signal peptide) and anchoring to the cell membrane via an N-terminal P L C-terminal transmembrane anchor domain derived from platelet growth factor receptor (PDGFR) C-terminal mymyc anchor domain the initial 5 constructs can also include a constitutive minimal promoter and minimal I L-2 promoter, a rapamycin regulated promoter based on hd1, a tetracycline responsive component (TRE) promoter or a bidirectional TRE (frp) promoter, the promoter in figure 3A promoter containing coding for fusion with the coding sequence encoding GFP 2 promoter under the hfp promoter and the hfp promoter encoding GFP 9-encoding site, and optionally a promoter region under the expression of the hfp gene promoter map 3A GFP 9-expressing ppt gene promoter, the hfp promoter, and a promoter region under the expression site of the hfp gene expression cassette promoter, including a GFP 3C promoter map 7-2 promoter, a GFP gene expression site under the expression site of the hfp gene expression site under the pfp gene expression site of the hfp gene expression vector expressing GFP gene expression site.

To generate packaging cell lines with landing sites integrated into the genome, HEK293S cells were co-transfected with 5 plasmids at equimolar concentrations (fig. 3A-3E) plus 5 μ g of ex vivo transcribed piggybac transposase mRNA or 5 μ g of a plasmid with a promoter for expressing piggybac transposase in the presence of PEI at a PEI to DNA (w/w) ratio of 2:1 or 3:1 or 2 μ g to 5 μ g piggybac transposase protein using a cationic peptide mixture, transfected cells were selected for 2 to 5 days with — puromycin in the presence of 100nm rapamycin and 1 μ g/m L doxycycline, followed by fluorescence activated cell sorting to collect cells expressing GFP and RFP.

HEK293S cells with constructs from FIGS. 3A-3E integrated into the genome were then transfected with constructs containing a tricistronic polynucleotide encoding the DAF signal sequence/anti-CD 3scFvFc (UCHT1)/CD14 GPI anchor linkage site (SEQ ID NO:287), the DAF signal sequence/CD 80 ectodomain and the DAF signal sequence/I L-7/DAF (SEQ ID NO:286) capable of binding to the CD28/CD16B GPI anchor linkage site (SEQ ID NO:286), and a transposon sequence flanked by polynucleotide regions for integration into the HEK293S genome (FIG. 4). after transfection, the cells were amplified for 2 days in the absence of rapamycin and doxycycline and selection of pure lines that are red in composition, followed by transient transfection of positive pure lines with constructs expressing the positive transfection DNA and RFP coding region to remove the remaining pure sequences of the DNA and RFP coding region, while transfection of the construct containing the BipOL promoter and one of the constitutive red marker sequences was selected.the polynucleotide sequences are inserted into the genome for the resulting polynucleotide sequences for the integration of the GFP-encoding polypeptide and the resulting polynucleotide sequences are analyzed for the integration of the remaining protein sequences in the genome for the sequence map for the presence of the GFP expression of the resulting genomic DNA sequence.

Example 2. production of lentiviral vectors and retroviral packaging.

Transfection of the retroviral packaging stable cell line generated in example 1 with a construct for expression of Flp recombinase (fig. 4C) and a construct containing a polynucleotide sequence encoding CAR and lymphoproliferative component I L R α -insPPC α under the control of the CD3Z promoter (which is inactive in HEK293S cells), wherein CAR and I α R α -insPPC L are separated by a polynucleotide sequence encoding a T2A ribosome skip sequence, and I L R α -insPPC L has an acyclovir riboswitch controlled ribonuclease a CAR-containing construct further comprising cPPT/CTS and RRE sequences and a polynucleotide sequence encoding HIV-1 Psi. the successful integration of the CAR-containing construct is flanked by FRT sites on the entire polynucleotide sequence on the CAR-containing construct to be integrated into the genome-the successful integration of the CAR-containing construct results in the successful integration of the CD-containing construct being thus removed by transient transfection of the construct for expression of Flp recombinase in a vial containing CD 5-expressing CD 19C 3-expressing CD 19C dna-expressing a dna construct is followed by a long-expressing CD 19C 3g expressing CD 15C-expressing a CD 15C dna-expressing a C-expressing a dna-expressing a heavy chain constant.

Example 3. efficiency of transduction of fresh isolated and unstimulated human T cells by retroviral particles pseudotyped with VSV-G and expressing anti-CD 3 scfvffc on their surface.

Recombinant lentiviral particles were generated by transient transfection of 293T cells (L enti-X) with separate lentiviral packaging plasmids encoding gag/pol and rev, and a pseudotyped plasmid encoding VSV-G^TM293T, Clontech) was co-transfected with a packaging plasmid with a third generation lentiviral expression vector (containing a deletion in the 3' L TR resulting in self-inactivation) encoding GFP, an anti-CD 19 chimeric antigen receptor, and an eTAG herein designated F1-0-03 (fig. 5)^TM293 expression medium (ThermoFisher Scientific) to accommodate suspension culture. To be provided with1×10⁶Cells/ml (30m L) cells in suspension were seeded in a 125m L Erlenmeyer flask and immediately transfected with Polyethyleneimine (PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 1.5ml Gibco for 30m L cells^TMOpti-MEM^TMTo obtain lentiviral particles pseudotyped with VSV-G in a medium, the total DNA used (culture volume of 1. mu.g/m L) is a mixture of 4 plasmids having the following molar ratio 2 × genomic plasmid (F1-0-03), 1 × 0 plasmid containing Rev, 1 × plasmid containing VSV-G and 1 × plasmid containing gag/pol. to obtain lentiviral particles pseudotyped with VSV-G and expressing on their surface anti-CD 3-scFvFc-GPI, the total DNA used (culture volume of 1. mu.g/m 24 1) is a mixture of 5 plasmids having the following molar ratio 2 × genomic plasmid (F1-0-03), 19 plasmid containing Rev, 1 × plasmid containing VSV-G, 1 × plasmid containing anti-CD 53-scFvFc-GPI and 1 plasmid containing RescFvFc-861 plasmid containing 5 plasmids containing 1, × plasmid containing ×, × plasmid containing RescFvFc-GPI, × plasmid containing 5 plasmid containing CD 8672, × plasmid containing 5 plasmid containing RescFvFc-Fc-GPI, × plasmid containing 5 plasmid containing DNA and expressing the above the molar ratio^TMOpti-MEM^TMMedium dilution PEI to 2 μ g/m L (culture volume, 2:1 ratio to DNA.) after a 5 minute room temperature incubation, the two solutions were mixed together and incubated at room temperature for 20 minutes, the final volume (3ml) was added to the cells followed by a 125rpm ramp with 8% CO₂The cells were incubated at 37 ℃ for 72 hours. The anti-CD 3-scFvFc-GPI-containing plasmid comprises scFv derived from OKT3 or UCHT1 and GPI-anchor linker sequences. The UCHT1scFvFc-GPI vector encodes a peptide (SEQ ID NO:278) comprising a human Ig kappa signal peptide (amino acid 1 to amino acid 22 of NCBI GI CAA 45494.1), fused to UCHT1scFv (amino acid 21 to amino acid 264 of NCBI GI CAH 69219.1), fused to human IgG1Fc with an A to T at position 115 (amino acid 1 to amino acid 231 of NCBI GI AEV 43323.1), human IgG1Fc fused to human DAF GPI-anchor linker(amino acid 345 to amino acid 381 of NCBI GI NP-000565). The OKT3scFvFc-GPI vector encodes a peptide (SEQ ID NO:279) comprising the human Ig kappa signal peptide (amino acid 1 to amino acid 22 of NCBI GI CAA 45494.1) fused to OKT3scFv (SEQ ID NO:285), the OKT3scFv fused to human IgG1Fc (amino acid 1 to amino acid 231 of NCBI GI AEV 43323.1), the human IgG1Fc fused to human DAF GPI anchor linker (amino acid 345 to amino acid 381 of NCBI GI NP-000565). The CD80-GPI vector encodes a peptide (SEQ ID NO:280) comprising a human CD80 signal peptide and the ectodomain (amino acid 1 to amino acid 242 of NCBI GI NP-005182) fused to a human CD16b GPI-anchor linker sequence (amino acid 196 to amino acid 233 of NCBI GI NP-000561).

After 72 hours, the supernatant was collected and clarified by centrifugation at 1,200g for 10 minutes. The clarified supernatant was poured into a new tube. Lentiviral particles were precipitated by centrifugation at 3,300g overnight at 4 ℃. The supernatant was discarded and the lentiviral particle pellets were resuspended at an initial volume of 1: 100X-Vivo ^TM15 medium (L onza) lentivirus particles were titrated using serial dilutions and flow cytometry analysis of GFP expression in 293T and Jurkat cells 72 hours after transduction.

Peripheral Blood Mononuclear Cells (PBMCs) were first isolated from fresh Blood in ACD (acid citrate) tubes from donor 12F and donor 12M or from the buffy coat layer of donor 13F, collected and distributed using San Diego Blood Bank, CA. SepMate-based execution according to manufacturer's instructions^TM50(Stemcell^TM) Ficoll-Paque

(GEHealthcare L ife Sciences) gradient density separation of PBMCs on SepMate according to each^TMTubes the 30m L blood or buffy coat diluted in PBS-2% HIFCS (heat inactivated fetal calf serum) was stratified, after centrifugation at 1,200g for 20 minutes at room temperature, the PBMC layers were collected, pooled and washed three times with 45m L PBS-2% HIFCS, and centrifuged at 400g for 10 minutes at room temperature, then the pellets were incubated in 10m L RBC lysis buffer (Alfa Aesar) for 10 minutes at room temperature, and incubated with 45m L RBC lysis buffer (Alfa Aesar)PBS-2% HIFCS washes were added twice more and centrifuged at 400g for 10min at room temperature. Final washes were performed in the following transduction and culture media: X-Vivo of donor 13F ^TM15 or donor 12F and donor 12M RPMI-1640+ 10% HIFCS, No additional steps were taken to remove monocytes after isolation, fresh and unstimulated PBMCs were resuspended in respective media to 1 × 10⁶Final concentration of/m L, and transduction of the PBMCs with previously revealed lentiviral particles (two or three replicates)₂X-Vivo at donor 13F ^TM15 Medium or 14 hours conduction in donor 12F and donor 12M RPMI-1640+ 10% HIFCS was typically performed in a1 ml/well 12-well disc format at MOI 1. for kinetic experiments, 0.5 × 10⁶PBMC/m L was in 7m L final solution at MOI 1, with 125rpm and 8% CO₂Was transduced by incubation in 125m L shake flasks at 37 ℃ for 2 to 20 hours after incubation with lentivirus for the selected time, with donor 13F X-Vivo ^TM15 Medium or donor 12F and donor 12M PBS + 2% HIFCS washes three times and finally 5% CO at 37 ℃₂X-Vivo at donor 13F ^TM15 Medium or donor 12F and donor 12M in RPMI-1640+ 10% HIFCS at 1 × 10⁶At cell density of/m L, beginning on day 3 post-transduction, and from there every 2-3 days, the media particle size was doubled and supplemented with I L-2 to a final concentration of 100U/m L samples were collected on different days post-transduction (day 3 to day 17) to assess the transduction efficiency of each type of lentivirus produced using GFP expression levels.

On different days after transduction, lentiviral particles pseudotyped with VSV-G with or without OKT3 antibody at 1. mu.g/ml (Biolegend), lentiviral particles pseudotyped with VSV-G and visualized on their surface anti-CD 3scFvFc-GPI, or lentiviral particles pseudotyped with VSV-G and visualized on their surface anti-CD 3scFvFc-GPI and CD80-GPI, 100. mu. L cells were collected and analyzed for GFP expression in the CD3+ cell population using flow cytometry.

Fig. 6A and 6B show histograms for the percentage (%) of CD3+ GFP + cells and histograms for the absolute number of cells per well of the CD3+ GFP + population, respectively, in the total CD3+ population at day 3, day 6, day 9, day 13 and day 17 after 14 hours of transduction of fresh isolated and unstimulated PBMC from donor 12M with the indicated lentiviral particles. Each bar represents the mean +/-SD of the replicates. Figures 6A and 6B show that lentiviral particles were pseudotyped with VSV-G and anti-CD 3-scfvffc was visualized on the surface of the lentiviral particles to efficiently transduce freshly isolated and unstimulated PBMCs. anti-CD 3 scFv's derived from OKT3 or UCHT1 were effective in the form of scFvFc-GPI.

FIGS. 7A and 7B show histograms for the percentage (%) of CD3+ GFP + cells and histograms for the absolute number of cells per well of the CD3+ GFP + population on day 3 and 6, respectively, after 14 hours of transduction with the indicated lentiviral particles from donor 13F fresh isolated and unstimulated PBMC, Please that "A" is the result using VSV-G pseudotyped lentiviral particles (three replicates), "B" is the result using VSV-G pseudotyped lentiviral particles with the addition of OKT3 Ab (1 μ G/m L) to the transduction medium (two replicates), "C" is the result using VSV-G pseudotyped lentiviral particles that exhibit on their surface GPI anchored UCHT1scFvFc and GPI anchored lentiviral particles (three replicates), and "D" is the result using UCGPI anchored VSV 1scFvFc on their surface and GPI anchored lentiviral particles that are GPI anchored GPI 2 (three replicates) and "D" is the result using a replicate anti-VSV-Fc-G viral particles with the results when GPI anchored VSV-Fc rod on their surface (three replicates) and the results are also shown as a 14, two replicates, a 14, and a plot for anti-VSV-G transduction medium.

Fig. 8A and 8B show histograms for the percentage (%) of CD3+ GFP + cells and histograms for the absolute number of cells per well of the CD3+ GFP + population, respectively, in the total CD3+ population at day 3, day 6, and day 9 after transduction of fresh isolated and unstimulated PBMCs from donor 12M with the indicated lentiviral particles for the indicated time (2-20 hours) (i.e., contact of cells with lentiviral particles in the transduction reaction mixture). Transduction was performed in a tray or shaker flask as indicated. Each bar represents the mean +/-SD of two replicates of lentiviral particles pseudotyped with VSV-G ("[ VSV-G ]"); other experiments were not repeated. Fig. 8A and 8B show that fresh isolated and unstimulated PMBC can be efficiently transduced with lentiviral particles pseudotyped with VSV-G and exhibiting anti-CD 3 scfvffc and CD80 on their surface in as little as 2 hours.

Notably, subsequent experiments were performed using lentiviral particles generated by transduction of freshly isolated resting PBMC for 2.5 hours in the presence of soluble anti-CD 3 antibody (100ng/ml) using Rev, VSV-G, gag/pol and F1-0-03 alone (instead of the vector encoding anti-CD 3 or CD 80). Transduction was achieved using 3 different soluble anti-CD 3 antibody clones. Transduction efficiencies measured by CD3+ GFP + cells on day 6 for CD3 antibody clones HIT3A, OKT3 and UCHT1 were 4.31%, 3.27% and 0.52%, respectively. Background transduction efficiency in the absence of soluble antibody was 0.06%.

Example 4 transduction efficiency of freshly isolated and unstimulated human T cells by retroviral particles containing a VSV-G pseudotyping module and a membrane-bound activation module, and optionally a Vpu protein.

293T cells (L enti-X) were transiently transfected with separate lentiviral packaging plasmids encoding gag/pol and rev, and a pseudotyped plasmid encoding VSV-G^TM293T, Clontech) to produce recombinant lentiviral particles. A third generation lentiviral expression vector encoding GFP, an anti-CD 19 chimeric antigen receptor, and eTAG (FIG. 5), herein designated F1-0-03, was co-transfected with a packaging plasmid. Cells were passed through Freestyle^TM293 expression Medium (ThermoFisher Scientific) to accommodate suspension culture with continuous growth 1 × 10⁶Cells/ml (30m L) cells in suspension were seeded in a 125m L Erlenmeyer flask and immediately transfected with Polyethyleneimine (PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 1.5ml Gibco for 30m L cells^TMOpti-MEM^TMIn a medium. To obtain lentiviral particles pseudotyped with VSV-G [ VSV-G]The total DNA used (1. mu.g/m L culture volume) was a mixture of 4 plasmids with a molar ratio of 2 × genomic plasmid (F1-0-03), 1 × Rev-containing plasmid,1 × plasmid containing VSV-G and 1 × plasmid containing gag/pol to obtain lentiviral particles pseudotyped with VSV-G and exhibiting anti-CD 3-scFvFc-GPI on their surface [ VSV-G + UCHT1]]The total DNA used (1. mu.g/m L culture volume) was a mixture of 5 plasmids with a molar ratio of 2 × genomic plasmid (F1-0-03), 1 × Rev-containing plasmid, 1 × VSV-G-containing plasmid, 1 × anti-CD 3-scFvFc-GPI-containing plasmid and 1 × gag/pol-containing plasmid to obtain lentiviral particles pseudotyped with VSV-G, rendered anti-CD 3-scFvFc-GPI and containing the helper protein Vpu [ VSV-G + UCHT1+ VpuCH]The total DNA used (1. mu.g/m L culture volume) was a mixture of 6 plasmids with a molar ratio of 2 × genomic plasmid (F1-0-03), 1 × Rev containing plasmid, 1 × VSV-G containing plasmid, 1 × anti-CD 3-scFvFc-GPI containing plasmid, 1 × Vpu-CH containing plasmid and 1 × gag/pol containing plasmid PEI alone^TMOpti-MEM^TMTo 2. mu.g/m L (culture volume, 2:1 ratio to DNA.) after a 5 minute incubation at room temperature, the two solutions were mixed together and incubated at room temperature for 20 minutes, the final volume (3ml) was added to the cells, followed by a concomitant 125rpm with 8% CO₂UCHT1scFvFc-GPI vectors encode peptides (SEQ ID NO:278) including scFv derived from UCHT1 and GPI-anchor linker sequence derived from DAF (CD55) fused to UCHT1scFv (amino acids 21 to QRT 264 of NCBI GI No. CAH69219.1), human IgG1Fc (amino acids 1 to 231 of NCBI GINo. AEV43323.1), amino acids 345 to QRD 865 of NCBI GI No. CAH69219.1), Vpu-CH containing plasmids encoding peptides including Vpu-CH 583639-CC isolates (amino acids 1-84, Gene: AGF30946.1) derived from HIV-1M 167-CC isolates (amino acids 1 to 84, MVA 5836L IKRIRERAEDSGNESDGEIEE L D of Vpu sequence of AGF MFDFIARVDYRVGVVA L (DMDD 53) of NCBI NO: AGF GPI).

After 72 hours, the supernatant was collected and clarified by centrifugation at 1,200g for 10 minutes. The clarified supernatant was poured into a new tube. Lentiviral particles were precipitated by centrifugation at 3,300g overnight at 4 ℃. The supernatant was discarded and the lentiviral particles were addedPellet resuspended in 1:100 initial volume of X-Vivo^TM15 medium (L onza.) lentivirus particles were titrated by serial dilution and analysis of GFP expression by flow cytometry in 293T and Jurkat cells 72 hours after transduction.

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coat, collected and distributed using San Diego Blood Bank, CA. SepMate-based execution according to manufacturer's instructions^TM50(Stemcell^TM) Ficoll-Paque

(GE Healthcare L ife Sciences) gradient density separation of PBMCs from SepMate^TMThe tube will be diluted in X-Vivo ^TM15 medium of 30m L buffy coat after centrifugation at 1,200g for 20 minutes at room temperature, the PBMC layers were collected, pooled and washed with 45m L X-Vivo ^TM15 media washes three times and centrifugation at 400g for 10min at room temperature then pellet incubated in 10m L RBC lysis buffer (Alfa Aesar) for 10min at room temperature and with 45m L X-Vivo ^TM15 media washes an additional two times and centrifuge at 400g for 10min at room temperature. No additional steps were taken to remove monocytes. After isolation, fresh and unstimulated PBMCs were resuspended in X-Vivo ^TM15 to 1 × 10⁶Final concentration of/m L, and transduction of the PBMCs with previously revealed lentiviral particles (two replicates)₂In the X-Vivo^TM15 in medium for 3 days. Transduction is generally in the form of a1 ml/well 12-well disc for [ VSV-g]At MOI 10 of and against [ VSV-G + UCHT1]And [ VSV-G + UCHT1+ Vpu-CH]At MOI 1 (two replicates). After 3 days of transduction, samples were collected and analyzed by FACS for absolute cell counts and GFP expression in CD3+ populations.

Fig. 9A and 9B show histograms of the percentage (%) of CD3+ GFP + cells in the total CD3+ population 3 days after transduction of fresh isolated and unstimulated PBMC with the indicated lentiviral particles (fig. 9A) and the absolute cell count per well of the live CD3+ population (fig. 9B), respectively. Each bar represents the mean +/-SD of two replicates. Figure 9A shows that the addition of an anti-CD 3-scfvffc-GPI moiety (UCHT1) to VSV-G-pseudotyped lentiviral particles resulted in higher transduction efficiency on freshly isolated and unstimulated PBMCs compared to [ VSV-G ] alone. FIG. 9A also shows that the addition of Vpu-CH can further improve the transduction efficiency of the double pseudotyped vector [ VSV-G + UCHT1 ]. Figure 9B shows that the portion of UCHT1 enhances cell stimulation at absolute cell counts per microliter compared to VSV-G alone. The addition of Vpu and UCHT1 similarly enhanced cell stimulation at absolute cell counts compared to VSV-G alone.

Example 5 transduction of unstimulated human PBMCs by 4 hour exposure to retroviral particles pseudotyped with VSV-G and expressing anti-CD 3scFvFc on their surface was inhibited by antiviral drugs.

In this example, unstimulated human PBMCs were efficiently transduced by 4 hours of brief exposure to retroviral particles pseudotyped with VSV-G and not revealing UCHT1 scfvffc-GPI on their surface alone or in combination with CD 80-GPI. The presence of the combination of the antiviral drugs dapiprinin (reverse transcriptase inhibitor) and dolutegravir (integrase inhibitor) was added to one sample to inhibit transduction.

In Freestyle^TM293T cells (L enti-X) adapted to suspension culture in 293 expression Medium (Thermo Fisher Scientific)^TM293T, Clontech). Cells were transiently transfected with PEI having a genomic plasmid and separate packaging plasmids encoding gag/pol, rev, and a pseudotyped plasmid encoding VSV-G as described in example 3. For some samples, the transfection reaction mixture also included a plasmid encoding UCHT1scFvFc-GPI alone or in combination with another plasmid encoding CD80-GPI as further described in example 3. The genomic plasmid is a third generation lentiviral expression vector encoding GFP, an anti-CD 19 chimeric antigen receptor, and eTAG (FIG. 5), referred to herein as F1-0-03. F1-0-03 did not include lymphoproliferative components.

Pbmc were prepared from buffy coat as described in example 3 without any additional step to remove monocytes after separation would contain 1 × 10⁶1ml of X-Vivo15 of unstimulated PBMCs were seeded into each well of a 96-well plate. The viral particles are added at an MOI of 1 andat 37 ℃ and 5% CO₂The plates were incubated for 4 hours. After 4 hours of transduction, cells were washed 3 times in DPBS + 2% HSA before resuspending in 1ml X-Vivo15 in each well and at 37 ℃ and 5% CO₂And (5) cultivating. For the antiviral drug treated samples, dapiprinine and dolutegravir were added to a final concentration of 10 μ M during transduction and subsequent incubation. Exogenous interleukins are not added to the sample at any time. Samples were collected on day 6 to determine transduction efficiency based on GFP expression as determined by FAC analysis using forward and side scatter based lymphocyte gates.

Fig. 10A and 10B show histograms for the percentage (%) of GFP + cells at day 6 after transduction of unstimulated PBMC (fig. 10A) and the absolute number of GFP + cells per well (fig. 10B). These histograms show that unstimulated human PBMCs can be transduced by 4 hours of exposure to VSV-G-pseudotyped lentiviral particles encoding F1-0-03 and that either UCHT1scFvFc-GPI alone or in combination with CD80-GPI was visualized. Furthermore, transduction efficiency of PBMCs by these lentiviral particles was significantly reduced by the presence of a combination of drugs that inhibited reverse transcription and integration, as demonstrated for VSV-G-pseudotyped lentiviral particles displaying UCHT1 scfvffc-GPI. This example shows that genetic modification and transgene expression of these PBMCs is not pseudo-transduced, but rather the result of transduction, after brief 4 hours of exposure to these retroviral particles, in which viral transgenic RNA is reverse transcribed, integrated into the genome of PBMCs and expressed.

Example 6. unstimulated PBMCs were incubated with retroviral particles pseudotyped with envelopes derived from VSV-G, BaEV or Mu L V and optionally expressing anti-CD 3 scfvffc on their surface for 4 hours of transduction efficiency.

In this example, lentiviral particles pseudotyped with various envelope proteins were incubated with unstimulated human PBMC for 4 hours and transduction efficiency was evaluated.

In Freestyle^TM293T cells (L enti-X) adapted to suspension culture in 293 expression Medium (Thermo Fisher Scientific)^TM293T, Clontech). Using plasmids having genome and encodingFor some samples, the transduction reaction mixture also included a plasmid encoding UCHT1scFvFc-GPI the genomic plasmid used for the samples in this example was F1-3-219 or F1-3-451 described in other examples herein the pseudotyped plasmid used for the samples in this example encoded envelope proteins from VSV-G (SEQ ID NO:548), BaEVwt (SEQ ID NO:535), Mu L V (SEQ ID NO:538) (sometimes referred to as M L V), a modification of the BaEV envelope in which fusion inhibitory R peptides were removed by truncation of the BaEVwt before methionine residue 546(SEQ ID NO:536) or valine residue 547(SEQ ID NO:537), or where anti-CD 3scFv from UCHT1 was fused to the amino terminal end of the Mu L V envelope U-L (MuID NO: 539).

Experiments using lentiviral particles encoding F1-3-219 and F1-3-451 were performed on different days, and PBMCs from different donors were used, but the same protocol was followed.on day 0 PBMCs were prepared from the buffy coat as described in example 3 without any additional step to remove monocytes. after separation, this would contain 1 × 10⁶1ml of X-Vivo15 of unstimulated PBMCs were seeded into each well of a 96-well plate. Unless otherwise indicated, viral particles were added at an MOI of 1 and at 37 ℃ and 5% CO₂The plates were incubated for 4 hours. After 4 hours of transduction, cells were washed 3 times in DPBS + 2% HSA before resuspending in 1ml X-Vivo15 in each well and at 37 ℃ and 5% CO₂And (5) cultivating. Exogenous interleukins are not added to the sample at any time. Samples were collected on day 3 or day 6 to determine transduction efficiency based on GFP expression as determined by FAC analysis using forward and side scatter based lymphocyte gates.

For the first transduction testing of VSV-G and baboon envelope proteins, the transduction efficiency of lentiviral particles encoding F1-3-219 was measured on day 6 fig. 11A shows F L AG +% and fig. 11B shows the number of F L AG + cells per microliter of lentivirus pseudotyped as indicated each of the lentiviral particles including those with VSV-G or baboon envelope proteins were able to promote transduction compared to untransduced cells, it is possible that the higher transduction rate by the VSV-G pseudotyped lentiviral particles in this example is the result of the chimeric lymphoid tissue proliferative component encoded by F1-3-219, relative to that in example 5, the viral particles pseudotyped with VSV-G and expressing UCHT1 scfvcfc-GPI and pseudotyped with BaEV Δ r (ha) showed the highest transduction efficiency in this experiment.

For testing the first transduction of VSV-G and baboon envelope proteins, the transduction efficiency of lentiviral particles encoding F1-3-451 was measured on day 6 FIG. 12A shows CD3+ F L AG +% and FIG. 12B shows the live CD3+ F L AG + cell% of lentiviruses pseudotyped as indicated each of the lentiviral particles including those with VSV-G or murine leukemia virus envelope proteins were able to promote transduction compared to untransduced cells.

Example 7. time course of retroviral transduction by exposure to unstimulated PBMC for 4 hours or less than 1 minute.

In this experiment, recombinant lentiviral particles were incubated with unstimulated PBMCs for a time between 4 hours and less than 1 minute, and examined for their ability to transduce PBMCs and promote their survival and/or proliferation in vitro in the absence of any exogenous interleukins.

Method of producing a composite material

In Freestyle^TM293T cells (L enti-X) adapted to suspension culture in 293 expression Medium (Thermo Fisher Scientific)^TM293T, Clontech.) recombinant lentiviral particles were produced.PEI transiently transfected cells with genomic plasmid and separate packaging plasmids encoding gag/pol, rev, as well as pseudotyped plasmid encoding VSV-G as described in example 3 for some samples, the transduction reaction mixture also included a plasmid encoding UCHT1scFvFc-GPI as further described in example 3. in this example 2 genomic plasmids were used.the first plasmid included a Kozak sequence, a CD8a signal peptide, a F L AG marker, and anti-CD 19: CD8: CD3z CAR followed by a triple termination sequence (F1-3-253). the second plasmid included a Kozak sequenceColumn, CD8a signal peptide, F L AG marker and anti-CD 19: CD8: CD3z CAR, T2A and C L E D L3A-4 (E013-T041-S186-S051), followed by a triple termination sequence (F1-3-451).

On day 0, Ficoll-Paque was used according to the manufacturer's instructions

(GE Healthcare L ife Sciences) and SepMate^TM-50(Stemcell^TMTechnologies) enriched PBMCs from buffy coat (San Diego Blood Bank) from 2 donors with density gradient concentration no additional steps were taken to remove monocytes PBMCs were diluted to 1ml per X-Vivo15 (L ONZA)1 × 10 after isolation⁶Not adding anti-CD 3, anti-CD 28, I L-2, I L-7, or other exogenous interleukins prior to transduction to activate or otherwise stimulate lymphocytes, lentiviral particles were added directly to unstimulated PBMCs at an MOI of 1, cells of the transduction mixture were washed 3 times with DPBS + 2% HSA at 37 ℃ and 5% CO₂The transducer was incubated for 4 hours, 2 hours, 30 minutes, 15 minutes, 7.5 minutes, 5 minutes, 2.5 minutes or not incubated at all. Next, the cells in each well were resuspended in 1ml of X-Vivo15 and at 37 ℃ and 5% CO₂And (5) cultivating. For samples treated with antiviral drugs, dapiprinin or dolutegravir was added to a final concentration of 10 μ M during transduction and at 37 ℃ and 5% CO₂The transduction reactions were incubated for 4 hours, after three washes, supplemented with drugs at the same concentrations in resuscitation medium, no exogenous interleukins were added to the samples at any time, the samples were collected on day 6, and transduction efficiency based on F L AG expression was determined by FACS analysis using forward and side scatter based lymphocytogates.

Results

In this example, an incubation period of less than 1 minute was found to promote transduction of unstimulated PBMCs as effectively as an incubation period of 4 hours fig. 13 shows that CD3+ F L AG + absolute cell counts (per microliter) at day 6 after the indicated period of unstimulated PBMCs transduced by different recombinant lentiviral particles from 1 donor, the ability of each of the recombinant lentiviral particles to transduce PBMCs was similar over all incubation periods, this was particularly evident for lentiviral particles expressing anti-CD 3 scfvcfc-GPI and had a higher transduction efficiency compared to their non-anti-CD 3 scfvcfc-GPI expressing counterparts for all incubation periods examined the total number of transduced PBMCs was more confirmed in those samples transduced by [ F1-3-GU ] 253 modified transduction samples indicated by D L E encoded in F3982-3-451 to promote survival and/or proliferation of these cells than in those transduced samples by [ F1-3-GU ] 253 modified by the indicated in F1-3-glu, and the results of the expression of retroviral genes demonstrated by the second transduced PBMC integration inhibitors (pseudorubicin) as observed in the transduced PBMC integration map, retroviral gene expression of the transduced PBMC.

Example 8 construction of the lymphoproliferative component of the I L-7 receptor.

A series of constitutively active I L receptor (I L R) transmembrane mutants from T-cell lymphoblastic leukemia (243InsPPC L (SEQ ID NO: 82); 246InsKCH (SEQ ID NO: 101); 241InsFSCGP (SEQ ID NO: 102); 244InsCH L (SEQ ID NO: 103); and 244InsPPVCSVT (SEQ ID NO: 104); all from Shochat et al, human 2011, J.Exp.Med. volume 208No.5901-908) were synthesized by overlapping oligonucleotide synthesis (DNA2.0, Newark, California) a series of constitutively active I L receptor transmembrane mutants (I L R) from Shocht et al, inserted immediately into constitutively expressing lentiviral vectors 243 following the 2A ribosome skip sequence via the synthetic constitutive active I L R transmembrane evolution, followed by anti-CD 3 zeta expression including CD 6858 handle (SEQ ID NO:79) and cassette peptide (SEQ ID NO:74) were transfected into the constitutively expressing lentiviral backbone 243 following the cDNA sequence of the cDNA sequence encoding construct, followed by CD 3. expressing CD 3. zeta expression vector, followed by transfection with the CD 293 DNA encoding CD III promoter DNA encoding CD III and amplification of the cDNA encoding cDNA for cell growth using the same vector, amplified by the same PCR with the expression vector using the expression vector, PCR amplification method for cell growth promoter DNA encoding CD III, PCR, and transfection of the expression vector, amplified expression vector using the expression vector, PCR, the expression vector, amplified with the expression vector, amplified plasmid DNA encoding CD III promoter DNA encoding cDNA encoding DNA sequence of the cDNA encoding cDNA, cDNA encoding cDNA.

Example 9 activity of I L-7 receptor lymphoproliferative/survival modules in PBMCs was tested.

To test the ability of the I L-7R α variant to mediate interleukin-independent survival of T cells, thirty milliliters of human blood was drawn into blood collection tubes with Acid Citrate (ACD) as an anticoagulant following the manufacturer's instructions through Ficoll-Pacque^TM(General Electric) whole blood was processed using density gradient concentrations to obtain Peripheral Blood Mononuclear Cells (PBMCs). Mixing aliquots of PBMC with X-Vivo ^TM15 Medium (L onza) was aseptically transferred together to the wells of 12-well tissue culture dishes to a final concentration in a final volume of 1m L containing 50 million viable cells/ml recombinant human interleukin-2 (I L-2) (Novoprotein) was also added to some samples at a concentration of 100IU/ml, activated anti-CD 3 Ab (OKT3, Novoprotein) was added at a concentration of 50ng/ml to activate PBMC for virus transduction, PBMC was incubated overnight in a standard humidified tissue culture incubator at 37 ℃ and 5% carbon dioxide after overnight incubation, a preparation of lentiviral particles containing the test construct to be tested (FIG. 14A) was added to individual wells at5 reinfection (MOI), the plates were incubated in a standard humidified tissue culture incubator at 37 ℃ and 5% carbon dioxide after overnight incubation, HSA was collected from each of the wells of 12-well dishes and was centrifuged to obtain HSA-serum samples which were resuspended in PBS + 2% overnight serum-PBS 15 (ViVOX) and washed once in HSA-serum pellets^TMIn culture medium, and transferred to

6-well gas permeable cell culture device (Wilson Wolf). Addition of additional X-Vivo^TM15 medium to bring the final volume of each well to 30 ml. . Transfer of matched control samples of each of the constructs to

6-pore gas permeable cell cultureWells of device (Wilson Wolf) and addition of additional media to bring final volume to 30ml with 100IU/ml I L-2 for some control samples, incubate at 37 ℃ and 5% carbon dioxide in a standard humidified tissue culture incubator

The device was used for 7 days.fresh I L-2 was added to the control sample containing I L-2 during every 2 to 3 days of incubation.matched test samples without I L-2 were not supplemented.samples were removed on day 7 to track cell number and viability during amplification (Countess, ThermoFisher).

FIG. 14A provides a schematic of the I L7R α constructs tested, these constructs inserted into the recombinant lentiviral particle genome replication-deficient recombinant retroviral particles were used to transduce PBMCs FIG. 14A shows a schematic of wild type I L7R α (SEQ ID NO:229) consisting of Signal Sequence (SS), extracellular domain (ECD), Transmembrane (TM) and intracellular domain (ICD). "1" indicates the site of the type III domain of the fiber web protein; "2" indicates the site of the WSXWS motif, "3" indicates the Box 1 site, "4" indicates the site of phosphorylation of Protein Kinase C (PKC), and "5" indicates the Box 2 site.

Variant "a" is I L-7R L with the instpc L at position 243 (shocha et al 2011, j. exp. med. volume 208No.5901-908) but without the S185C mutation, expressed on transcripts with GFP polypeptide, GSG linker and P2A ribosomal skip sequence fused to its N-terminus variant "B" is I L-7R α instpc L with GFP polypeptide, GSG linker and P2A ribosomal skip sequence fused to its N-terminus and Myc-tagged between signal sequence and extracellular domain, variant "C" is similar to variant "B" except its intracellular domain is truncated at position 292, variant "D" is similar to variant "a" except its intracellular domain is truncated at position 292, variant "E" is a variant with N-terminus truncated so that no signal sequence and most of the extracellular domain (residues 1 to 228) are absent, variant "a" is similar to the variant "a" B "is a variant" B "with N-terminal amino acid deletion of the T linker 3636, N-7R L, which is expressed on transcripts with GFP polypeptide, GSG linker, fused to its N-terminus, GSG linker, gsc-7R-7, 96, and N-7T-7T-7 variants, which have amino acid deletion sequences, numbered, respectively, based on the deletion sequence of the amino acid sequence of the cell.

As shown in figure 14B, PBMCs required I L-2 for survival in vitro as illustrated in figure 14B, at day 7 post-transduction, untransfected PBMCs had about 80% viability in the presence of I L-2 and 0% viability in the absence of I L-2. at day 7 post-transduction, PBMCs with the full-length version of I L-7R L0 InsPPC L (I636-7R L variants a and B in figure 14A) had more than 20% viability in the absence of I L-2, indicating that constitutive activity I8-7R L InsPPC L receptor expression had survival activity in these cells, hi addition, on day 7 post-transduction, the expression of I L-7R 359 receptor with truncated endodomain (ICD L-7R 84 survival) had survival activity in comparison to wild-type I L-7 receptor, when these cells had final activity as illustrated in figure 14B 4614B, the mutant variants of ICD 4614B 4614-7B had increased survival in figure 14B, and when these variants had final activity in figure 14B 4614A.

Example 10 analysis of intracellular signaling domains from lymphoproliferative components and whole chimeric constructs screened from duplicate chimeric pools.

This example confirms that endodomains from most candidate proliferation and/or survival genes reported to promote lympho-or myelo-proliferation and/or survival under certain conditions are effective in promoting proliferation and/or survival of PBMCs when cultured between day 7 and at least day 21 post-transduction in the absence of exogenously added interleukins (such as I L-2). in addition, at time points 21 days and beyond, endodomains and corresponding genes were identified that surprisingly promote especially noteworthy expansion.

Materials and methods

Data preparation

The data used for this analysis was a subset of constructs in a set of 8 replicate pools, each of the 4 pools prepared according to pool 3.1A ("PBMC fed" in) (3.1.1A to 3.1.4a) and pool 3.1B (PBMC not "fed") of example 12 herein (3.1.1B to 3.1.4B). Differential enrichment between day 7 and day 21 was analyzed for all 8 pools, and additional time points were analyzed as follows: pool 3.1.1 a-day 28 and day 35; pool 3.1.1 b-day 35; pool 3.1.4 a-day 28, day 35 and day 42; and pool 3.1.4 b-day 28. All constructs with one (only the first endodomain tested) or two endodomains (any second endodomain in the test endodomains plus pool) tested were analyzed at each time point compared to no endodomains (linker and terminator) for the same ectoand transmembrane domains.

Function/analysis

Differential enrichment of constructs containing a particular endodomain portion compared to constructs containing any other endodomain portion was subjected to the Mann-Whitney-Wilcoxon test, where differential enrichment was defined as the difference between the enrichment of constructs with endodomains and their corresponding portions with the same ectodomain and transmembrane domains and no endodomains (log 2 transition ratios between normalized counts at day 21 and normalized counts at day 7, each increased by a false count of 1). The P value can be adjusted for the false discovery rate (Benjamini-Hochberg) for each set of tests (when considering one set per library test) and the false discovery rate set to 0.1(Sober et al Sci Rep.2016, 12/8/v; 6: 38439; and Biostatics.2017, 4/1/v; 18(2): 275-.

Results

We sought to confirm that the intracellular signaling domains of various candidate genes reported to promote cell proliferation and/or survival of lymphoid or myeloid cells under certain conditions can provide effective intracellular signaling domains for lymphoproliferative components included in the compositions and methods provided herein, thus, chimeric lymphoproliferative components were constructed that combine a first intracellular signaling domain with various extracellular and transmembrane domains (and in most cases, a second intracellular domain) these chimeric lymphoproliferative component (ce) constructs including, from amino to carboxy terminus, a first extracellular domain (P), a transmembrane domain (P) and one or both of them, a first intracellular signaling domain (P) and a second intracellular signaling domain (P) as shown in fig. 15, CSF 1S 150, S119, S150, S1, S20, S18, S, and S18, S, and S, and S.

In the 3.1 library, the "second" intracellular domain (P4) is intended to be a regulatory domain, although several of these P4 intracellular domains are from genes that have also been reported to promote proliferation and/or survival of lymphoid or myeloid cells under certain conditions, and thus can provide effective proliferation and/or survival signals in the absence of the "first" intracellular signaling domain (P3) on lymphoid tissue proliferative components the second intracellular signaling domain (P4) from genes (with identifiers of the parts provided in parentheses and sequences of the parts provided in table 7) CD3D (S037), CD3E (S038), CD3G (S039), CD27(S047), mutant CD 42 CD28(S048), CD28(S049), CD40(S050 and S051), CD79A (S052), CD79B (S053), fc461 (S3545), fcks 464 (S464), CD28(S049), CD40 (S051), tnfs 6342) and tnfs 599, tnfs 18, tnfs 5923, tnfs 18, and tnfs 5923 are included in addition in the lymphoid tissue proliferative components including tnfs 5923, tnfs 599, tnfs 6324, tnfs 18, tnfs 599, and tnfs 599.

As discussed in example 12 for library 3.1, the ectodomain containing variants of c-Jun including leucine zipper motifs was used with eTAG and a linker containing 0, 1,2, 3 or 4 alanine (E006, E007, E008, E009 and E010), transmembrane domains or combinatorial handles and TM domains from the following genes (with the identifiers of the portions provided in parentheses and the sequences provided in Table 7) CD (T001), CD3 (T002), CD3 (T3), CD 004, CD 005 (T006), CD8 (T007), CD8 (T008), CD 009 (T009), CD 06010 (T011), CD79 (T012), CD79 (T013), CR F (T014 and T), CSF2 (T017 and T018), EPOR 063 (T0651) and 0651, RB 0651, CD 0651, RB 067T 034, RB 037T 0351, RB 034T 0351, FR 037T 034 (04), FR 037T 04), FR 034T 04, FR No. 7 (No. 7T, No. 03, No. 7T, No. 03, No. (No.), FR) and No. 7T), No. 7T, No.5, No. 7T, No. 3 (No. 04), No. 7T, No. 03, No. 3 (No. 7), No. 7, No. 4, No. 03, No. 3 (No. 03), No. 3 (No. 7), No. 3 (No. 03), No. 7), No. 3 (No. 03), No. 3 (No. 04), No. 3 (No. 4), No. 7), No. 4, No. 3 (No. 03), No. 3 (No. 7), No. 3 (No. 7), No. 3 (No. 7), No. 7, No. 03, No. 7, No. 103), No. 7, No. 3 (No. 7), No. 3 (No. 7), No. 3 (No. 03), No. 7), No. 3 (No..

In addition to the repeats of pool 3.1 discussed above and further analyzed in this example, a total of twenty pool screens have been performed, confirming that the various candidate endodomains can effectively promote T cell in vitro expansion in the absence of any exogenous T cell growth stimulating agent of the culture medium (e.g., exogenous I L-2.) under various conditions similar to those provided in examples 11 and 12 and using PBMCs from different donors, the constructs include one or two endodomains and a transmembrane domain as well as an extracellular domain as provided in table 7.

To confirm that many different intracellular domains that drive proliferation and/or survival of bone marrow or lymphocytes under certain conditions can serve as the first intracellular signaling domain in the lymphoproliferative component provided herein to facilitate amplification of transduced PBMCs in the absence of any exogenously added interleukins or exogenously added mitogens (e.g., anti-CD 3 or anti-CD 28) during culture, eight library screens were performed on intracellular P3 and P4 domains according to the methods provided for library 3.1 in example 12, with 4 duplicate libraries according to 3.1A ("fed" with PBMCs) and 4 duplicate libraries according to 3.1B (not "fed" with PBMCs). Cells were cultured for 21 days after transduction in the absence of exogenous interleukins or other T cell stimulators and the results analyzed. Amplification at day 21 and later time points was determined by measuring enrichment as the ratio between normalized counts (per million counts) at day 21 or later and normalized counts for day 7 for that construct. Cell expansion was analyzed at day 21, day 28, day 35 and day 42 for each available time point within the pool replicate compared to day 7. Constructs were ranked in terms of the level of amplification obtained at the test time point compared to day 7.

Table 25 provides constructs that ranked the top 100 constructs in terms of amplification at a time point relative to day 7in any of these eight replicate pool screens at least one time point. Quantification of cell expansion revealed that each extracellular domain (part P1) and transmembrane domain (part P2) as well as all second intracellular domains (part P4) were represented in at least one of the first 100 constructs in at least one of these eight pool screens. Of the 79 first intracellular signaling domains (portions P3) tested in the 49 genes of these screens, only 6 intracellular signaling domain portions were not present in the first 100 constructs in any of the eight duplicate bank screens, and different intracellular domain fragments from two of the genes were found in the first 100 constructs. Thus, intracellular signaling domains from only 3 tested genes are not represented in the first 100 constructs. Constructs without intracellular signaling domains (i.e., constructs in which P3 is the linker (X001) and P4 is the stop codon (X002)) were not found in the first 100 parts. In fact, none of the constructs without intracellular signaling domains were sequenced in the top 350 (top sequence 353 in bank 3.1.1 b) in any of the eight replicate bank screens, thereby verifying the effect of these lymphoproliferative component (i.e., driver) screens in identifying effective lymphoproliferative component constructs, and in particular in the intracellular signaling domains that facilitate amplification of these cultures ex vivo or in vitro under these conditions.

These data support that a relatively large number of endodomains from genes reported to promote cell proliferation or survival under certain conditions in lymphoid or myeloid cells (including those cells successfully tested in these eight repertoire screens) can be used as the first signaling endodomains for lymphoproliferative components in the methods and compositions provided herein, five genes not identified to provide the first 100 enrichments in either of these screens (six endodomains P3 portions) are I L R (portions S110 and S113), I L RA (portion S144), I L RC (portion S146), I L RE (portion S149), and IFN L R1 (portion S087), either in gene constructs, or in methods used for detection, these P3 portions may present technical problems not identified in the first 100 of any of the repeats, and therefore, it is not possible to conclude from this data that these endodomains are not able to form the effective intracellular signaling domains in the first 100 pools, but that the first signaling endodomains are also found in the first 100 of these first library 3, I L, and that are also found in the first 100 of these previous pools 3, I3517, and no further summary is found in the first library 3.

Without being limited by theory, the results that all extracellular domains (P1) and all transmembrane domains (P2) were present in at least one of the first 100 constructs from at least one bank screen are consistent with the hypothesis that extracellular domains and transmembrane domains support in targeting one or both intracellular signaling domains in these lymphoproliferative component constructs. Since the ECDs tested in the screen are known to form dimers, and the transmembrane domains are known to act as transmembrane polypeptides that can direct the intracellular signaling domain into an active conformation, all ECDs and TMs tested in the screen are expected to be effective at least some of the time when paired with an effective intracellular signaling domain.

It is believed that in the complex lymphoproliferative component screens of those analyzed in such examples, as exemplified in more detail in examples 11 and 12 herein alone, the most potent endodomains and complete chimeric constructs will be shown in at least one screen out of eight screens as the first 100 hits out of the following identified numbers of the constructs in each library: 3.1.1a73,556; 3.1.1b 93,768; 3.1.2a 91,572; 3.1.2b 115,860; 3.1.3a 91,918; 3.1.3b99,772; 3.1.4a 31,177; 3.1.4b 66,873. However, many valid endodomains are not shown as the first 100 hits in all or even most screening. Without being limited by theory, it is believed that there are many factors that contribute to whether a lymphoproliferative component is relatively effective in a given experiment in which cultures are produced from PBMCs transduced with a large complex mixture of lentiviruses encoding candidate lymphoproliferative components that compete with one another for amplification, and transduced PBMCs are obtained from different donor individuals in each "fed" experiment and each "unfed" experiment.

Portions containing the following genes are present in the first 100 constructs of at least one of the P3 and pool 3.1 repeats (table 25) on the construct containing X002 at P4 in CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, I L1R 1, I L1 RAP, I L01R L11, I L22 RA, I L32 RG, I L45 RA, I L56R, I L69R, I L710 RB 710, I L811 RA, I L912 RB1, I L12 RB2, I L013 RA2, I L115 RA, I L17 RD, I L21L 23L RA, I L RA, L EPR, MP L, MyD L or osmr. portions are present in the first 100, S199, S198, S168, S198, S170, S199, S170, S115S 199, I3631 RA, I L S170, S198S, S170, S168, S198S, S170, S115 RA, I L RA, I367 RA, I L S29S 115RA, S115S 170S 29S, S170S, S170S, S29S 170S 29S, S170S, S and S29S 168S 29S.

The portion containing the following genes was present on the construct containing X001 at P3 at P4 and in the first 100 constructs of at least one of the pool 3.1 repeats (table 25): CD40, CD79B, FCGR2C or FCGRA 2. The following parts were present at P4 on the X001 containing construct at P3 and in the first 100 constructs of at least one of the pool 3.1 repeats (table 25): s037, S039, S050, S051, S052, S080, S212, or S213.

Next, statistical analysis of the data from 8 pool 3.1 replicate pool screening experiments was performed to identify particularly effective intracellular P3 and P4 domains. Relative to the cell count on day 7 (i.e., DNA barcode count assuming equal cell counts), the analysis began at time points on day 21 and later, as discussed above for the previous 100 pool analysis.

We used statistical analysis to identify particularly potent intracellular domains (i.e., part P3 and part P4) independent of their intracellular partners. Differential enrichment values were calculated as the difference between the enrichment of constructs containing the intracellular portion of interest and the enrichment of constructs with the same extracellular and transmembrane portions but without the intracellular domain, where enrichment was expressed as the log base 2, the ratio between the normalized count value (in million counts) at a particular time point increased by false counts of 1 and the normalized count value (in million counts) at day 7 increased by false counts of 1. Statistical analysis was performed by performing a Mann-Whitney-Wilcoxon test on each first endodomain (P3) compared to all other possible P3 fractions. This test calculates the sum of the rankings for the construct containing the particular P3 portion and all other constructs in the dataset, and performs a chi-square test to determine if the ranking distributions are significantly different. A Benjamini-Hochberg correction was applied to obtain a p-value adjusted for the false discovery rate for each individual pool. This analysis was first performed on all constructs not related to part P4. When several parts represent variants of the same gene, the analysis is also performed using parts grouped by the gene alone. The same analysis was then performed only for constructs with a stop codon at P4 (part X002).

For 8 pool 3.1 repeats, table 1 provides an identifier of the most efficient first intracellular signaling domain (P3) gene and portion using statistical analysis when analyzed with termination at P3 only, asterisks after the number of days in table 1 indicates that the P3 gene or portion is significant to the indicated level, and when analyzed with any P3 for a given P3, the number of bars after the number of days indicates that the P3 gene or portion is significant to the indicated P level, the absence of asterisks or tic after the number of days indicates that the indicated significance and significance level is for the P3 portion or gene of both the analysis with all P3 portions of the given P3 and the analysis for termination at P3 of the given P3, the following genes are obtained for the first intracellular signaling domain portion identified in the circle shown by this statistical analysis (P <0.1) as the most efficient construct (S06s) and the statistical analysis of the most efficient CSF 1S 150, the mr < S > S < 72 > S < 200, the most efficient gene (S < S > S < 1 >) and the most efficient gene shown in the circle shown by this library 3, S < 72, mr < S < 200), the statistical analysis (S < 72), the statistical analysis) and the list of the cells shown by ifs < S < 72, no more than the number of the indicated by the P3, no more than the indicated by the first intracellular signaling domain (S72, no more than the indicated by the P3, no more than the number of the indicated by the P3, No. 7, No. S72, No. 7, no more than the indicated by the P3, no more than the number of the indicated by the number of the P3, no more than the number of the number.

The following first intracellular domain genes are represented by a first intracellular signaling domain portion that is most efficient by this statistical analysis of at least 2 pools (P <0.1), with part numbers in parentheses CSF2RB (S057), CSF3R (S062, S063, and S064), I L2 RB (S105), I L2 RG (S106), I L6 ST (S117), I L27 RA (S168 and S169), I L31 RA (S170), MP L (S186), and MyD88(S190, S191, S192, S194, S196, S197, S198).

The following first intracellular domain genes are represented by a first intracellular signaling domain portion that is most effective even when the statistical cutoff for P is less than 0.05 by this statistical analysis of at least 2 libraries, CSF2RB (S057), CSF3R (S062, S063 and S064), I L RB (S105), I L ST (S117), I L27 RA (S168 and S169), I L31 RA (S170), MP L (S186) and MyD88(S190, S865, S192, S194, S196, S197, S198). finally, the following first intracellular domain genes are most effective by this statistical analysis of at least 2 libraries, even when the statistical cutoff for P is less than 0.05, where the libraries are not fed-in/unfed pairs from the same repeat CSF 583, I L, I634 RA, I L RA and MP 88.

TABLE 1 most potent P3 part and genes from pool 3.1 repeat screen.

Analysis of the P4 Domain

Statistical analysis of the P4 domain was conducted as indicated above for the P3 domain, except that the tested P4 domain was analyzed using any of the P3 domains or without the P3 domain (a linker (X001) at portion P3). For the 8 pool 3.1 replicates, table 2 provides the identifiers of the most potent second intracellular signaling domain (P4) genes and parts using statistical analysis. Asterisks after the number of days in table 2 indicate that the P4 gene or portion is significant to the indicated level when analyzed with only the linker at P3, and the tic marks after the number of days indicate that the P4 gene or portion is significant to the indicated P level when analyzed with any P3 for a given P4. The absence of an asterisk or tic after the number of days indicates that the significance and level of significance for the indicated days was obtained for the P4 portion or gene with both the analysis of all P3 portions of the given P4 and the analysis of the linon at P3 of the given P4. The following genes with partial numbers in parentheses are represented by the second intracellular signaling domain portion identified in the constructs shown to be most effective by this statistical analysis (P < 0.1): CD27(S047), CD40(S050 and S051), CD79B (S053), TNFRSF4(S211), TNFRSF8(S212), TNFRSF9(S213), and TNFRSF18 (S215). The following genes are represented by a second intracellular signaling domain portion that is most effective in this statistical analysis by at least 2 pools: CD40(S050 and S051) and TNFRSF8 (S212). The following genes with partial numbers in parentheses are represented by the second intracellular signaling domain portion identified in the construct shown to be most effective by this statistical analysis (P < 0.05): CD27(S047), CD40(S050 and S051), TNFRSF4(S211), TNFRSF8(S212), TNFRSF9(S213), and TNFRSF18 (S215). Notably, all of the statistically most significant second intracellular signaling domains are derived from TNF receptor family members.

Table 2 most potent P4 part and gene from pool 3.1 repeat screen.

Further analysis was performed to identify the most potent second intracellular signaling domain (P4) partner for each first intracellular signaling domain. To perform this analysis, a construct with two endodomains was compared to its equivalent with only one endodomain (same ectodomain, transmembrane domain and first endodomain). Differential enrichment was calculated as the difference between the enrichment values between the two constructs, where enrichment was expressed as the logarithm of the ratio between the normalized counts (in million counts) at the time point of interest and the normalized counts (in million counts) at day 7 (each increased by false count 1), based on 2. The differential expression values for constructs with a particular second endodomain compared to other second endodomains were subjected to a Mann-Whitney-Wilcoxon test for each representative combined combination of extracellular portion, transmembrane portion, and first intracellular portion to identify portions associated with significant changes in the ordering distribution. The P value was adjusted using the Benjamini-Hochberg method for each test set, and the false discovery rate was set to 0.1 or 0.05.

For 8 library 3.1 repeats and library 3.0 (example 12), Table 3 provides the identifiers of the most effective first and second intracellular signaling domain (P) partners using this statistical analysis, the following pairs of genes having the first from the P portion and the genes from the P portion are represented by the first and second intracellular signaling domain portions identified on the same construct most effective by this statistical analysis (P <0.1) CSF2 and TNFRSF, CSF2 and CD, CSFR and CD79, IFNAR and TNFRSF, I1 and CD79, I3 RA and CD 010, I RA and I111 RA and FCGRAS, I213 RA and TNFRSF, I RAP 427 and CD3, I318 and FCRA, and CD5, and CD 063, I and CD79, I, II, III A, III, I, III, and CD3, preferably, and III, preferably, the first from the first and CD 053, the most effective second intracellular signaling domain (P) portions and the first and the corresponding genes, the following pairs of the first and the second intracellular signaling domain portions of the first and the second intracellular signaling domain (P) and the first and the second intracellular signaling domain (P053.186, the second intracellular signaling domain (P) portions and the first and the second intracellular signaling domain (P) portions and the second intracellular signaling domain (P) and the first and the second intracellular signaling domain portions of the first and the second intracellular signaling domain portions of the first and the second intracellular signaling domain (P portions of the first and the second intracellular signaling domain (P) portions of the first and the second intracellular signaling domain portions of the first, the first and the second intracellular signaling domain portions of the first and the first, the first portions of the first, the second intracellular signaling domain, the first, the second intracellular signaling domain, the first, the second intracellular signaling domain, the first, the second intracellular signaling domain, the first, the second intracellular signaling domain, the first, the second intracellular signaling domain, the second, the first, the.

Thus, the intracellular signaling domain from the listed P4 gene appears to be particularly effective in co-stimulating the proliferative signal provided by the intracellular signaling domain of the listed P3 partner gene.

TABLE 3 part P4 modified part P3

This example confirms that most of the first endodomains tested were effective in driving expansion of transduced PBMCs between day 7 and day 21 post transduction in the absence of exogenous interleukins when cultured ex vivo. In addition, various endodomains and combinations of first and second endodomains were identified to be particularly effective in driving amplification under these conditions for 21 days and later time points. Example 11 identification of candidate chimeric polypeptide lymphoproliferative components.

In this example, two libraries (library 1 and library 2) of candidate (putative) chimeric lymphoproliferative components were assembled into viral vectors from an extracellular-transmembrane blocking sequence library, an intracellular blocking sequence library, and a barcode library according to constructs encoding chimeric polypeptides provided in FIGS. 16-17 proliferation of library-transduced PBMCs was performed over time by extracting genomic DNA from PBMCs at one week intervals and amplifying barcode regions for DNA sequencing to estimate the number of cells in these mixed PBMC cultures containing each of the test candidate chimeric lymphoproliferative components.

Two pools were prepared and analyzed in this study, constructs of pool 1 (which was used in the screening assays identified as pools 1A, 1.1A and 1.1B) were flanked by CARs at the 5' end of the candidate chimeric polypeptide, while constructs of pool 2 (which was used in the screening assays identified as pools 2B and 2.1B) did not include CARs (fig. 16 and 17), fig. 16 provides a schematic of a non-limiting exemplary transgene expression cassette containing a polynucleotide sequence encoding the CAR of pool 1 and a candidate chimeric lymphoproliferative component (C L E) driven by the EF-1 α promoter and Kozak type sequence (GCCGCCACC (SEQ ID NO:519)) in the lentiviral vector backbone, CAR labeled with F L AG and containing ASTR for CD19, the stem and transmembrane portion of CD8, and intracellular activation domain from CD3z each candidate lymphoproliferative component of pool 1 includes 3 modules 1 extracellular/transmembrane module (P1-2) and one coding for the last termination codon (P) of wpt 3-t module (P-t) found after the last nucleotide deletion of the coding codon (P-t-codon (P-t-block) 521, 5 (P-t-codon (P-t-.

The CAR of pool 1 and P1-2 were separated by a polynucleotide sequence encoding a T2A ribosome skipping sequence.

FIG. 17 provides a schematic of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding each candidate lymphoproliferative component of Bank 2C L E. candidate C L E. library 2 driven by the EF-1 α promoter and a Kozak type sequence (GCCGCCACC (SEQ ID NO:519)) in a lentiviral vector backbone, including 3 modules, one extracellular/transmembrane module (P1-2) and 2 intracellular modules (P3 and P4). P1-2 module also encodes a recognition and/or elimination domain (TAG). triple termination sequence (TAATAGTGA (SEQ ID NO:520)) isolated from the DNA barcode (P5) P4. WPRE (GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:521)) is present between the last termination codon (4 bp starting after the last nucleotide of the last termination codon) and 3' L TR (83 nucleotides after the last nucleotide of the WPRE) starting.

The assembled candidate chimeric polypeptide portions of the two libraries follow the exact same design with 3 positions (1 extracellular/transmembrane module (P1-2) and 2 intracellular modules (P3 and P4), one of which may be a premature stop signal) including the GGS linker encoded by the nucleic acid sequence between each of the domains as a construct component product the nucleic acid encoding the complete or variant form of the Myc or eTAG (SEQ ID NO:284) recognition and/or elimination domain (referred to as "eTAG" or "Myc Tag" in table 7) is inserted into the 5 'of the extracellular domain of each truncated interleukin receptor (or select L MP1 construct) as part of the P1-2 module so that the encoded candidate chimeric polypeptide includes the recognition and/or elimination domain in the frame with the extracellular domain, the signal sequence inserted into the 5' end of the coding sequence in library 1 and the 5 'end of the recognition and/or elimination domain of the coding sequence of library 2 in library 5' of coding sequence of library CD coding sequence insert in CD 11, CD coding sequence of CD8 library, CD coding sequence of CD8, CD coding sequence of CD8, CD coding sequence of CD8, CD coding sequence of CD8, CD coding sequence of CD8, CD coding sequence of CD8, CD8 promoter, CD8 promoter, CD 8.

Synthesis of viral vectors

The individual portions of the library elements are designed and synthesized as DNA blocks with compatible type IIs enzyme (AarI) overhangs and linkers encoding glycine-serine sequences at the 5 'and 3' ends of the portions. These fractions were then individually cloned into universal plasmid vectors. The assembly of each part was completed as one assembly in which 0.04pmol of the part, 0.04pmol of the barcode mixture and 0.04pmol of the plasmid vector backbone were added to 10ul of the reaction, followed by 1 unit of AarI enzyme, 10 units of T4 DNA ligase and 1 Xligase buffer (50mM Tris-HCl, 10mM MgCl. sub.₂1mM ATP, 10mM DTT) to obtain optimal ligase activity. The decomposition and ligation cycles were performed using the following thermal cycles: at 37 deg.C for 2min and 16 deg.C for 5 min; incubating at 50 ℃ for 5min for final decomposition; incubate at 80 ℃ for 10min to inactivate AarI, and finally maintain at 4 ℃ for 55 cycles. The assembled support is then purified by conventional ethanol precipitation. The resulting purified vector was electroporated (Electro Micropulser, Bio-rad) into Top10 electrocompetent cells (Thermo Fisher scientific) and directly recovered in liquid culture containing the antibiotic for selection and amplification. The cultures were incubated at 37 ℃ for 10 to 12 hours and subsequently harvested and used ZymoPURE-EndoZero^TMPlasmid Gigaprep kit purified plasmids.

Lentiviral particle production

Recombinant lentiviral particles were generated by transient transfection of 293T cells (L enti-X) with separate lentiviral packaging plasmids encoding gag/pol and rev, and a pseudotyped plasmid encoding VSV-G^TM293T, Clontech). Synthetic viral vectors encoding candidate chimeric polypeptides with or without co-expressed chimeric antigen receptors are co-transfected with packaging plasmids. Cells were passed through Freestyle^TM293 expression Medium (ThermoFisher Scientific) to accommodate suspension culture with continuous growth 1 × 10⁶Cells/ml (200m L) at 1L Erlenmeyer flasks were seeded with cells in suspension and immediately transfected with Polyethyleneimine (PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 10ml Gibco for 200m L cells^TMOpti-MEM^TMTo obtain lentiviral particles pseudotyped with VSV-G, the total DNA used (culture volume of 1. mu.g/m L) was a mixture of 4 plasmids with a molar ratio of 2 × combination of viral vector, 1 × plasmid containing Rev, 1 × plasmid containing VSV-G and 1 × plasmid containing gag/pol PEI individually PEI was added to 10ml of Gibco^TMOpti-MEM^TMTo 2. mu.g/m L (culture volume, 2:1 ratio to DNA.) after a 5 minute room temperature incubation, the two solutions were mixed together and incubated at room temperature for 20 minutes, the final volume (20ml) was added to the cells, followed by a concomitant 125rpm with 8% CO₂The cells were incubated at 37 ℃ for 48 hours.

After 48 hours, the supernatant was collected and clarified by centrifugation at 1,000g for 10 minutes, then the viral supernatant was filtered through a low protein bound Millex-HV 0.45 μm PVDF filter (Millipore, catalog No. S L HVR 25L S), the clarified supernatant was poured into a new tube, 1 volume of L enti-X PEG (Takara, 4 ×) was mixed with 3 volumes of the clarified supernatant, and incubated overnight at 4 ℃.

Transduction and culture of PBMC

All human blood (101ml) from healthy donors was collected into 200ml bags containing CDP anticoagulant (Nanger; S-200). Ficoll-Pacque was used with a Sepax 2S-100 device (Biosafe; 14000) following the manufacturer' S instructions and kit CS900.2 (Biosafe; 1008)^TM(General Electric) blood was processed by density gradient centrifugation to obtain Peripheral Blood Mononuclear Cells (PBMC). the yield of viable PBMC was 7.3 × 10⁷1.5 × 10⁷Each PBMC individuallyFull OpTsizer added to two G-Rex 100M cell culture devices (Wilson Wolf, 81100S) to final volume of 30ml^TMCTS^TMT cell expansion SFM (supplemented with 26ml OpTsizer according to manufacturer's instructions)^TMCTS^TMT cell expansion supplement (Thermo Fisher, A10484-02), 25ml CTS^TMImmune cell SR (ThermoFisher, A2596101) and 10ml CTS^TMGlutaMAX^TMOpTsizer of I supplement (Thermo Fisher, A1286001)^TMCTS^TMT cell expansion basal Medium 1L (Thermo Fisher, A10221)) to a final concentration of 0.5 × 10⁶Recombinant human interleukin-2 (I L-2) (Novoprotein) at a concentration of 100IU/ml was also added together with activated anti-CD 3 Ab (OKT3, Novoprotein) at a concentration of 50ng/ml to activate PBMCs for viral transduction G-Rex devices were used at 37 ℃ and 5% CO₂Next, incubate overnight in a standard moist tissue incubator. After overnight incubation, a preparation of lentiviral particles containing the construct encoding the chimeric polypeptide to be tested (library 1 or library 2) was added to individual G-Rex devices under 5-superinfection (MOI). G-Rex devices (Bank 1 and Bank 2) were placed at 37 ℃ and 5% CO₂Next, incubate overnight in a standard moist tissue incubator. After overnight incubation, additional complete OpTsizer was added^TMCTS^TMT cells expand SFM to reach a final volume of 500ml for each G-Rex device, no additional I L-2 or other exogenous interleukins were added during this or following cell culture step

The device was at 37 ℃ and 5% CO₂At day 7, cells were collected by centrifugation from two G-Rex devices transduced with either pool 1 or pool 2 and resuspended in fresh complete OpTsizer without I L-2^TMCTS^TMT cell expansion in SFM 1.25 × 10⁷ Pool 1 cells and 2.54 × 10⁷The library 2 cells were placed in a complete OpTsizer containing 500ml without I L-2^TMCTS^TMT cell expansion on a novel G-Rex 100M device of SFM. Day 0 frozen donor matched P thawing a vialBMC(1.46×10⁷PBMC) were added to G-Rex devices containing cells transduced with pool 1 to provide CD19+ B cell activation of CD19 ASTR. In these screens, cells fed with PBMCs are designated with a after the pool number, e.g. a screen performed using cells transduced with pool 1 and fed with PBMCs is referred to herein as pool 1A. Pool 2 received no day 0 PBMC. In these screens, cells not fed with PBMCs were designated with B after the pool number, e.g. a screen performed using cells transduced with pool 2 and not fed with PBMCs is referred to herein as pool 2B. The G-Rex device was placed at 37 ℃ and 5% CO₂At day 14, cells were collected from two G-Rex devices (Bank 1A and Bank 2B) by centrifugation and resuspended in fresh complete OpTsizer without I L-2^TMCTS^TMT cell expansion in SFM these cells were each placed in a complete OpTsizer containing 30ml without I L-2^TMCTS^TMT cells expanded individual wells of a 6-well G-rex disk (Wilson-Wolf; 80240M) of SFM. A vial of thawed day 0 frozen donor-matched PBMCs was added to the wells containing pool 1A cells to provide CD19+ B cell activation of CD19 ASTR. The G-Rex device was placed at 37 ℃ and 5% CO₂Overnight incubation in a standard moist tissue incubator at day 21, cells were collected from each well of a 6-well G-rex plate, and the treatment at day 14 was repeated, samples (1 × 10) were collected on different days (day 7, day 21, day 28, and/or day 35) after transduction (day 21, day 28, and/or day 35)⁵To 4-5 × 10⁶Individual cells), and using GenElute according to the kit protocol^TMMammalian genomic DNA miniprep genomic DNA was purified. In some cases, Ficoll-Pacque is used^TM(General Electric) density gradient centrifugation to purify the sample to remove dead cells prior to genomic DNA extraction. Purified genomic DNA was sequenced using Illumina HiSeq to generate paired-end 150bp reads. Typically, a subset of ten million reads is extracted from each indexed fastq file and analyzed using a barcode reader, which is a custom R-script that extracts barcode sequences based on the presence of constant regions. Purified genomic DNA was also tested on the PacBio sequencing SystemIn order to obtain a longer read length to associate the barcode with the construct. These screens were repeated and the pools were designated 1.1A, 1.1B and 2.1B, where a and B indicate whether cells as discussed above were fed with PBMCs.

Data analysis

Once HiSeq data for all time points are obtained, the barcode count table is merged based on the barcode ID the counts are normalized to "counts per million": 1 × 10 using the following equation⁶Raw counts/sum (raw counts of all barcodes in the sample), which is called prevalence value. Barcodes with an average prevalence of 0.33cpm or less per time point were rejected. Full-length construct-barcode associations obtained at select time points by SMRT sequencing (pacifiic Biosciences) were used to identify the modules of interest. Non-competent constructs with no 3 parts or ambiguous constructs with more than one building cell were filtered out. The enrichment value for each component was calculated as (normalized count at final time point + 1)/(normalized count at initial time point + 1). Candidate chimeric polypeptide genes were identified as constructs with an enrichment above the respective pool-specific threshold.

Results

Chimeric polypeptide candidates are indicated to have 3 test domains including an extracellular and transmembrane domain (P1-2), a first intracellular domain (P3) and a second intracellular domain (P4) (fig. 16 and 17). furthermore, the constructs include DNA barcodes to aid in analysis and identification of the constructs using the next generation of sequencing.in addition, most constructs as shown in table 7 include nucleic acid sequences encoding recognition and/or elimination domains in boxes with extracellular and transmembrane domains whereas, recognition and/or elimination domains of those constructs encoding extracellular or transmembrane fragments of L MP1 are designed for a total of 226,840 possible conceptual combinations based on 20 different extracellular-transmembrane domains, 106 possible P3 domains and 107 possible P4 domains, one of which is a stop codon, after assembly of viral vectors, generation of lentiviral vectors, the concept of P008 and the concept of P3 domains, and the results for the expression of the constructs are shown in table 8, and the results are provided for the expression of the constructs as a detailed information on day 7, the expression of PBMC 7, No. 7B 7, No. 5B 7, no more than the expression of the constructs found in cd8 constructs, no more than the expression of cd8 constructs, No. 7 c expression of cd8, No. 7 c, No. 5c, No. 7 c, No. 5c found in the expression of.

The candidate extracellular and transmembrane domains are selected from known mutant receptors, such as interleukin or hormone receptors that have been reported to be constitutively active when expressed in at least some cell types.the ligand binding domain is not included in the candidate extracellular and transmembrane domains.mutations in such receptor mutants occur in the transmembrane or juxtamembrane regions.exemplary candidate extracellular and transmembrane domains include I L RAIns PPC L, L MP1, CR L F2F 232C, CSF2RB V449 2, CSF3R T640N, EPOR L C I252C, GHR E260CI270C, I L RA F523C and MP L S505N. the candidate extracellular and transmembrane domains include the wild-type viral protein L MP1, a human transmembrane protein known to activate cell signaling with ligands when targeted to rafts or when fused to CD40 (Kaykas et al: 2001J: 2641).

The identities of potential polypeptides occupying the first and second intracellular domains of the candidate chimeric polypeptide are selected to be identified as identical. These candidate endodomains are intracellular signaling domains of genes known to promote proliferation, survival (anti-apoptosis) and/or provide costimulatory signals that enhance differentiation status, proliferative potential or resistance to cell death in at least some cell types. Some of the intracellular domains of candidate chimeric polypeptides are known to activate JAK1/JAK2 signaling and STAT 5. Endodomains from some of the genes listed in table 7 are included in the library. Exemplary intracellular domains from these genes are provided at positions P3 and P4 of tables 8-12. Detailed information about these P3 and P4 domains is provided in table 7.

For pool 1A, which included constructs encoding chimeric polypeptides and CARs, 172 optimal candidate chimeric polypeptides that promoted PBMC proliferation to the greatest extent (and also could survive) between day 7 and the last day indicated on the table were identified when cultured in the absence of I L-2 (see table 8, where the enrichment was calculated as L og2 (normalized count on the last day +1 indicated in the table) -log 2 (normalized count on day 7 + 1)).

For pool 1A, some chimeric polypeptide constructs promoted cell proliferation between day 7 and the last day indicated on the table for each extracellular and transmembrane mutant domain tested at position P1-2. When all the results for all constructs deduced from the same position P1-2 mutant were combined, all the tested extracellular and transmembrane domains produced a cell enrichment of greater than 1 (i.e. promoted cell proliferation) between day 7 and the last day indicated on the table. Examples of extracellular and/or transmembrane domains, or portions and/or mutants thereof, present in constructs that promote cell proliferation are provided in table 8. Examples of extracellular and/or transmembrane domains or portions thereof and/or mutants present in constructs that promote cell proliferation in a repeat screen referred to as library 1.1A are provided in table 10. Examples of extracellular and/or transmembrane domains or parts thereof and/or mutants present in constructs promoting cell proliferation in a repeat screen called pool 1.1B (which includes transduction of cells not fed to PBMCs with pool 1) are provided in table 11.

For pool 1A, intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, I L2 RA, I L3 RA, I L13 RA2, TNFRSF8 and TNFRSF9 or mutants thereof known to have signaling activity (if such mutants are present in the constructs provided in table 8) promote proliferation between day 7 and the last day indicated on the table when data are found at the first intracellular domain position of the candidate chimeric polypeptide (P3) with all of the sequence counts of the constructs in the first intracellular domain (P3) from that gene are considered in combination, this conclusion is based on the enrichment of the sequence counts of the constructs in the population of PBMC cells in mixed culture, when the results of all constructs of one gene are combined, such that for pool 1A, this domain count present on the last day indicated in the table is at least as great as compared to the 7 th day 3 times the highest degree of presence of the construct in the first intracellular domain in the first cell panel or mutant thereof is referred to as a 1. the mutant is provided in the first cell panel 1A or a mutant which is not present in the same panel or a panel which is referred to provide a panel B.

For pool 1A, the intracellular domains from CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B, CR L F2, FCGR2C, ICOS, I L2 RA, I L13 RA1, I L13 RA2, I L15 RA, TNFRSF9, and TNFRSF18 known to have signaling activity, or mutants thereof if such mutants are present in the table, promote PBMC proliferation between day 7 and the last day indicated on the table when found at the second intracellular domain position of the candidate chimeric polypeptide (P4), when considering in combination all data with the second intracellular domain from that gene (P4) this conclusion based on the enrichment of the sequence count of the constructs in the mixed culture PBMC cell population when combining all results for one gene, such that the results for pool 1A, the presence of this second intracellular domain on day 35, or a mutant thereof in the second intracellular domain position referred to as the second intracellular domain count in table 1. or mutant, the second intracellular domain is included in the second cell fraction of the construct 1.9 or a fraction of the same which is referred to provide a factor which is present in the second cell count in the second cell.

More specifically, for the library 1A, among 5,996 constructs having MP L S505N (MP L protooncogene), having serine 505 mutated to asparagine as P1-P2 modules, 165 positive constructs are defined as constructs having an enrichment greater than two for the library 1A, among 436 constructs having CD3D (CD3d molecule) as P3 module, 24 positive constructs, among 528 constructs having CD3E (CD3e molecule) as P3 module, among 17 positive constructs, among 261 constructs having CD 83 (CD 83 molecule) as P3 module, among 261 constructs having CD3 as P3 module, among 1,022 constructs having CD3 (CD 3) as P7372 module, among 3 constructs having CD3 positive modules (CD 75) as CD 75 module P1) as CD 75, among 3 positive constructs having CD 75 module P1 module as CD 75 module P positive modules (CD 75) as CD 75 module P1 as CD 75) as CD 75 module P positive modules, among 3 modules (CD 75) as CD 75 modules, among 3 modules) as CD 75 modules, among 3 positive constructs having CD 75 modules (CD 75 as CD 75 modules, among 3 positive modules) as CD 75 modules, among 3675 modules (CD 75 modules) as CD 75 modules, among 3 positive constructs, among 3660 positive modules (CD 75 modules) as CD 75 modules, among 3675 modules, as CD 75 modules, as CD8 modules, as CD8 modules among 3675 modules, CD8 modules, as CD8 modules, CD8 modules among 3 modules, CD8 modules among 3675 modules among 3 positive constructs, CD8 modules among 3 positive constructs (CD8 modules, CD8 modules among 3 positive constructs, CD8 modules among 3 modules among 3675 modules, CD8 modules among 3 modules, CD 8.

For purposes of repeat screening, constructs with particularly noteworthy enrichment were log greater than 2 for two of the duplicate pools₂(normalized count data on last day + 1)/(normalized count data on day 7 +1)) values. Constructs meeting this cut-off for pool 1 and pool 2 are listed in tables 20 and 21. For pools 1A and 1.1A, constructs M001-S116-S044, M024-S192-S045, M001-S047-S102, M048-S195-S043, M012-S216-S211 and M030-S170-S194 had a particularly noteworthy enrichment in the two screenings。

Additional information regarding the first and second endodomains in constructs with particularly noteworthy enrichments in the screen of both library 1A and library 1.1A is provided in table 20, including the genes from which the first and second endodomains are derived, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif when a construct with endodomains from I L6R, MYD88, CD27, TNFRSF18 or I L RA is present in the first endodomains (P3), and a construct with endodomains from CD8B, I L R L, CD8A, TNFRSF4 or MYD88 is present in the second endodomains (P4), when a construct with endodomains from I361, I3684 or tnfr 465 is present in both library 1A and 1.1A when a construct with endodomains from tnfr 461, a construct with particularly noteworthy enrichments (table 20), when a construct with endodomains from tnfr 466, a construct with intracellular domains from tnfr 461, and/or a construct with two endodomains from tnfr 465, especially noteworthy enrichments in table 1A 465 and 1.

In pool 2B, which included constructs encoding chimeric polypeptides and which did not encode CARs, 167 optimal candidates were identified that promote PBMC proliferation between day 7 and the last day indicated on the table when cultured in the absence of I L-2 (see table 9, where the enrichment was calculated at L og2 (normalized count of last day +1 indicated on the table) -log 2 (normalized count of day 7 + 1)).

For pool 2B, CSF3R T640N in position P1-2 promoted cell proliferation better than the other extracellular and transmembrane (P1-2) domains tested between day 7 and the last day indicated on the table, yielding an enrichment factor greater than 2.5 when all results from all constructs of the extracellular and transmembrane domains were combined. Examples of extracellular and/or transmembrane domains, or portions thereof and/or mutants thereof, present in constructs that promote the highest degree of cell proliferation are provided in table 9. Examples of extracellular and/or transmembrane domains, or portions thereof and/or mutants thereof, present in constructs that promote the highest degree of cell proliferation in the repeat screen known as pool 2.1B are provided in table 12.

For pool 2B, the intracellular domains from CD8A, CD40, IFNAR1, I L31 RA and MyD88 known to have signaling activity, or mutants thereof if such mutants are present in the constructs provided in the table, promote PBMC proliferation between day 7 and the last day indicated on the table when found at the first intracellular domain position of the candidate chimeric polypeptide (P3), when data for all constructs with the first intracellular domain from that gene (P3) are considered in combination, this conclusion is based on enrichment of sequence counts of constructs in a mixed culture PBMC cell population, such that when the results for all constructs of one gene are combined, this domain count present on day 28 is at least 2 times greater than that on day 7 for pool 2B, examples of the first intracellular domain or portion thereof and/or mutant present in the construct that promotes maximal degree of cell proliferation are provided in table 9.

For pool 2B, the endodomain from CD27 or a mutant thereof known to have signaling activity, if such mutant is present in the constructs provided in the table, promoted PBMC proliferation between day 7 and the last day indicated on the table when found at the second endodomain position of the chimeric polypeptide candidate (P4), when the data for all constructs with the second endodomain from that gene (P4) were considered in combination. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed culture PBMC cell population, when the results for all constructs of one gene are combined, such that this domain count is present at day 28 at least 11-fold more than day 7 for pool 2B. Examples of the second endodomain, or a portion thereof, and/or a mutant present in the construct that promotes maximal cell proliferation are provided in table 9. Examples of the second endodomain, or portions thereof, and/or mutants present in constructs that promote the greatest degree of cell proliferation in a repeat screen called pool 2.1B are provided in table 12.

More specifically, for pool 2B, 59 out of 6,909 constructs with CSF3R T640N (colony stimulating factor 3 receptor with threonine 640 mutated to asparagine) as the P1/P2 module of pool 2B were positive, defined as constructs with enrichment greater than two for pool 2B, of 184 constructs with IFNAR1 (interferon α and β receptor subunit 1) as the P3 module of pool 2B, 3 were positive, of 1,258 constructs with CD40(CD40 molecule) as the P3 module of pool 2B, 17 were positive, of 851 constructs with CD27(CD27 molecule) as the P4 module of pool 2B, 11 were positive, of 63418 constructs with CD27(CD27 molecule) as the P4 module of pool 2B, of 6356, 5912 of the P5912 constructs with I L as the P3 module of pool 2B, of 6356 positive constructs with I L as the P L module of pool 2B, of 5912.

For both libraries 2B and 2.1B, constructs M007-S049-S051, M007-S050-S039, M012-S050-S043, M012-S161-S213, M030-S142-S049, M001-S145-S130, M018-S085-S039, M018-S075-S053, M012-S135-S074, and M007-S214-S077 had particularly noteworthy enrichments in both screenings. Constructs with particularly noteworthy enrichment for the purpose of repeat screening were log₂Those constructs having a value of greater than 2 (normalized count data on the last day + 1)/(normalized count data on day 7 + 1).

Further information on the first and second endodomains in constructs with particularly noteworthy enrichments in the screening of both pool 2B and pool 2.1B is provided in table 21, including the first and second endodomains derived gene, whether the first and/or second endodomains are an interleukin receptor, and whether the first and/or second endodomains have at least one ITAM motif when constructs with endodomains from CD28, CD40, I L22 RA1, I L13 RA2, I L17 RB, IFNGR2, FCGR2C, I L11 RA or TNFRSF14 are present in the first endodomains construct (P3), and when constructs with endodomains from CD40, CD3G, CD gr 8A, TNFRSF9, CD28, I3610 RB, CD 79L 10, fc1 or GHR L have an intein the first endodomains (ifr L), and when constructs with two ifra domains from ifra L B L, particularly display ifra 2 receptor ifra with an intein the second endodomains from the first and when constructs with ifra L B, and when constructs with two ifra domains from ifra L, particularly display ifra L B, ifr receptor ifra L B, and when constructs with two ifr epitopes from ifr receptor ifra L B L, ifr domains from the first and/or GHR L are present in the ifr 19 constructs (L).

Example 12 identification of candidate chimeric polypeptide lymphoproliferative components.

In this example, two libraries (library 3 and library 4) of candidate (putative) chimeric lymphoproliferative components were assembled into viral vectors from an extracellular-transmembrane blocking sequence library, an intracellular blocking sequence library, and a barcode library according to constructs encoding chimeric polypeptides provided in fig. 15 and 18 proliferation of library-transduced PBMC was performed over time by extracting genomic DNA from PBMC at one week intervals and amplifying barcode regions for DNA sequencing to estimate the number of cells in these mixed PBMC cultures containing each of the test candidate chimeric polypeptides.

Two pools, pool 3 (used in the screening assays identified as pools 3A, 3B, 3.1A and 3.1B) flanked by CARs at the 5' end of the chimeric polypeptide, while pool 4 (used in the screening assays identified as pools 4B and 4.1B) did not include CARs in this study figure 15 provides a schematic of a non-limiting transgene expression cassette containing a polynucleotide sequence encoding a CAR and a candidate C L e.car of pool 3 driven by the EF-1 α promoter and Kozak type sequence (GCCGCCACC (SEQ ID NO:519)) in the lentiviral vector backbone labeled with F L AG and containing ASTR for CD19, the stem and start of the transmembrane portion of CD8, and the intracellular activation domain from CD3 z. each candidate lymphatic component of pool 3 comprises 4 extracellular (P1), transmembrane module (P2) and 2 intracellular activation modules (P3 and P73742) present after the last nucleotide recognition codon (wpt) of the last codon (P) of the coding for the last codon (P6342) of the amplified gene (wpr) and the final coding nucleotide (WPRE) of the terminal codon (P63520) of the final coding sequence of SEQ ID No. DNA (P9) is also present between the last trec codon (P9 AG 14) and the last codon (P9 bp) of the terminal codon (P9B).

The CARs of pool 3 and P1 were separated by a polynucleotide sequence encoding a T2A ribosome skipping sequence.

FIG. 18 provides a schematic of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding each of the candidate lymphoproliferative components of Bank 4C L E. the candidate lymphoproliferative components of Bank 4 driven by the EF-1 α promoter and a Kozak-type sequence (GCCGCCACC (SEQ ID NO:519)) in a lentiviral vector backbone, including 4 modules, an extracellular module (P1), a transmembrane module (P2), and 2 intracellular modules (P3 and P4). P1 also encodes a Myc recognition and/or elimination domain.A triple termination sequence (TAATAGTGA (SEQ ID NO:520)) is present between the last termination codon (4 bp starting after the last nucleotide of the last termination codon) (GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:521)) and a 3' L TR starting 83 nucleotides after the last nucleotide of the WPRE) isolated from a DNA barcode (P5).

The assembled candidate chimeric polypeptide portions of the two libraries follow identical designs with 4 positions (1 extracellular module (P1), 1 transmembrane module (P2), and 2 intracellular modules (P3 and P4), one of which may be a premature termination signal) (fig. 15 and 18). A nucleic acid encoding the Myc Tag or complete or variant form of the eTAG recognition and/or elimination domain (referred to as "eTAG" or "Myc Tag" in table 7) is inserted at the 5 'end of the CAR-encoding sequence in pool 3 and at the 5' end of the c-Jun domain in P1 in pool 4. The signal sequence is inserted at the 5 'end of the CAR-encoding sequence in pool 3 and at the 5' end of the recognition and/or elimination domain-encoding sequence of pool 4. General design and construction of libraries, including barcodes, is as disclosed in example 11 herein, except that the P1 and P2 domains are as described in more detail later in this example. Furthermore, clearance domains are on some constructs as indicated in table 7, and when present are eTag variants or Myc tags.

Synthesis of viral vectors and lentivirus production

Vectors were synthesized as disclosed in example 11 and lentiviral particles for each library were generated.

Transduction and culture of PBMC

For pool 3A, whole human blood (89.1ml) from healthy donors was collected into 200ml bags containing CDP anticoagulant (Nanger; S-200). Ficoll-Pacque was used with a Sepax 2S-100 device (Biosafe; 14000) following the manufacturer' S instructions and kit CS900.2 (Biosafe; 1008)^TM(General Electric) blood was processed by density gradient centrifugation to obtain Peripheral Blood Mononuclear Cells (PBMC). the yield of viable PBMC was 8.4 × 10⁷Ten vials of 5 × 10 cells per vial⁶Cells of individual PBMCs were frozen for later use in feeding CD19+ B cells of a pool 3A sample containing CD19-CAR 3 × 10⁷One PBMC was added to one G-Rex 100M cell culture device (Wilson Wolf, 81100S) for complete OpTsizer at a final volume of 60ml^TMCTS^TMT cell expansion SFM (supplemented with 26ml OpTsizer according to manufacturer's instructions)^TMCTS^TMT cell expansion supplement (Thermo Fisher, A10484-02), 25ml CTS^TMImmune cell SR (Thermo Fisher, A2596101) and 10ml CTS^TMGlutaMAX^TMOpTsizer of I supplement (Thermo Fisher, A1286001)^TMCTS^TMT cell expansion basal Medium 1L (Thermo Fisher, A10221)) to a final concentration of 0.5 × 10⁶Recombinant human interleukin-2 (I L-2) (Novoprotein) at a concentration of 100IU/ml was also added together with activated anti-CD 3 Ab (OKT3, Novoprotein) at a concentration of 50ng/ml to activate PBMCs for viral transduction G-Rex devices were used at 37 ℃ and 5% CO₂Next, incubate overnight in a standard moist tissue incubator. After overnight incubationA preparation of lentiviral particles containing the construct encoding the chimeric polypeptide to be tested (library 3) was added to the G-Rex device under a 5-superinfection (MOI). The G-Rex device was placed at 37 ℃ and 5% CO₂Next, incubate overnight in a standard moist tissue incubator. After overnight incubation, additional complete OpTsizer was added^TMCTS^TMT cells expand SFM to reach a final volume of 500ml, no additional I L-2 or other exogenous interleukins were added during this or following cell culture step

The device was at 37 ℃ and 5% CO₂At day 7, cells were collected by centrifugation from G-Rex devices transduced with pool 3 and resuspended in fresh complete OpTsizer without I L-2^TMCTS^TMT cell expansion in SFM 5 × 10⁷ Individual pool 3 cells were plated in a complete OpTsizer containing 500ml of cells without I L-2^TMCTS^TMT-cell expansion on a novel G-Rex 100M device of SFM and labeled library 3A. 1.1 × 10⁸ Individual pool 3 cells were plated in a complete OpTsizer containing 500ml of cells without I L-2^TMCTS^TMT cell expansion SFM on a new G-Rex 100M device and labeled pool 3b in these screens, cells not fed with PBMCs were designated with a after pool number, e.g., using cells transduced with pool 3 and fed with PBMCs, screen referred to herein as pool 3 a. vial thawed donor matched PBMCs day 0 (2 × 10)⁷Individual PBMCs) were added to G-Rex devices containing cells transduced with pool 3A to provide CD19+ B cell activation of CD19 ASTR. Pool 3B received no day 0 PBMC. In these screens, cells not fed with PBMCs were designated with B after the pool number. For example, screening using cells transduced with pool 3 and not fed with PBMCs is referred to herein as pool 3B. The G-Rex device was placed at 37 ℃ and 5% CO₂At day 14, cells were collected from two G-Rex devices (Bank 3A, Bank 3B) by centrifugation and resuspended in fresh complete OpTsizer without I L-2^TMCTS^TMT cell expansionIn SFM, these cells were placed individually in a complete OpTsizer containing 30ml of cells without I L-2^TMCTS^TMT cell expansion of SFM in individual wells of 6-well G-Rex plates (Wilson-Wolf; 80240M) A vial of thawed, day 0 frozen donor-matched PBMC (5 × 10)⁶PBMC) were added to the pool 3A G-Rex containing pool to provide CD19+ B cell activation of CD19 ASTR. The G-Rex device was placed at 37 ℃ and 5% CO₂The cells were then incubated in a standard moist tissue incubator. This procedure was repeated on days 21, 28, 35 and 42, except that two vials of frozen day 0 PBMCs were added to the pool 3A on day 21 and one vial was added on days 28, 35 and 42. Pool 3B was not cultured for the past 21 days. These screens were repeated except that the Myc tag on the P1 portion was replaced with eTag (as shown in tables 7, 15 and 16) and designated as pools 3.1A and 3.1B, where the a and B indicate whether the cells as discussed above were fed with PBMCs.

Cells from pool 3A were prepared for analysis by flow cytometry (400g, 5 min) on day 49. Cells were collected by centrifugation and layered on Ficoll (Ficoll-Paque Premium (GE, cat #17-5442-02)) according to the manufacturer's instructions. 1.7 x 10⁶The resuspended cells were resuspended in 450. mu.l FACs staining buffer (554656, BD.) the resuspended cells were aliquoted into 9 eppendorf tubes, each containing 50. mu.l or 1.8 × 10⁵2.5. mu.l of human Fc block (564220, BD) was added to each tube and incubated for 10 minutes at room temperature the following antibody formulation was added to 50. mu.l of the sample to stain CD 564220, F564220 AG Tag (CAR +), 2.5. mu.l of anti-CD 564220-BV 421 antibody (pure line OKT 564220, Biolegend), 2.5. mu.l of anti-CD 564220-BV 510 antibody (pure line RPA-T564220 (Biolegend)), 2.5. mu.l of anti-CD 564220-PE-Cy 564220 antibody (pure line OKT 564220 (564220, Biolegend)), 2.5. mu.l of anti-CD 564220-785 (564220, Biolegend) and 0.5. mu.l of anti-F564220 Tag (anti-DYDDDKD, 564220, Clogen) for preparing mouse IgG 5. mu.5. mu.g of the sample, CD 564220, mouse IgG 421, mouse IgG 5. mu.g of the staining mouse with the anti-CD 564220, mouse IgG 421, mouse IgG 5. mu.g, mouse IgG 5. mu.5. mu.g of staining mouse (pure line of anti-CD 564220, mouse IgG 421, mouse IgG 5. mu.g of anti-CD 564220, mouse IgG 3, mouse IgG 421, mouse IgG 5. mu.g of staining antibody (pure line of mouse 564220, mouse IgG 3. mu.5. mu.g of mouse 564220, mouse), and mouse antibody staining CD 564220. mu.5. mu.00317 Biolegend), 2.5 μ l BV510 IgG (mouse IgG1, 400171, Biolegend), 2.5 μ lBV785 IgG (mouse IgG1, 400169, Biolegend) and 10 μ l PE IgG (mouse IgG1, 555749, BD) were used for IgG control staining samples. No antibody was added for unstained controls. The samples were incubated on ice for 30 minutes. The samples were centrifuged (400g, 5 min) and the supernatant removed. The samples were washed twice with 0.5ml FACS staining buffer. Stained cell pellets were resuspended in 125 μ l FACS staining buffer and then fixed by addition of 125ul BD fixation buffer (554655 BD). The samples were incubated with the fixation buffer for 15 minutes at 4 ℃. The samples were washed twice with 500 μ l FACS staining buffer and resuspended in 0.4ml FACS staining buffer. FAC analysis of samples was performed in a Novocyte flow cytometer (acesabiscociences) and analyzed using a lymphocytogate based on forward and side scatter.

For pool 4B, whole human blood (70ml) from healthy donors was collected into 200ml bags containing CDP anticoagulant (Nanger; S-200). Ficoll-Pacque was used with a Sepax 2S-100 device (Biosafe; 14000) following the manufacturer' S instructions and kit CS900.2 (Biosafe; 1008)^TM(General Electric) blood was processed by density gradient centrifugation to obtain Peripheral Blood Mononuclear Cells (PBMC). the yield of viable PBMC was 4.9 × 10⁷4.9 × 10⁷One PBMC was added to one G-Rex 100M cell culture device (Wilson Wolf, 81100S) for complete OpTsizer at a final volume of 98ml^TMCTS^TMT cell expansion SFM to a final concentration of 0.5 × 10⁶Recombinant human interleukin-2 (I L-2) (Novoprotein) at a concentration of 100IU/ml was also added together with activated anti-CD 3 Ab (OKT3, Novoprotein) at a concentration of 50ng/ml to activate PBMCs for viral transduction G-Rex devices were used at 37 ℃ and 5% CO₂Next, incubate overnight in a standard moist tissue incubator. After overnight incubation, a preparation of lentiviral particles containing the construct encoding the chimeric polypeptide to be tested (library 4) was added to the G-Rex device under 5-superinfection (MOI). The G-Rex device was placed at 37 ℃ and 5% CO₂Next, incubate overnight in a standard moist tissue incubator. After overnight incubation, addAdditional full OpTsizer^TMCTS^TMT cells expand SFM to reach a final volume of 500ml, no additional I L-2 was added during this or following cell culture step after isolation from PBMC, the cells were washed with water

The device was at 37 ℃ and 5% CO₂At day 7, cells were collected by centrifugation from G-Rex devices transduced with library 4 and resuspended in fresh complete OpTsizer without I L-2^TMCTS^TMT cell expansion in SFM 1.63 × 10⁸ Individual pool 4 cells were plated in a complete OpTsizer containing 500ml cells without I L-2^TMCTS^TMT cells expanded on the new G-Rex 100M device of SFM and labeled as pool 4. Pool 4 received no day 0 PBMC. Thus, in these screens, pools were designated with B after the pool number, e.g., the screen performed using cells transduced with pool 4 and not fed with PBMCs was referred to herein as pool 4B. The G-Rex device was placed at 37 ℃ and 5% CO₂At day 14, cells were harvested from the G-Rex device (pool 4) by centrifugation and resuspended in fresh complete OpTsizer without I L-2^TMCTS^TMT cell expansion in SFM these cells were each placed in a complete OpTsizer containing 30ml without I L-2^TMCTS^TMT cells expanded individual wells of a 6-well G-rex disk (Wilson-Wolf; 80240M) of SFM. The G-Rex device was placed at 37 ℃ and 5% CO₂The cells were then incubated in a standard moist tissue incubator. The pool 4 samples were incubated over the past 21 days. These screens were repeated except that the Myc tag on the P1 portion was replaced with eTag (as shown in tables 7 and 17) and designated pool 4.1B, where the B indicates that the cells as discussed above were not fed with PBMCs.

Samples (7 × 10) were collected on different days post transduction (days 7 and 21)⁴To 4-8 × 10⁶Individual cells), and using GenElute according to the kit protocol^TMMammalian genomic DNA miniprep genomic DNA was purified. In some cases, Ficoll-Pacque is used^TM(General Electric) Density gradient centrifugation to purify samplesDead cells were removed prior to genomic DNA extraction. Purified genomic DNA was sequenced using Illumina HiSeq to generate paired-end 150bp reads. Typically, a subset of ten million reads is extracted from each indexed fastq file and analyzed using a barcode reader, which is a custom R-script that extracts barcode sequences based on the presence of constant regions. Purified genomic DNA was also sequenced on the PacBio sequencing system to associate the barcode with the construct.

Data analysis

Data analysis was performed as disclosed in example 11, except that the time points were different.

Results

In this experiment, chimeric polypeptide candidates were designed with 4 test domains, including an ectodomain (P1), a transmembrane domain (P2), a first endodomain (P3), and a second endodomain (P4) (fig. 15 and 18). As explained in example 11, the constructs included DNA barcodes to aid in analysis and identification of the constructs using next generation sequencing. In addition, all constructs include nucleic acid sequences encoding recognition and/or elimination domains in frame with the extracellular domain. The constructs in pool 3, but not pool 4, encoded a CAR upstream of the chimeric polypeptide candidate that was structurally identical to the CAR used in pool 1 (example 11).

The ectodomains used in pools 3 and 4 are c-Jun variants comprising the leucine zipper motif known as the dimeric motif (NM-002228-3) (see, e.g., Chinenov and Kerppola, oncogene, 2001, 30.4; 20(19): 2438-52). A spacer having between 0 and 4 alanine residues comprises a candidate chimeric polypeptide between the c-Jun ectodomain and the transmembrane domain. Without being limited by theory, the sirnas can influence signaling of an intracellular domain linked to a leucine extracellular domain via a transmembrane domain by altering the orientation of the linked transmembrane and/or intracellular domain.

Candidate transmembrane domains are selected from single transmembrane domains of known wild-type and mutant type I receptors, such as interleukins, hormones, co-stimulatory or activating receptors which have been reported to be constitutively active when expressed in at least some cell types.mutations in such receptor mutants selected from pool 3 and pool 4 occur in transmembrane regions.exemplary transmembrane domains of pools 3A, 3B, 3.1A, 3.1B, 4B and 4.1B can be identified in tables 13 to 18. the headings of tables 13 to 18 are as follows.blockaksequence is the code for the P1, P2, P3 and P85 4 domains from table 7 used in each construct; normd _ D7 is the normalized count at day 7; Enrich x is the enrichment on day XX compared to day 7, wherein XX is the number indicated in the table, e.g. Enrich _ D21 is the enrichment on day 21 compared to day 7 and enr _ D35 is the enrichment on day 35 compared to day 7, wherein XX is the day 2 is the enrichment of the construct as shown in table 7, see the same table where P637 is included in the first and the intracellular domain of this construct (see P-B) which is shown in the publication under the same table where P637, the publication No. seq id No. 7, No. 7 is included in the publication No. 7 (see P-7).

A total of 5 possible conceptual combinations were designed based on the Jun leucine zipper ectodomain and 0-4 threonine spacers separating it from 82 different transmembrane domains, 81 potential P3 domains, one of which was a 23 amino acid spacer, and 21 potential P4 domains, one of which was a stop codon. Not all conceptualized constructs were detected after 7 days of viral vector assembly, lentiviral particle production, transduction of PBMC, and culture. For pool 3A and pool 3B, 68,951 constructs were present after PBMC transduction and 7 days of culture. For pool 4B, 50,652 constructs were present after PBMC transduction and 7 days of culture. Detailed information about the analyzed candidates can be determined from table 7 and tables 13 to 18. The coding system of the constructs was the same as explained in example 11, as set forth in the preceding paragraphs.

For pools 3A, 3B, 3.1A, 3.1B, 4B and 4.1B, constructs that promote PBMC cell proliferation were identified between day 7 and the last day indicated on the table some polypeptides in the specific module of C L E were between the best hits in pools 3A, 3B and 4 these C L E promoted proliferation to the maximum extent, for example, CD40 or ICOS at position P2, MP L, MyD88, L EPR or IFNAR2 at position P3, and/or exemplary constructs containing CD40, CD79B or CD27 at position P4 were the first 5 most common chimeric polypeptides (see tables 13, 14 and 17) in at least 2 of the 3 pools 3A, 3B and 4B, with MP L being the most common chimeric polypeptide at the interval P3 for all 3 pools (see tables 13, 14 and 17) and MP L being the most common polypeptide from the best chimeric polypeptides (see P358, MP L) and IFNAR 73748 among these.

In pool 3A and pool 3B comprising constructs encoding chimeric polypeptides and CARs and transduced PBMCs supplemented with (pool 3A) or without (pool 3B) fresh untransduced PBMCs, 126 and 127 optimal candidates (see tables 13 and 14) that promote PBMC proliferation between day 7 and the last day indicated on the tables were identified when cultured in the absence of I L-2, respectively, the optimal candidate lists of tables 13 and 14 were generated by combining the first 100 most prevalent candidates on day 21 based on an initial middle term data analysis with the first 100 most enriched candidate constructs between day 21 and day 7 based on an initial middle term data analysis, the initial middle term data was generated by coding the portion of the encoded library such that 66 of the first 100 constructs and 538 of the first 1000 constructs were decoded for pool 3A, and 77 of the first 100 constructs and 464 of the first 1000 constructs were decoded for pool 3B.

For pool 3A, when considering the data for all constructs with transmembrane domains from that gene in combination (data not shown), transmembrane domains (P2) or mutants thereof from CD40, CD8B, CR L F2, CSF2RA, FCGR2C, ICOS, IFNAR1, IFNGR1, I L RB, I L R1, I L RAP, I L RA, L EPR and PR L R are known to promote signaling activity in certain cell types (if such mutants are present in the constructs provided in the table), to promote proliferation of PBMCs between day 7 and day 21. this conclusion is based on the enrichment of the sequence counts of the constructs in the PBMC cell population in mixed culture, when the results for all constructs of one gene are combined, such that for pool 3A, the normalized count at day 21 plus the normalized count between one and day 7 plus the log CSF count plus the log ratio is calculated as the ratio of those for the bottom of the constructs found in table 583A, CD 463B 5, or mutant as the bottom of the enrichment of the cell type reported as enriched pool 3B 463, CD 463B, CD 463, CD2, and/or rpa, and/or rpb 463, or rpb, which are reported as the same.

For pool 3A, when promotion of PBMC proliferation is found at the first endodomain position (P3) of the candidate chimeric polypeptide modules herein between days 7 and 21, when data from all constructs with the first endodomain from that gene are considered in combination (data not shown), the first endodomain (P3) from I L RD, I L RE, I L1 RAP, I L R and MP L, or mutants thereof, are known to promote signaling activity in certain cell types (if such mutants are present in the constructs provided in the table), this conclusion is based on enrichment of sequence counts of the constructs in a mixed culture PBMC cell population, when the results for all constructs of one gene are combined, such that the enrichment calculated on a 2-base logarithm of the ratio of normalized counts on days 21 plus one and normalized counts on days 7 for pool 3A is at least 2 for pool 3B, the results for certain cell types known to promote signaling activity in certain cell types are found in the first endodomain position (P3) and/or mutants thereof are known to promote signaling activity in the first endodomain in the cell type of cells of the cell type (if such a 351 and/or mutant is present in the mutant in the first endodomain of the same time of the cell type given in the series B3513 and/or the same type of the series of the same.

For pool 3A, when PBMC proliferation was found to be promoted at the second endodomain position (P4) of the candidate chimeric polypeptide modules herein between days 7 and 21, the second endodomain (P4) from CD27, CD3G, CD40 and CD79B, or mutants thereof, were known to promote signaling activity in certain cell types if such mutants were present in the table, when data for all constructs with the second endodomain from that gene were considered in combination (data not shown). This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed culture PBMC cell population, when the results for all constructs of one gene are combined, such that the enrichment calculated as the log base 2 of the ratio between the normalized counts at day 21 plus one and the normalized counts at day 7 plus one for pool 3A is at least 2. For pool 3B, the second endodomain from CD40 or a mutant thereof (if such mutants are present in the table) known to promote signaling activity in certain cell types is best expressed when analyzed in this manner. Examples of the second endodomain, or portion thereof, and/or mutants present in the constructs that promote the greatest degree of cell proliferation for pools 3A and 3B at days 35 and 21, respectively, are provided in tables 13 and 14. Examples of the second endodomain, or portions thereof and/or mutants, present in the constructs promoting maximal cell proliferation in the repeat screens referred to as pools 3.1A and 3.1B are provided in tables 15 and 16.

With respect to specific genes, of the 798 constructs with CD 8(CD 8 molecule) as the P moiety, 111 positive, defined as constructs with enrichment greater than two (log) for library 3A or 3B at day 21, of the constructs with CD (CD molecule) as the P moiety, 159 positive, of the constructs with CR F (interleukin receptor-like factor 2) as the P moiety, 175 positive, of the 593 constructs with CSF2 (colony stimulating factor 2 receptor subunit) as the P moiety, 108 positive, of the 714 RB constructs with FCGR2 (Fc fragment of IgG receptor IIc (gene/pseudogene)) as the P moiety, 88 positive, of the 788 constructs with ICOS (inducible T cell co-stimulator) as the P moiety, 108 positive, of the constructs with ifr (interferon and receptor subunit 1) as the P moiety, of the ifr (interferon and receptor subunit 1), of the positive constructs with ifr (interferon and receptor subunit 1) as the P moiety, of the ifr (interferon and receptor subunit 1) as the P moiety, of the 99 positive, of the constructs with ifr, 18 positive, of the prr receptor subunit, of the prr, 18 positive, of the prr, prr.

Of the 1,259 constructs with I L1 RAP (interleukin 1 receptor accessory protein) as part of P3, 158 positive, defined as constructs with an enrichment greater than two for either pool 3A or 3B at day 21, of the 200 constructs with I L17 RD (interleukin 17 receptor D) as part of P3, 31 positive, of the 11 constructs with I L17 RE (interleukin 17 receptor E) as the P3 module, 3 positive, of the 538 constructs with I L23R (interleukin 23 receptor) as the P3 module, 86 positive, of the 1,531 constructs with MP L (MP L protooncogene, thrombopoietin receptor) as the P3 module, 509 positive.

Of the 3,124 constructs with CD3G (CD3g molecule) as the P4 moiety, 458 positive, defined as constructs with an enrichment greater than two for pool 3A or 3B at day 21. Of the 3,293 constructs with CD27(CD27 molecule) as the P4 moiety, 443 were positive. Of the 5,163 constructs with CD40(CD40 molecule) as the P4 module, 724 were positive. Of the 4,486 constructs with CD79B (CD79b molecule) as the P4 module, 663 were positive. Notably, for any construct, as this is a competitive assay, "negative" means that the construct does not compete in promoting proliferation, and the non-construct is not capable of promoting proliferation.

For purposes of repeat screening, constructs with particularly noteworthy enrichment were those with a log greater than 2 in both screens repeated₂Those of the value of ((normalized count data on last day + 1)/(normalized count data on day 7 + 1)))Bodies, as listed in tables 21-24 for pools 3 and 4 duplicate pools. For libraries 3A and 3.1A, constructs E008/E013-T041-S186-S050, E006/E011-T077-S186-S211, E007/E012-T021-S186-S051, E009/E014-T041-S186-S053, E007/E012-T073-S186-053, E006/E011-T017-S186-S051, E006/E011-T031-S186-S211, E006/E011-T011-S186-S050, E006/E011-T011-S186-S047, E007/E012-T001-S186-S, E006/E011-T041-S186-S051, E008/E-T028-S186, E009/E014-T023-S023, E006/E011-S186-S04216, E014-S186-S199, E006-S186-S046, E014-S199, E011-S023-S186-S04216, E007/E012-T006-S058-S051, E009/E014-T076-S186-S211 and E007/E012-T001-S186-S047 all had particularly noteworthy enrichments in both screenings, with portions P1 spaced by slashes herein comprising the different labels of libraries 3A and 3.1A as shown in tables 7, 13 and 15.

Additional information about the first and second endodomains in constructs with particularly noteworthy enrichments in the screen of both library 3A and library 3.1A is provided in table 22, including the genes from which the first and second endodomains are derived, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif when a construct with endodomains from MP L, OSMR or CSF2RA is present in the first endodomains (P3), and a construct with endodomains from CD40, TNFRSF4, CD79B, CD27, FCGR2A or TNFRSF18 is present in the second endodomains (P4), particularly noteworthy enrichments (table 22) are shown in both libraries 3A and 3.1A when a construct with endodomains from cellular receptors MP L, OSMR 2 or TNFRSF 2 is present in the second endodomains (P3527), particularly noteworthy enrichments from ifsa 3A construct with intracellular endodomains from cellular receptors L, OSMR 2 or tnfr 2 are present in second endodomains from table 3A 3, particularly noteworthy enriching construct with two ITAM 3A (P638) and CD 9 when a construct with intracellular domains from tnfa 3, CD 9, CD 3A, CD 9, CD2, CD 3A, CD 9, CD 3.1b 3b 3.3, CD 9, and a construct with intracellular domains containing intracellular domains from tnfr 27, a construct with an ITAM domain containing.

For libraries 3B and 3.1B, the constructs E007/E012-T017-S186-S051, E007/E012-T073-S186-S053, E008/E013-T028-S186-S047, E006/E011-T011-S186-S047, E007/E012-T082-S176-S214, E006/E011-T046-S186-S052、E008/E013-T029-S186-S052、E009/E014-T011-S186-S053、E008/E013-T032-S186-S039、E007/E012-T034-S186-S051、E007/E012-T041-S192-S213、E006/E011-T014-S069-S213、E006/E011-T022-S186-S053、E006/E011-T023-S115-S075、E006/E011-T029-S106-S213、E006/E011-T032-S155-S080、E006/E011-T041-S186-S216、E006/E011-T057-S135-S080、E006/E011-T072-S191-X002、E006/E011-T077-S186-S216、E006/E011-T080-S141-S080、E007/E012-T001-X001-S214、E007/E012-T007-S059-S211、E007/E012-T016-S186-S052、E007/E012-T031-S186-S053、E007/E012-T044-S102-S052、E007/E012-T044-S142-X002、E007/E012-T055-S069-S053、E007/E012-T063-S176-S216、E007/E012-T065-S157-S075、E008/E013-T008-S085-X002、E008/E013-T011-S085-S048、E008/E013-T021-S109-X002、E008/E013-T021-S168-S211、E008/E013-T032-S064-S214、E008/E013-T037-S170-S215、E008/E013-T038-S176-S048、E008/E013-T039-S137-S216、E008/E013-T041-S141-S053、E008/E013-T045-S177-S048、E008/E013-T048-S109-S074、E008/E013-T073-S199-S075、E009/E014-T001-S157-S074、E009/E014-T005-S196-S049、E009/E014-T011-S130-X002、E009/E014-T013-S155-X002、E009/E014-T017-S186-S076、E009/E014-T021-S142-S080、E009/E014-T023-S082-S076、E009/E014-T038-S196-S037、E009/E014-T055-S186-S052、E009/E014-T060-S175-S053、E009/E014-T070-S085-S212、E008/E013-T026-S054-S213、E009/E014-T007-S120-S053、E007/E012-T045-S186-S211、E008/E013-T073-S186-X002、E008/E013-T074-S186-X002、E007/E012-T055-S186-S053、E008/E013-T036-S186-S053、E007/E012-T017-S058-S053、E008/E013-T030-S189-S080、E006/E011-T029-S081-S047、E009/E014-T044-S194-S050、E006/E011-T028-S121-X002、E008/E013-T028-S186-S053、E009/E014-T078-S142-S213、E009/E014-T041-S186-S051、E008/E013-T006-S186-S050、E006/E011-T028-S186-S075、E006/E011-T040-S120-S038、E007/E012-T044-S115-S211、E009/E014-T039-S176-S075、E007/E012-T028-S186-S050、E008/E013-T031-S202-S050、E007/E012-T072-S192-S053、E006/E011-T065-X001-S051、E007/E012-T030-S062-X002、E007/E012-T073-S186-X002、E009/E014-T056-S186-S053、E008/E013-T046-S137-X002、E006/E011-T016-S136-S076, E007/E012-T032-S142-S037, E007/E012-T065-S120-S215, E009/E014-T077-S186-S047, E009/E014-T001-S126-S051, E006/E011-T030-S121-S039, E008/E013-T006-S176-S213, E009/E014-T032-S130-S215, E008/E013-T041-S186-S039, E009/E014-T021-S186-S047, E008/E013-T026-S137-S214, E007/E012-T029-S116-S075, E/E008-T026-S106-S049 and E012-S075 have two particularly remarkable screening degrees in enrichment, where the portions of P1 separated by the slashes herein include the different labels of libraries 3B and 3.1B as shown in tables 7, 14 and 16. Constructs with particularly noteworthy enrichment for the purpose of repeat screening were log₂((normalized count data on last day + 1)/(normalized count data on day 7 +1))

Further information about the first and second intracellular domains in the constructs with particularly noteworthy enrichment in both library 3B and library 3.1B is provided in table 23, including genes derived from the first and second intracellular domains, whether the first and/or second intracellular domain is an interleukin receptor, and whether the first and/or second intracellular domain has at least one ITAM motif from MP L, L EPR, MYD L, EPOR, I L RA, I L RG, I L RAP, I L gr, I L RA L, CSF 2L, I L RB, I L R L, I L RA 713, I L RA 820RB, IFNGR L, I3693 RA, I L RA, CSF3 CSF 72, I36031 RB, I L RB, I L RA L, I L RB, I L RA L, I L, when the constructs with two CD3, CD 72, CD3, CD 72, CD3, CD 72, CD3, a, CD 72, CD3, a CD L, CD3, CD L, CD3, a CD L, a CD3, CD L, CD3, a CD L, a CD L, a CD L, a CD L, CD 3675, a CD L, a CD L, CD 3675, CD L.

FACs analysis showed that pool 3A expanded cells on day 49 were predominantly CD3+, CD8+, CD56+ and F L AG-Tag +. the table immediately below shows the percentage of lymphocytes that stained positive for the indicated markers and marker combinations.

Table 4 percentage of lymphocytes staining positive for the indicated markers and marker combinations.

For pool 4B, which included constructs encoding chimeric polypeptides without CAR and transduced PBMCs not supplemented with fresh untransduced PBMCs, 154 optimal candidates that promoted maximal PBMC proliferation between day 7 and the last day indicated on the table were identified when cultured in the absence of I L-2 (see table 17).

For pool 4B, when promotion of cell proliferation of PBMCs was found at the transmembrane domain position (P2) of the candidate chimeric polypeptide modules herein between day 7 and the last day indicated on the table, the transmembrane domains (P2) from CD40, CD8B, CSF2RA, I L18R 1, and I L3 RA, or mutants thereof, when considered in combination with data for all constructs with a transmembrane domain from that gene, are known to promote constitutive signaling activity in certain cell types (if such mutants are present in the constructs provided in the table), this conclusion is based on the enrichment of sequence counts of constructs in the mixed culture PBMC cell population, such that when the results of all constructs are combined for one gene, the enrichment calculated as a base 2 logarithm of the ratio of the normalized counts of the last day plus one and the normalized counts of day 7 for pool 4B, the fraction of the transmembrane domains present in pool 4B, or the fraction of transmembrane domains thereof, is referred to as a transmembrane domain promoter, or mutant, which is present in pool 4B, is referred to as a transmembrane domain promoter, or a mutant, which is present in pool 4B, or a fraction of the list, is referred to as a mutant, or a mutant, which is provided in table, or a fraction of a mutant, in table, or a fraction of a table, 1.

For pool 4B, when promotion of cell proliferation of PBMC was found at the first endodomain position (P3) of the candidate chimeric polypeptide modules herein between day 7 and the last day indicated on the table, the first endodomain (P3) from CR L F2, CSF2RA, IFNGR2, I L4R, MP L and OSMR, or mutants thereof, when considered in combination to have data from all constructs from the first endodomain of that gene, are known to promote signaling activity in certain cell types (if such mutants are present in the constructs provided in the table), this conclusion is based on enrichment of sequence counts of the constructs in a mixed culture PBMC cell population, when the results for all constructs of one gene are combined such that for pool 4B, the enrichment calculated as log base 2 in the ratio between the normalized count for the last day indicated on the table plus one and the normalized count for day 7 plus one is greater than 0, the first endodomain in pool 4B and the mutant in the construct providing a proliferation promoting or mutant in the first endodomain in the pool 4B, or the mutant in the example of table 1.

For pool 4, when promoting PBMC proliferation was found at the second endodomain position (P4) of the candidate chimeric polypeptide modules herein between day 7 and the last day indicated on the table, the second endodomain (P4) from CD27, CD40 and CD79B, or mutants thereof, were known to promote signaling activity in certain cell types if such mutants were present in the constructs provided in the table, when data for all constructs with the second endodomain from that gene were considered in combination. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed culture PBMC cell population, when the results for all constructs of one gene are combined, such that for pool 4B, this domain count is present on the last day more than on day 7. Examples of second endodomains or portions and/or mutants thereof present in the constructs that promote cell proliferation in library 4B are provided in table 17. Examples of second endodomains or portions and/or mutants thereof present in constructs that promote cell proliferation in a repeat screen called pool 4.1B are provided in table 18.

For libraries 4B and 4.1B, constructs E007/E012-T078-S154-S047, E008/E013-T062-S186-X002, E008/E013-T055-S186-S050, E009/E014-T057-S186-S050, E007/E012-T077-S054, E007/E012-T034-S135-S211, E009/E014-T071-X001-S216, E009/E014-T011-S141-S037, E008/E013-T041-S186-S037, E006/E011-T038-S106-S039, E006/E011-T011-S121-X002, E007/E-T007-S085-S215, E006/E06-T008-S011-S186-S011, E I-S012-S011, E007-S186-S011, E006/E011-T041-S186-S047, E008/E013-T045-S186-S051, E008/E013-T003-S104-S216, E006/E011-T019-S186-S053, E008/E013-T071-S064-S080, E006/E011-T021-S054-X002, E006/E011-T003-S135-X002, E009/E014-T020-S199-S213, E008/E013-T027-S121-S211, E009/E014-T032-S195-X002, E009/E014-T050-S171-X002, E008/E013-T069-S002, E008/E-T026-S038, E014-S038-S014-S037, E014-S057-S012-S057, E008/E072-S057, E072-S057, E008/E013-T046-S142-S080, E006/E011-T065-S186-S076, E006/E011-T062-S069-X002, E007/E012-T047-S098-X002, E009/E014-T069-S099-S048, E008/E013-T039-S141-S050, E006/E011-T052-S130-S052, E008/E013-T041-S186-X002, E007/E012-T019-S120-X002, E008/E013-T045-S186-S053, E006/E011-T003-S170-S039, E007/E012-T047-S8-S051, E007/E013-T069-S06109-S014, E011-T047-S011-S048-S039, E007/E012-S047-S051-S011-S054, E011-S053 and E011-S011, wherein the two sieves are particularly suitable for enriching the strains of the strains, where the portions of P1 separated by the slashes herein include the different labels of libraries 34B and 4.1B as shown in tables 7, 17 and 18. Construction with particularly noteworthy enrichment for the purpose of repeated screeningThe body is of log₂Those constructs having a value of greater than 2 (normalized count data on the last day + 1)/(normalized count data on day 7 + 1).

Further information about the first and second endodomains in constructs with particularly noteworthy enrichment in the screening of both Bank 4B and Bank 4.1B is provided in Table 24, including the first and second endodomains-derived genes, whether the first and/or second endodomains are interleukin receptors, and whether the first and/or second endodomains have at least one ITAM motif, constructs with endodomains from I18R, MP, CR 0F, I111 RA, I213 RA, I32 RG, I47, CSF3, I52 RA, OSMR, MYD, I631 RA, IFNAR, I76, I2, I RA, RB, I91R, I10 RB or I03 RA are present in the first endodomains (GR), and constructs with GR from CD, CD79, TNFRSF, CD3, CD, FCF, FCOS, 2, CSF2, GR, CSF2 GR, and when the constructs with GR from the first endodomains of I18R, MP, CR 0F, I111 RA, I213 RA, I47 GR, and when constructs with GR from the two endodomains from the ITFR, CD79, CD3, CD79, CD3, CD79, CD3, III.

For pool 4B, of the 572 constructs with CD8B (CD8B molecule) as the P2 moiety, 21 were positive, defined as constructs with enrichment greater than two for pool 4B, of the 897 constructs with CD40(CD40 molecule) as the P2 module, 50 were positive, of the 551 constructs with CSF2RA (community stimulating factor 2 receptor α subunit) as the P2 module, 25 were positive, of the 732 constructs with I L3 RA (interleukin 3 receptor subunit α) as the P2 module, 47 were positive, of the 738 constructs with I L18R 1 (interleukin 18 receptor 1) as the P2 module, 44 were positive, of the 738 constructs with MP L (MP L protooncogene, platelet receptor) as the P3 module, 305 were positive constructs of the 1,683 positive constructs with P3 module.

In the initial metaphase analysis of pool 3A on day 21, 66 of the first 100 hits and 538 of the first 1000 hits were decoded. Further coding provides deep coding and identification of 99 of the first 100 hits and 992 of the first 1000 hits of library 3A and data was collected on day 35 and analysis was updated on day 21. In the initial analysis of pool 3B on day 21, 77 of the first 100 hits and 464 of the first 1000 hits had been decoded in the initial metaphase analysis. Further coding provides depth coding and identification of the first 100 hits and 708 of the first 1000 hits for bank 3B. Optimal constructs for libraries 3A and 3B are shown and in tables 13 and 14, respectively, along with day 35 data for library 3A and day 21 data for library 3B.

In pool 3A and pool 3B comprising constructs encoding chimeric polypeptides and CARs and transduced PBMCs supplemented with (pool 3A) or without (pool 3B) fresh untransduced PBMCs, 124 optimal candidates for pool 3A on day 35 that promote the greatest degree of PBMC proliferation between day 7 and the last day indicated on the tables were identified when cultured in the absence of I L-2 (i.e., no I L-2 was added to the culture medium during the culture period after initial transduction), and 131 optimal candidates for pool 3B on day 21 (see tables 13 and 14, respectively) were identified, the optimal candidate lists of table 13 (day 35 pool 3A) and table 14 (day 21 pool 3B) were enriched by combining the 100 most prevalent candidates before day 21 (pool 3B) or day 21 or 35 (pool 3A) with the constructs between day 21 and day 7 (pool 3B) or day 7 and day 3A day 100 before day 21 or 35 (pool 3A) after deep decoding.

In particular modules of C E, as seen in the table of best hits using deep decoding results, certain polypeptides are among the best hits at least one of pools 3A and 3B and at least one of the time points of day 21 and day 35 (pool 3A) or day 21 (pool 3B), for example, exemplary constructs containing, among the first 5 most common chimeric polypeptides (see tables 13 and 14), CD (specifically, for example, a transmembrane domain having the codon T011), ICOS (specifically, for example, a transmembrane domain having the codon T028), FCGR2 (specifically, for example, a transmembrane domain having the codon T024), PR R (specifically, for example, a transmembrane domain having the codon T077), I03 RA (specifically, for example, a transmembrane domain having the codon T041), I16 ST (specifically, for example, a transmembrane domain having the codon T045), in P position, I18R (specifically, for example, a transmembrane domain having the codon T077), I18R (specifically, for example, a transmembrane domain having the codon T041), I16 ST (specifically, for example, a transmembrane domain having the codon T045), I18R (specifically, for example, a transmembrane domain having the codon T03p × 52), or for a specific intracellular domain having the coding sequence of tnfs, such as well as for example, a domain having the coding sequence of tnfs equivalent coding sequence of a coding sequence, such as a sequence of a sequence.

The additional observations noted P2_ CD40, P2_ ICOS, P3_ MP L, P4_ CD40, and P4_ CD79B, which are shown as the most common portions in each of tables 13 and 14, P4_ CD27, which is shown as the most common portion in tables 13 and 14, CSF2RB, which occurred 4 times in the first 123 most enriched and repeated the most at P2, but mutation V449E did not occur in these optimal hits (table 13), thus, in some embodiments herein, the chimeric polypeptide comprises the transmembrane domain constructs identified in any of the ones shown in tables 13 and 14, except for the transmembrane domain mutation V449E of CSF2RB, eight MyD88 variants, which occurred at least once in the P3 positions in the first 123 of library 3A (table 13), the two CD40 variants tested in the library were shown at least once in the first 123 of the library 3A list of P3 constructs (table 13) and P8653 in the first library 123 of the list of P8227 and P4.

For gene analysis of the gene, for pool 3A, a mutant from CD4, CD8B, CD40, CR L F2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, I L1R 1, I L01 RAP, I L12 RG, I L23 RA, I L35 RA, I L46 ST, I L RA, I L RB 610RB, I L RA L, I L RA, I L RB 17, I L RC, I L RE, I L R L, I L18, I L RA, I L R L, I L PR 72RA, I L PR 72, and/R72 a mutant from this group, when the expression of the mutant in a cell type is calculated as a cross-membrane-proliferation promoting ratio between the last cell population of the number of the cell population of the expression of the cell type (or the cell line of which has been enriched with the cell line or the cell line of the line of.

For pool 3A, the first intracellular domain (P3) from CSF2RA, IFNAR1, I L1 RAP, I L4R, I L ST, I L111 RA, I L RB2, I L RA, I L RD, I L RE, I L R1, I L R, I L R, MP L and MyD88, or a mutant thereof, known to promote signaling activity in certain cell types (if such mutant is present in the constructs provided in the table), promotes PBMC proliferation between day 7 and the last day indicated on the table when found at the first intracellular domain position of the candidate chimeric polypeptide module herein (P3), this conclusion is based on the enrichment of the sequence counts of the constructs in the PBMC population cultured mixed cells, such that when the results for a set of constructs are combined, the results for all constructs are present in table 3A, the results for a set of constructs are calculated as the ratio of the first intracellular domain counts in table 583A 1, the last intracellular domain of the construct of table plus the mutant when the results are present in table 3B 2, the results are calculated as the ratio of the first intracellular counts in the first intracellular domain of the normalized cell type of the library 3, and/or the mutant is calculated as the ratio of the second intracellular domain of the second intracellular signal activity of the second mutant when the second intracellular domain of the second cell type of the same.

For pool 3A, the second intracellular domains from CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4 and TNFRSF8 (P4) or mutants thereof (if such mutants are present in the constructs provided in the tables) that are known to promote signaling activity in certain cell types promote PBMC proliferation between day 7 and day 21 or 7 and the last day indicated on the tables when found at the second intracellular domain position (P4) of the candidate chimeric polypeptide modules herein, when the data for all constructs with the second intracellular domain from that gene are considered in combination. This conclusion is based on enrichment of the sequence counts of the constructs in the mixed culture PBMC cell population such that when the results of all constructs are combined for one gene, the enrichment calculated as the log base 2 of the ratio between the normalized counts on the last day plus one and the normalized counts on day 7 plus one indicated on the table is at least 2 for pool 3A. For pool 3B, the second endodomain from CD40 or a mutant thereof (if such mutants are present in the table) known to promote signaling activity in certain cell types, although not achieving a 2-fold log2 enrichment, was best expressed when analyzed in this manner. Examples of second endodomains, or portions thereof and/or mutants present in constructs that promote maximal cell proliferation, for pools 3A and 3B are provided in tables 13 and 14. Examples of the second endodomain, or portions thereof, and/or mutants present in the constructs promoting cell proliferation in the repeat screens referred to as pools 3.1A and 3.1B are provided in tables 15 and 16.

EXAMPLE 13 characterization of Individual lymphoproliferative elements

In this example, the selected chimeric lymphoproliferative component (C L E) identified in the bank screens described in examples 11 and 12 was evaluated separately to further analyze the C L E identified in the bank 2B screen, activated PBMCs were transduced overnight with lentiviral particles encoding C L E alone and cultured in the absence of exogenous interleukins to further analyze the C L E identified in the bank 3A screen, activated PBMCs were transduced overnight with lentiviral particles encoding C L E flanked at the 5' end with anti-CD 19CAR, and donor-matched CD19+ B cells were added to the culture every 7 days in the presence, but cultured in the absence of exogenous interleukins.

The recombinant retroviral particles were in 293T cells (L enti-X)^TM293T, Clontech), which is adapted to Freestyle^TM293 expression Medium (ThermoFisher Scientific) was cultured in suspension in serum-free, chemically defined medium. As explained in example 4, the use of a plasmid with a genome and 3 lentimorbidities encoding gag/pol, revThe selected C E identified in the library screening assays described in examples 11 and 12 are regenerated from their portions (P-2, P and P or P, P and P) respectively and inserted into the same transgene expression cassette as their first identified library, the modular portions of each of the selected C E are shown in fig. 19 and 20 and the gene names and amino acid sequences are shown in table 7 fig. 17 and 15 show cassettes of library 2 and library 3 respectively, cells transduced with lentiviral particles containing constructs identified by the library screening assay are referred to as "D1" immediately following library number, for example, cells transduced with lentiviral particles containing constructs from library 2 are referred to as D32. thus, C7E with the prefixes "D42" and "D53" are constructed as in the non-transmembrane cassettes shown in fig. 17 and 15 as "CD 5" and "DC-6" comprise the same transgene expression cassette encoding PBMC 1-7C 7-4, as shown in fig. 17 and 15, and the non-carboxy-terminal human C7-C-7C-5-7C-5 (CD-5) and non-4-C-7-5-4-C-5-7-5-7-4-C5-4-5-4-6-5-7-4-5-4-5-4-5-7-5-7-4-5-4-7-5-4-6-5-4-6-5-6-7-5-4-5-7-5-4-6-5-6-5-4-6-4-6-5-6-7-5-6-4-6-7-4-6-7-5-6-4-5-6-7-4-6-4-6-4-7-6-5-6-5-6-5-7-6.

The constructs from pool 2B used in this example were D L2-1 (M024-S190-S047), D L2-2 (M025-S050-S197), D L2-3 (M036-S170-S047), D L2-4 (M012-S045-S048), D L2-5 (M049-S194-S064), D L2-6 (M025-S190-S050), and D L2-7 (M025-S190-S051).

The constructs from library 3A used in this example were D L3A-1 (E013-T047-S158-S080), D L3A-2 (E011-T024-S194-S039), D L3A-3 (E014-T040-S135-S076), D L3A-4 (E013-T041-S186-S051), D L3A-5 (E013-T064-S058-S212), D L3A-6 (E013-T028-S186-S051), D L3A-7 (E014-T015-S186-S051), D L3A-8 (E011-T016-S186-S051), D L3A 3-9 (E011-T073-S186-S050) and D L3A-10 (E011-T863-S186-S013).

On day 0, according to the manufacturer's instructions, by using Ficoll-Paque

(GE Healthcare L ife Sciences) density gradient centrifugation followed by lysis of erythrocytes from buffy coat (San Diego Blood Bank) enriched PBMC 1.5 × 10⁶PBMCs were treated in a 3ml complete OpTsizer supplemented with 100IU/ml (I L-2) and 50ng/ml anti-CD 3 antibody (317326, Biolegend)^TMCTS^TMT cell expansion SFM was seeded in each well of G-Rex 6 well plates (WilsonWolf, 80240M) to activate PBMCs for virus transduction. At 37 ℃ and 5% CO₂Following overnight lower transduction, lentiviral particles encoding the constructs described above were added directly to activated PBMCs at an MOI of 5 and at 37 ℃ and 5% CO₂Then, the mixture is cultivated overnight. The following day, use the complete OpTsizer^TMCTS^TMT cells expand SFM to reach a medium volume of 30ml in each well and the dish is returned to the incubator, where or following cell culture steps are not added I L-2, I L-7, or other exogenous interleukins.cells from each well are collected on day 7 to determine cell number, percent survival, and percent transduced cells, defined as the percentage of F L AG-Tag + or E-Tag + cells by FACS analysis^TMCTS^TMT cells were expanded in SFM and 0.5 × 10 of each sample was added⁶Cells were completely OpTsizer at 30ml^TMCTS^TMT cell expansion SFM reseeded into wells of G-Rex 6 well plates previously will contain 0.5 × 10⁶Cold-stored day 0 donor-matched PBMCs of individual CD19+ B cells (as determined by FACS on day 0) were added to the "D L3A-" culture to provide CD19+ B cell activation of CD19 ASTR, harvest on day 35This procedure was repeated on days 14, 20 and 28 before the cells.

Results

PBMCs transduced with lentiviral particles encoding C L E were cultured for 35 days in the absence of exogenous interleukins proliferation is expressed as fold expansion calculated by dividing the total number of cells by the number of cells inoculated at that time point PBMCs transduced with each of the individually selected C L E shown in fig. 19 were proliferated more than negative controls including non-transduced PBMC and PBMC transduced with vectors encoding eTag but not C L E (fig. 19) furthermore, the following C L E exhibited fold expansion greater than 23 at days 14, 21, 28 and 35, while for these time points the fold expansion of the negative controls was almost 0: D L-7, DC2-6, D L-6, D L-2, D L-1 and DC2-5, CAR was amplified with vectors encoding anti-CD 19 and different C L E (shown in fig. 20) transduced constructs and was transduced in the absence of fresh donor cells added every day but did not express more than the fold expansion of PBMC 3-transfected cells in the presence of exogenous C057, when the PBMC 35-transfected cells were proliferated with PBMC 35-transfected with PBMC 35E-expressing the vector encoding no more than the fold expansion of PBMC 20, when PBMC 20-transfected cells expressed as negative controls (fig. 20) and no PBMC 20, no PBMC 20-expressing no fold expansion of PBMC 20-3-expressing PBMC 20, no PBMC 20-expressing the fold expansion, no PBMC-expressing PBMC-3-9-3-expressing the fold expansion was expressed as fold expansion was shown by PBMC-9 when PBMC 9-expressing the fold expansion was analyzed by the fold expansion in PBMC-9-20-9-expressing PBMC-20S-20-expressing the vector expressing PBMC-20 (fig. C-20S-20-3S-3S-20-3S-3S-3-20, no-9-expressing the fold expansion and no-3 vector.

Example 14 expression of C L E to promote survival and/or expansion of PBMCs.

In this example, 14 unique recombinant lentiviral particles exposed to unstimulated human PBMC were individually assessed for their ability to promote transduction and subsequent survival and/or proliferation of PBMC when cultured in vitro for 21 days in the absence of exogenously added interleukins, the retroviral particles in this example were pseudotyped with VSV-G, expressing anti-CD 3 scfvcfc-GPI on their surface, and containing a transgene expression cassette encoding 1 of anti-CD 19CAR and 14 unique C L E.

Method of producing a composite material

In Freestyle^TM293 expression Medium (Thermo Fisher Scientific) 293T cells adapted to suspension culture in serum-free chemically defined Medium (L enti-X)^TM293T, Clontech) recombinant lentiviral particles, PEI transiently transfected cells with a genomic plasmid and a plasmid encoding gag/pol, rev, a pseudotyped plasmid encoding VSV-G and a plasmid encoding UCHT1scFvFc-GPI as described in example 4 the genomic plasmid comprises a Kozak sequence, a CD8 signal peptide, an F AG marker and anti-CD: CD3 CAR, T2 and C E, and subsequently a triple termination sequence as shown in FIG. 15. anti-CD deletion co-stimulatory domain 13 different C0 Es were identified as being more enriched or more throughout on day 35 in library 3.1.1A, and were re-generated by identifying 1C 1E from library 3A with its portions (P, P and P) D23.1A-1 (E-T006-S194-S211), D33.1A-062 (E-T062-S063-010), T-S3-S0310, S3-03, S-D-3-10S 3-D-3-10, S3-D-3-D-3-D-10, S-D-3-D-15, S-3-D-3-D-15, S-3-D-3-15, S-D-15, S-3-D-3-D-3-D-3-D-15, S-D15, S-3-D-3-D15, S-D-3-D-15, S-D-3-D-3-D-S-D3-D3-D-S-D-S-D3-15, S-D3.

On day 0, according to the manufacturer's instructions, by using Ficoll-Paque

(GEHealthcare Life Sciences) density gradient centrifugation followed by lysis of red Blood cells from leukopenia system (L RS-WBC) (San Diego Blood Bank) enriched PBMC.. No additional steps were taken to remove monocytes⁷Individual PBMCs were frozen for later use in feeding CD 19-CAR-expressing cells from each donor were also left for expression profiling by FACs the remaining PBMCs were diluted to 1.0 × 10 in intact OpTzerTM CTSTM T cell expanded SFM⁶Viable cells/ml, and 5ml were added to 50ml conical tubes per sample no anti-CD 3, anti-CD 28, I L-2, I L-7, or other exogenous interleukins were added prior to transduction to activate or otherwise stimulate lymphocytes, lentiviral particles were added directly to unstimulated PBMCs in conical tubes at an MOI of 1 and at 37 ℃ and 5% CO₂The cells were incubated for 4 hours. Cells were then washed 3 times in DPBS + 2% HSA and transferred to 30ml of intact OpTzerTM CTSTM T cell expansion SFM

Cells from each well were collected on day 7 to determine cell number, percent viability, and percent transduced cells, defined as the percentage of F L AG-Tag + cells analyzed by FACS using the lymphocyto gate

In the wells of a 6-well plate. Pre-refrigerated day 0 donor-matched PBMCs were added to each sample at a ratio of 1 transduced cell to 1 day 0 donor-matched CD19+ B cell (the percentage of CD19+ B cells in PBMCs was determined by FAC at day 0). Whole OpTmizer (TM) CTSTM T cell expansion SFM was then added to each well to bring the volume to 30ml, and at 37 ℃ and 5% CO₂Transduced cells were incubated. On day 14, 15ml of spent medium was replaced with fresh medium and additional day 0 PBMCs were added at the same 1:1 ratio. Cells were again assayed on days 14 and 21 to determine cell number, percent viability andexpression of FACS surface markers.

Results

The lentiviral particles in this example were pseudotyped with VSV-G, expressing anti-CD 3 scfvffc-GPI on their surface, and containing a transgene expression cassette encoding anti-CD 19CAR and 1 of 14 unique C L E prior to washing cells 3 ×, the lentivirus was exposed to unstimulated and presumably quiescent PBMC for 4 hours and cultured in the absence of any exogenously added interleukins analysis of PBMC at day 7 confirmed that each of the lentiviral particles comprising C L E and expressing anti-CD 3 scfvffc-GPI was effective in transducing PBMC, furthermore, analysis of the total number of CAR cells at days 7, 14 and 21 demonstrated that the in vitro presence of live CAR + CAR cells and expansion of transduced PBMC with transduced constructs encoding anti-CD 19 and C L E was better able to promote expansion of live CAR + CAR cells and/or the percentage of transduced PBMC cells between days 7 and 7 of C3 + 3-253 on days with transduced constructs (F1-3-253) displayed on days as well as the percentage of transduced PBMC on days 7, 14 and 21 days with transduced constructs indicated by the following table 3-253.

TABLE 5 cell count analysis.

As supported by the data shown in table 5, transduction of PBMCs with anti-CD 19 CARs and tested C L E provided improved expansion and/or survival at days 7, 14, and 21 compared to untransduced control cells or control cells transduced with lentiviral particles without C L E.

C L E, particularly notable in this example for its ability to promote survival and proliferation of transduced PBMCs, are D L3.1.1A-2 (E010-T073-S186-S211), D L3.1.1A-7 (E010-T024-S197-S214), D L3.1.1A-8 (E009-T072-S186-039), D L3.1.1A-12 (E009-T071-S186-S211), and D L3A-4 (E013-T041-S186-S051).

Example 15 Peripheral Blood Mononuclear Cell (PBMC) isolation, transduction and expansion.

The following examples illustrate the use of a closed system for ex vivo processing of PBMCs prior to in vivo expansion.As an example, 30ml to 200ml of human blood is drawn from an individual into a blood collection bag with an acid citrate dextrose solution (ACD) as an anticoagulant.alternatively, blood is drawn into a blood collection tube, syringe or equivalent and transferred into an empty blood collection bag or IV bag according to manufacturer's instructions, whole blood is treated on a Sepax 2 cell processing system (BioSafe) using a pure cell kit (catalog # CS-900.2, Omnimamed). Peripheral Blood Mononuclear Cells (PBMC) are collected into a culture bag or alternatively in a syringe.sterile aliquot collection is used for cell counting to determine the number of surviving cells.10 IU/ml to 300IU/ml I L-2 (catalog #202-I L-010, R < 25 >) with a final volume of up to 200ml&D Systems) will be present in 0.1 × 10 in 0.1-VIVO 15 (Cat #08-879H, L onza) or CTS OpTsizer cell expansion SFM (Cat # A1048501, Thermo Fisher scientific) medium⁶To 1.0 × 10⁶PBMC at final concentration of individual surviving cells/ml were transferred to G-Rex100MCS gas permeable cell culture system device (Wilson Wolf) in addition to I L-2, CTS immune cells SR (catalog # a2596101, Thermo Fisher Scientific) may be added to the culture medium.

PBMC isolated from peripheral blood were loaded onto PA LL PBMC filter, washed once through a filter with 10ml of AIM V (Thermo Fisher Scientific) or X-VIVO 15 medium, then perfused at 37 ℃ with 5 ml/hr with 10ml to 60ml of lentiviral stock solution (prepared as in example 2), then washed again with AIM V, CTS OpTsizer T cell amplification SFM or X-VIVO 15 medium containing recombinant human DNase (Pulmozyme, Gentech), then washed with DNase-free ringer lactate (catalog # L7500, Braun), then back-perfused into a syringe through a filter⁵To 1 × 10⁶One cell/kg) reinfusionInto the subject.

Depending on the riboswitch contained within the retroviral genome, individual nucleoside analog antiviral drugs or nucleoside analog antiviral prodrugs (acyclovir, valacyclovir, penciclovir, and famciclovir) are administered to an individual. Any therapeutically effective dose (such as 500mg of the nucleoside analog antiviral drug or prodrug) can be administered to the subject orally three times a day. Treatment with the nucleoside analog antiviral drug or prodrug preferably begins before reinfusion (such as 2 hours ago), and may also begin at or some time after reinfusion. Treatment may continue for at least 1,2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, 120 days or for 5, 6, 9, 12, 24, 36, or 48 months or longer. Treatment may include administration of the nucleoside analog antiviral drug or prodrug once, twice, three times, or four times daily. After reinfusion and treatment initiation, the number of infected cells was determined by using qPCR to quantify the amount of viral genome by blood count at day 2, day 5, day 7, day 11, day 13, day 18, day 28, and day 56 post reinfusion. An individual experiencing fever or an interleukin release syndrome may have the dose or frequency of the nucleoside analog antiviral drug or prodrug reduced or stopped. If infected T cells fail to expand 10,000 to 100,000 fold on day 18, the dose or frequency of the nucleoside analog antiviral drug or prodrug may be increased. Clinical response of an individual can be measured via FDG PET imaging and continuous CT scanning. The oral dosage of the nucleoside analog antiviral drug or prodrug can be reduced or stopped after an extended remission period or in the event that excess T cells spread beyond 30% of the total peripheral T cell count.

Example 16. conceptual demonstration of various illustrative methods provided herein, including in vivo expansion of genetically modified lymphocytes.

This example provides an exemplary method for transducing PBMCs (including T cells) ex vivo and expanding these transduced PBMCs (including T cells) in vivo. Such methods include illustrative 4 hour transduction methods with illustrative recombinant lentiviral particles expressing certain illustrative chimeric lymphoproliferative components. Furthermore, such methods provide additional exemplary methods for transducing PBMCs (which are typically T cells in illustrative embodiments) with replication-defective recombinant retroviral particles produced by transfecting packaging cells with vectors encoding various components of the replication-defective recombinant retroviral particles in suspension in serum-free chemically-defined media for 4 hours.

Materials and methods

The recombinant retroviral particles were in 293T cells (L enti-X)^TM293T, Clontech), which is adapted to Freestyle^TMPEI transient transfection cells were transiently transfected as explained in example 4 using 1 of 3 genomic plasmids (detailed below) and 3 separate packaging plasmids encoding gag/pol, rev, and pseudotyped plasmid encoding VSV-G to produce lentiviral particles also showing membrane-bound activating components, a fourth packaging plasmid co-transfected as described in example 4 encoding a membrane-bound polypeptide capable of binding to CD3(UCHT1 scfvffc-GPI) the genomic plasmid is a third generation lentiviral expression vector containing a deletion in 3' L TR that results in self-inactivation, wherein the plasmid encodes the following:

(1) anti-ROR 2MRB-CAR T2A: eTag (F1-1-27): this genomic plasmid is identical to that shown in FIG. 5, except that the sequence encoding anti-CD 19CAR was replaced with MRB-ASTR having scFv that recognize human ROR2, CD8 stem and transmembrane sequences (SEQ ID NO:75), CD137(SEQ ID NO:1) and CD3z (SEQ ID NO: 13). The recombinant lentiviral particle encoding this construct was designated F1-1-27

(2) Flag-anti-ROR 2MRB-CAR T2A: C L E (F1-1-228 and F1-1-228U) the genomic plasmid corresponds to the one shown in FIG. 5, with the difference that the sequence encoding anti-CD 19 is replaced by a Flag tag covalently linked to the anti-ROR 2MRB-CAR described in (1) above, and GMCSFR ss: eTag is replaced by C L E, the C L E is a type I transmembrane protein comprising an extracellular dimerization module (P1), a transmembrane module (P2) and 2 intracellular modules (P3 and P4), the gene in position P1-P2-P3-P4 is MycTag 2A Jun-I L RA-MP L-CD40, the E of these modules is E041-T186-S L-S051 coding for the most significant genes of the coding for the retrovirus vector DNA coding for the DNA coding for the gene for the protein for the gene on day III on day I639-C9-III and the gene for the coding

(3) Flag-anti-CD 19CAR T2A: C L E (F1-3-219 and F1-3-219U) the genomic plasmid is identical to the anti-human CD19CAR shown in figure 5, except that the Flag tag is inserted between CD8ss and the CAR and the sequence encoding GMCSFRss: eTag is replaced by C L E the C L E is a type I transmembrane protein comprising an extracellular and transmembrane moiety covalently linked via a flexible linker to its cognate interleukin receptor and covalently linked to the intracellular domain of a different interleukin receptor, in particular, from the amino to carboxy terminus the extracellular and transmembrane portions of the plasmid encoding I L-7 (SEQ ID NO:511), a flexible linker (SEQ ID NO:53), I L-7R α (CD127) (SEQ ID NO:513) and the extracellular and transmembrane portions of I L-2R β (CD122) (SEQ ID NO:514) and the recombinant fc domain of this dna encoding a recombinant fc-expressing a recombinant fc-F-219 particle encoding gpif-25-fcf-219 and showing that the retroviral construct encodes GPI-3-7-C-5.

PBMC isolation, overnight transduction of PBMC following ex vivo stimulation, followed by 15 days ex vivo expansion of engineered lymphocytes

On day 0, Ficoll-Pacque was used by density gradient centrifugation on a Sepax 2S-100 device (Biosafe; 14000) using the CS-900.2 kit (Biosafe; 1008) according to the manufacturer' S instructions^TM(General Electric) PBMC were isolated from ACD peripheral blood of healthy volunteers informed of consent 3.0 × 10⁷Viable PBMCs were inoculated on 1L G-Rex (Wilson-Wolf) and used a complete OpTsizer supplemented with 100IU/ml I L-2 (Novoprotein, GMP-CD66), 10ng/ml I L-7 (Novoprotein, GMP-CD47) and 50ng/ml anti-CD 3 antibody (OKT3, Novoprotein)^TMCTS^TMT cell amplification SFM brought the volume to 60ml to activate the PBMCs (which include T cells and NK cells) for viral transduction. At 37 ℃ and 5% CO₂After next overnight incubation, lentiviral particles encoding anti-ROR 2MRB-CAR, F1-1-27 were directly incubated at an MOI of 5(440ul)Added to activated PBMC and at 37 ℃ and 5% CO₂Then, the mixture is cultivated overnight. After overnight incubation, by using a complete OpTsizer supplemented with NAC (Sigma)^TMCTS^TMT cell amplification SFM to achieve a total volume of medium in G-Rex of 100ml to feed cells to increase final concentration by 10mM and 100IU/ml recombinant human I L-2 and 10ng/ml recombinant human I L-7 at 37 ℃ and 5% CO with addition of 100IU/ml recombinant human I L-2 and 10ng/ml recombinant human I L-7 solution every 48 hours₂The G-Rex devices were then incubated in a standard moist tissue incubator before harvest, cells were expanded at day 15, these transduced cells were washed in freezing medium (70% RPMI 1640, 20% heat-inactivated FBS, 10% DMSO) and washed at 5.0 × 10⁷Two days prior to use in experimental group A in the examples below, 8 vials (4.0 × 10) were placed⁸) These cryopreserved F1-1-27 transduced PBMCs were thawed and placed in a complete OpTsizer containing 377ml of I L-2 (Novoprotein) supplemented with 100IU/ml, 10ng/ml I L-7 (Novoprotein) and sufficient NAC^TMCTS^TMT cell expansion SFM (supplemented with 26ml OpTsizer according to manufacturer's instructions)^TMCTS^TMT cell expansion supplement (Thermo Fisher, A10484-02), 25ml CTS^TMImmune cell SR (Thermo Fisher, A2596101) and 10ml CTS^TMGlutaMAX^TMOpTsizer of I supplement (Thermo Fisher, A1286001)^TMCTS^TMT cell expansion basal medium 1L (Thermo Fisher, A10221)) in G-Rex100M for 2 days to increase the final concentration by 10 mM. without prior ex vivo stimulation and without PBMC isolation under ex vivo cell expansion and advantageously rapid transduction of resting lymphocytes

All human blood from 2 healthy volunteers with informed consent was collected into multiple 100mm Vacutainer tubes (Becton Dickenson; 364606) containing 1.5ml of citrate dextrose solution A anticoagulant (ACD peripheral blood). For each volunteer, the following steps of pooling blood from the Vacutainer tubes (185.2ml for group B, 182.5ml for group C) and dispensing into 2 standard 500ml blood collection bags for PBMC enrichment via transduction were performed in a closed system.

Ficoll-Paque was used on a Sepax 2S-100 device (Biosafe; 14000) using the CS-900.2 kit (Biosafe; 1008)^TMDensity gradient centrifugation (General Electric) blood from 2 bags from each volunteer was processed sequentially using 2 wash cycles according to the manufacturer's instructions to obtain 45ml of isolated PBMCs per run. The wash solution used in the Sepax 2 treatment was standard saline (Chenixin Pharm) + 2% Human Serum Albumin (HSA) (Sichuan Yuana Shuyang Pharmaceutical). The final cell resuspension solution was 45ml complete OpTsizer^TMCTS^TMT cell expansion SFM (supplemented with 26ml OpTsizer)^TMCTS^TMT cell expansion supplement (ThermoFisher, A10484-02), 25ml CTS^TMImmune cell SR (Thermo Fisher, A2596101) and 10ml CTS^TMGlutaMAX^TMOpTsizer of I supplement (Thermo Fisher, A1286001)^TMCTS^TMT cell expansion basal medium 1L (Thermo Fisher, a 10221-03)). each treatment step on the Sepax 2 machine was approximately 1 hour and 12 minutes enriched PBMCs obtained from 2 treatment runs were pooled for group B and group C, respectively, and the cells were counted.

5.5 × 10⁷Freshly enriched live PBMCs were inoculated into each of 4 standard blood collection bags for group B and a full OpTmizer was used^TMCTS^TMT cell expansion SFM to reach a volume of 55ml to give a cell density of 1.0 × 10⁶1.12 × 10/ml⁸Freshly enriched live PBMCs were inoculated into each of 2 standard blood collection bags for group C and a full OpTmizer was used^TMCTS^TMT cell expansion SFM to 110ml volume to make cell density 1.0 × 10⁶Addition of No anti-CD 3, anti-CD 28, I L-2, I L-7, or other exogenous interleukins to activate or otherwise stimulate lymphocytes ex vivo prior to transduction Lentiviral particles were added directly to unstimulated PBMCs in a blood collection bag at an MOI of 1 by adding 0.779ml of F1-1-228 to one bag of group B PBMCs and 3.11ml of F1-1-228U to another bag of group B PBMCs, 0.362ml of F1-3-219 to one bag of group C PBMCs and 3.52ml of F1-3-219U to another bag of group C PBMCsIn the PBMC of the group. The transduction reaction mixture was gently massaged to mix the contents, followed by 37 ℃ and 5% CO₂The following was incubated in a standard moist tissue incubator for four (4) hours in a blood collection bag. PBMCs from each bag were then translated into 50ml confinal tubes (thereby removing cells from the closed system in this proof of concept assay) and washed 3 times in DPBS + 2% HAS and counted before being resuspended in 5ml DPBS + 2% HAS. The following table shows the duration and total time elapsed for each step in the process. These PBMCs were not subjected to additional treatment prior to their use in the assay in this example.

Table 6. elapsed time for each step after blood thawing and pooling.

In vitro transduction efficiency and Interleukin-independent survival/proliferation of PBMCs transduced by the above method

T cells and NK cells will be induced and as immediately below revealed with retrovirus contact 4 hours 2.0 x10⁶Each PBMC was inoculated in duplicate or triplicate into each well of a 6-well tissue culture plate for each sample. The discs were centrifuged and each sample was resuspended in 2ml complete OpTsizer^TMCTS^TMT cells expand in SFM. No interleukins were added. At 37 ℃ and 5% CO₂The plates were incubated in a standard moist tissue incubator for 6 days.half of the cell suspension (1ml) of each well was removed on day 3 and the remaining cells were removed on day 6 to determine the cell number, percent survival and percent transduced cells, defined as the percentage of F L AG-Tag + cells by FACS analysis.the total cell count on day 6 was doubled to account for half of the cells removed on day 3.

In vivo proliferation/survival and targeted killing of tumors by effector PBMCs in the above-described manner

Xenograft models using NSG or NOD Scid Gamma mice were selected to probe the ability of human PBMCs transduced with F1-1-27, F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U to survive, proliferate and kill cognate antigen expressing tumors in vivo. NSG is a mouse strain lacking mature T cells, NK cells, and B cells and is one of the most immune deficient mouse strains described to date. These cellular components of the immune system are typically removed to enable human PBMCs to be transplanted in the context of an innate, humoral, or adaptive immune response in the host. Because of the absence of the mouse extracellular common gamma chain, stable cytokine concentrations are typically achieved only after radiation or lymphodepleting chemotherapy in humans, which enables receptive transferred human cells to receive such cytokines. Also, these animals can be used to transplant tumor xenograft targets to test the efficacy of CARs in killing target expressing tumors. Although the presence of xenoreactive T cell receptor antigens in effector cell products ultimately leads to graft versus host disease, these models enable short-term assessment of animal pharmacology and acute tolerance.

Raji cells expressing endogenous human CD19 (ATCC, Manassas, VA) and CHO cells (ATCC, Manassas, VA) transfected to stably express human ROR2(CHO-ROR2) were used to provide antigen to stimulate CAR-effector cells and generate consistent target tumors to determine the efficacy of CAR-effector cells to kill cognate antigen expressing tumors. Raji cells and transgenic CHO variants grew rapidly with disseminated malignancy following subcutaneous administration in NSG mice in combination with Matrigel artificial substrates.

Mice were treated according to protocols approved by the institute for Biochemical and cell laboratory animal administration (Institutional animal Care and Use Committee). Subcutaneous (sc) tumor xenografts in female NOD-Prkdc^scidIl2rg^tm1Posteroventral establishment of/Begen (B-NSG) mice (Beijing Biocytogen Co. L td.) briefly, cultured Raji cells and cultured CHO-ROR2 cells were separately washed in DPBS (thermo Fisher), counted, resuspended in cold DPBS and incubated at a concentration of 0.5 × 10⁶Individual cells/200 microliter of an appropriate volume of Matrigel ECM (Corning; final concentration 5mg/m L) were mixed on ice, animals were prepared for injection using standard approved anesthesia and depilation (Nair) prior to injection, 200. mu.l of ECM containing cell suspensions of Raji and CHO-ROR2 cells, respectively, were injected subcutaneously to 9-week-old or 10-week-old youngMouse posterior lateral.

Intravenous (IV) administration 5 days after tumor inoculation, carried an average of 77mm by tail vein injection as follows³Volume of CHO-ROR2 tumor-containing mice NSG in group A received a vaccine containing 1 × 10 transduced with F1-1-27 lentivirus particles ⁷200 μ l DPBS (n ═ 4) or only 200 μ l DPBS (n ═ 2) of PBMC, NSG mice in group B received 0.85 × 10 containing lentiviral particles transduced with F1-1-228⁷200 μ l DPBS (n-2) from PBMC, containing 0.85 × 10 transduced with F1-1-228U lentiviral particles ⁷200 μ l DPBS (n ═ 2) or only 200 μ l DPBS (n ═ 2) for PBMC similarly, 5 days after tumor inoculation, only 200 μ l DPBS (n ═ 4) or 1 × 10 transduced with 200 μ l DPBS (n ═ 3) containing F1-3-219 lentiviral particles or 200 μ l DPBS (n ═ 3) containing F1-3-219U lentiviral particles by tail vein injection⁷Mice in group C (average 76 mm) bearing Raji tumors were administered Intravenously (IV) to PBMCs³Volume). It should be noted that PBMCs with F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U were transduced for administration to these mice using a protocol that favoured rapid transduction of resting lymphocytes prior to ex vivo activation and without ex vivo cell expansion. The total time from total human blood collection to IV administration to mice with transduced PBMC was 14.5 hours for F1-1-228 and F1-1-228U, and 11.5 hours for F1-3-219 and F1-3-219U.

Tumors were measured 2 times a week using a caliper and tumor volume was calculated using the equation (longest diameter × shortest diameter)²)/2. Approximately 100 μ l of blood was collected from each mouse on days 7, 14 and 21 for FACS and qPCR analysis. When mice were euthanized, blood, spleen and tumors were also collected, which were consistent with necropsy pointers from tumor burden.

Flow cytometry

Cells harvested for the in vitro culture-cells were centrifuged and resuspended in 0.5ml FACS staining buffer (554656, BD), -2.5 μ l of human Fc block (BD, 564220) were added to each sample and incubated at room temperature for 10 minutes-cells were stained on ice with 0.5 μ l of anti-F L AG Tag PE ((anti-DYKDDDDK) 637310, Biolegend) and 0.5 μ l of L ive/DeadFixable Green de cell stain (L34970, Thermo Fisher) for 30 min-cells were washed twice with FACS buffer, fixed in a 1:1 mixture of FACS buffer and BD Cytofix (554655, BD), treated with novocyte (ACEA), and the resulting data were analyzed using a live-valve door NovoExpress software (ACEA) based on forward and side scatter and live and Dead staining.

For cells obtained from blood-erythrocytes in freshly collected blood were lysed using lysis buffer (555899, BD) and the remaining cells resuspended in 100 μ l FACS staining buffer, 2.5 μ l human Fc block (BD, 564220) were added to each sample and incubated at room temperature for 10 minutes, cells stained with biotinylated cetuximab on ice for 30min stained cells were washed with FACS buffer and further stained with 5 μ l anti-human CD45-PE-Cy7 and 0.5 μ l anti-mouse CD45-FITC, 0.4 μ l SA-PE was added to samples from mice dosed with F1-1-27, F1-1-228, F1-1-228 and control transduced cells from these groups, 1 μ l anti-F L AG tagpe ((anti-DYKDDDDK) 637310, biogend) was added to samples from mice dosed with F2-219 min and control transduced cells from this group and the samples were dosed with FACS wash with fresh FACS staining buffer and incubated with FACS wash buffer (cacvyd) and wash with fresh cells with FACS wash-219, wash with b-21 μ l FACS staining buffer, and wash with FACS wash with fresh cells with FACS wash with 20, and wash with FACS wash with fresh gavagen-3-21, wash, and wash with FACS wash with b-21 μ l FACS staining software (afc).

qPCR

Genomic DNA was isolated from 50 μ l Blood samples using the QIAamp DNA Blood Mini kit (Qiagen 51106) and further washed using the QIAamp DNA Micro kit (56304) the TaqMan assay (Thermo Fisher) was performed on the isolated genomic DNA using a primer and probe set specific for the 5' L TR of lentivirus to detect transduced cells.

Results

Lentiviral particles F1-1-228 and F1-1-228U used in this experiment to encode MRB-CAR as ROR2 and C L E including MycTag and 2A Jun dimerization domain, I L RA transmembrane domain, and MP L and CD40 intracellular domains, and Lentiviral particles F1-2-219 and F1-3-219U encode CAR as CD19 and C L E encoding CD19 and C L E encoding I L covalently linked to the extracellular and transmembrane portions of I L-7R α (CD127) (SEQ ID NO:513) and the intracellular domain of I L-2R (CD 122). Uc 1-1-228-3-219U scFcol 1, and F1-3-219U also present on their surface via Pafcoll GPI-PvHT 1^TMDensity gradient centrifugation PBMCs were isolated from human blood. Fresh PBMCs were transduced with lentiviral particles in standard blood bags prior to ex vivo activation of cells. After 4 hours of transduction, cells were washed and used in the following experiments. The skilled artisan will appreciate that the entire process from blood collection to washing of the cells can be performed in a closed system. As a control, PBMCs were activated overnight, transduced with lentiviral particles encoding CAR without encoding lymphoproliferative component or UCHT1 scfvffc-GPI (F1-1-27), and cultured ex vivo for 15 days (F1-1-27).

Transduced PBMCs are cultured in vitro in the absence of interleukins. Fig. 21 shows transduction efficiency at day 6. UCHT1scFvFc-GPI increases the transduction efficiency of F1-1-228 and F1-3-219. Transduction efficiencies of resting PBMC were 34% and 73% when exposed to F1-1-228U or F1-3-219U for 4 hours, respectively. Figure 22 shows the total number of viable cells in these cultures between day 3 and day 6. Resting PBMC transduced with F1-1-228U or F1-3-219U survived and even proliferated between day 3 and day 6 in the absence of exogenous interleukins, while PBMC transduced with F1-1-228 or F1-3-219 did not survive and even proliferate. These results demonstrate that lentiviral particles displaying an activating module and encoding a lymphoproliferative module can transduce resting PBMCs within 4 hours, and that these transduced PBMCs can proliferate and survive in vitro when cultured for 6 days in the absence of exogenous interleukins.

Immunodeficient mice bearing ROR2 or CD19 tumors were given intravenously 1 × 10 expressing CARs to ROR2 or CD19, respectively⁷And (5) PBMCs. Detection of transduced PBMC in vivo over timeFigures 23A-23C show lentivirus copies per microgram of genomic DNA isolated from the blood of mice administered with CAR-expressing PBMC by qPCR as readouts of transduced cells figure 23A shows lentivirus copies per microgram of genomic DNA for F1-1-27 that do not encode lymphoproliferative components, decreasing from mean 884 at day 7 to below the lower limit of quantitation (LL OQ) at day 21 for the mean number of cells on day 7, figure 23B shows lentivirus copies for F1-1-228U to be below LL OQ at day 7 and day 14, but increasing from more than LL OQ on day 21 to mean 1,939 on day 25 figure 23C shows that when mice are euthanized, lentivirus copies for F1-3-219 increase from LL OQ at day 7 to 20,430 on day 14 when mice are euthanized, the number of cells in control samples F1-1-228 and F1-3-219 is still detected by the number of cells per gram FACS of cells transduced cells per gram of interest (agu-9634) as measured by FACS F-3-96 g of cells on ad 1 in the last FACS (agus-3-219).

PMBCs genetically modified to express lymphoproliferative components and anti-CD 19 CARs or anti-ROR 2 CARs were analyzed for anti-tumor activity. Mice bearing either the CHO-ROR2 tumor or the CD-19 expressing tumor were generated as provided above. Lymphocytes transduced with F1-1-228U or F1-3-219U killed tumors that expressed their antigen of interest in vivo (FIGS. 25A and 25B). In both cases, lymphocytes reduce tumor volume in a delayed manner. Without being limited by theory, this delay is consistent with the need for injected cell amplification, which takes time as observed by qPCR. Later time points could not be investigated in this experiment, since mice bearing ROR2 tumor reached an euthanasia index for tumor burden, and mice bearing Raji tumor developed graft-versus-host disease caused by a large number of transduced and expanded lymphocytes in the mice.

Together, these data demonstrate that retroviral particles that display an activating component and encode a lymphoproliferative component can transduce resting PBMCs within 4 hours, and that these transduced PMBCs can proliferate and survive in vivo two lymphoproliferative components, MycTag 2A Jun-I L Ra-MP L-CD 40 and I L-7-I L-7R α -I L-2R β, tested in this experiment exhibit the ability to promote survival and proliferation in vivo.

Example 17 functionality of miRNA inserted into the intron of the EF-1 α promoter.

Design four of the single

Gene fragments, each containing a miR-155 framework comprising miR-1555 'flanking sequences or "5' arms" (SEQ ID NO:256) and miR-1553 'flanking sequences or "3' arms" (SEQ ID NO: 260). For each

The only miRNA fragment targeting the CD3zeta mRNA transcript was used to replace the miR-155 stem-loop precursor. Each one of

Containing components designed to facilitate the coupling of all four

40bp overlap as single-stranded Assembly into the intron of the EF-1 α promoter use for performing

Assembly ultraWas assembled using the commercial kit of (NEBuilder, New England Biolabs, Inc.)

Synthetic EF-1 α promoter containing miRNA (in sequence SEQ ID NO: 255) and intron A are part of a transgene expression cassette that drives expression of GFP and eTag contained in a lentiviral vector backbone (the lentiviral vector backbone with GFP recognized by cetuximab and the exemplary eTag is referred to herein as F1-0-02; FIGS. 26A and 26B). SEQ ID N indicated in Table 26Each of O:255

And the nucleotide positions of their individual components are the positions of each "signature" in fig. 26B the proper assembly of the four mirnas into the lentiviral vector backbone was confirmed by the full sequencing of the modified EF-1 α promoter and intron regions.

TABLE 26 nucleotide positions characteristic of SEQ ID NO: 255.

Replication-defective lentiviral particles containing in their genome a nucleic acid encoding four mirnas against CD3 ζ were produced by transiently co-transfecting into suspension HEK293 cells a plasmid containing a nucleic acid encoding F1-0-02 modified to include four mirnas targeting CD3zeta mRNA transcripts, a plasmid encoding VSV-G, a plasmid encoding REV, and a plasmid encoding GAG-PO L, collecting lentiviral particle supernatant after 48 hours and precipitating with PEG for 24 hours, centrifuging the supernatant and resuspending pelleted lentiviral particles in complete PBMC growth medium without I L-2, calculating lentiviral particle titers using 48 hour transduction of Jurkat cells.

For transduction, PBMCs were thawed on day 0 and incubated with hrI L-2 at 100U/m L for 24 hours on day 1 PBMCs were activated via CD3/CD28 bound beads on day 2, cells were transduced with lentiviral particles containing a genome with a nucleic acid sequence encoding a miRNA at 10MOI, expanded until day 11 with fresh hrI L-2 added every two days, on days 7, 9 and 11, 1 million cells were harvested for FACS analysis.

Cells were stained with PE-conjugated OKT-3 antibody (Biolegend) for CD3 Epsilon surface expression levels were determined using the mean fluorescence intensity (MF) of PE in the GFP positive population (transduced cells) the expression levels of transduced cells were compared between retroviral particles derived from F1-0-02 and retroviral particles derived from F1-0-02, with the insertion of a nucleic acid sequence encoding a contiguously located CD3z miRNA into the EF-1 α promoter and intron a.

The results are shown in figure 27 this data demonstrates that continuous mirnas targeting CD3zeta encoded by the nucleic acid sequence within intron a of the EF-1 α promoter are effective at knocking out expression of the CD3 complex.

Example 18 position dependence of continuous inhibitory RNA inserted into the intron of the EF-1 α promoter.

Cloning

Four lentiviral vector constructs expressing mirnas were designed to test the processing of individual miRNA precursors in a structure comprising 4 consecutive miRNA precursors table 27 shows the names of the individual constructs and the location of miR-TCR α in each construct.

Construct	Position	1	Position 2	Position 3	Position 4
						TCRa-P1	miR-TCRa	miR-155	miR-PD-1	miR-CTLA-4
TCRa-P2	miR-155	miR-TCRa	miR-PD-1	miR-CTLA-4
					TCRa-P3	miR-155	miR-PD-1	miR-TCRa	miR-CTLA-4
TCRa-P4	miR-155	miR-CTLA-4	miR-PD-1	miR-TCRa

TABLE 27 constructs containing polycistronic miRNAs.

Each miRNA contains the specific order of the miR-155 frameworks used in example 17, such as the miR-1555 'arm (SEQ ID NO:256), miR-1553' arm (SEQ ID NO:260), loop (SEQ ID NO:258), and stem sequence as shown in Table 28A type II assembly method is used to arrive at the assembly of four miRNA fragments into their appropriate positions within the EF-1 α intron of a lentiviral vector construct (F1-0-02; provided in example 17 and shown in FIGS. 26A and 26B).

Table 28: sequences in miRNA constructs

Wherein:

267 as TCR α miRNA stem 1, ATATGTACTTGGCTGGACAGC

SEQ ID NO 268 ═ TCR α miRNA stem 2;, GCTGTCCACAAGTACATAT

269 ═ linker 1; CACATTGGTGCCGGATGAAGCTCTTATGTTGCCGGTCAT

270 ═ mir-155 stem 1 of SEQ ID NO; CTGTTAATGCTAATCGTGATA

271-mir-155 stem 2; TATCACGATTATTAACAG

272. catenin 2; GTTGCCGGAGTCTTGGCAGCGAGAGATCACTATCAACTAA

273 ═ PD-1miRNA stem 1; TACCAGTTTAGCACGAAGCTC

274 ═ PD-1miRNA stem 2; GAGCTTCGCTAAACTGGTA

275 ═ linker 3; GTGTTAATTGTCCATGTAGCGAGGCATCCTTATGGCGTGG

276 ═ CT L A-4miRNA stem 1; TGCCGCTGAAATCCAAGGCAA; SEQ ID NO: TGCCGCTGAAATCCAAGGCAA

277-CT L A-4miRNA stem 2, TTGCCTTGTTTCAGCGGCA and SEQ ID NO

Lentiviral particle production

Four constructs and controls F1-0-02 (which did not include the nucleic acid sequence encoding miRNA) were used to produce lentiviral particles in 30m L suspension cultures of 293T cells, lentiviral particles were harvested and concentrated using PEG precipitation functional lentiviral particle titers were obtained by transducing Jurkat cells at multiple dilutions (1:1000, 1:10000, 1:100000), incubating the lentiviral particles and cells for 2 days at 37 ℃, washing the cells with FACS buffer 2 × and analyzing GFP using flow cytometry other details on lentiviral particle production are provided in example 17 herein.

Transduction of

For transduction, PBMCs were thawed and recovered overnight in complete medium containing 100U/m L hrI L-2 1 × 10 was activated via exposure to CD3/CD 28-bound beads for 24 hours⁵Cells were transduced in two replicate wells with each of the four miRNA constructs or with control retroviral particles F1-0-02 at MOI 10 cells were supplied with 100U/m L hrI L-2 every 3 days and expanded until day 10.

FACS

Cells were pooled for FACS analysis, which confirmed cells transduced with replication-defective lentiviral vectors. The results show that approximately equal amounts of miRNA-containing virus were delivered to each well in the experiment.

Cell-p-ct miRNA RT-qPCR and analysis

RT-qPCR analysis was designed to detect expression and process miRNA precursors into mature processed mirs. Analysis was performed by first normalizing all miR-TCRa Ct values to the RNU48 internal control to generate Δ Ct values. Next, the Δ Ct value for each transduced sample was subtracted from the Δ Ct for the untransduced control to yield a Δ Δ Ct. This value represents the amount of processed miR-TCRa miRNA in each transduced sample relative to the untransduced control.

As shown in figure 28, RT-qPCR analysis successfully detected processed miR-TCR α in samples transduced with replication-deficient lentiviral particles containing miR-TCR α, furthermore, the results clearly indicate no significant effect in miRNA TCR α processing at any of the four locations tested.

Example 19 miRNA expression increases survival and/or proliferation of transduced cells expressing the CAR in vivo.

Method of producing a composite material

Library preparation

108 are

Gene fragments were used to generate libraries of constructs each containing 4miRNA precursors at consecutive positions 1(P1), 2(P2), 3(P3) and 4 (P4). Each one of

Specific for P1, P2, P3, or P4 and containing a miR-155 framework comprising a 5 'arm and a 3' arm as described in example 17, wherein a unique miRNA framework targeting mRNA transcripts corresponding to 1 to 27 different genes was used to replace the miR-155 stem-loop precursor. For clarity, the sequence of the miRNA fragment differs for each position P1 to P4, even among miRNA fragments targeting mRNA transcripts corresponding to the same gene. At each position

Contains a unique 40bp overlap sequence, and the type II assembly method is used to assemble four in their prescribed order

By these methods, total diversity of 531,441 unique constructs (27 miRNA at 27miRNA × P3 at 27miRNA × P2 at P1, 27miRNA × P4) is possible.

Pools of miRNA constructs were cloned individually into EF-1 α intron a of F1-1-315 and F1-2-314 to generate pool 315 and pool 314, respectively F1-1-315 also includes CD8a signal peptide, anti-ROR 2: CD28: CD3z CAR, T2A and etag, in addition to the EF-1 α promoter, similarly F1-2-314 includes CD8a signal peptide, anti-Axl: CD8: CD3z CAR, T2A and etag, fig. 26A and 26B show similar lentiviral vectors with EF-1 α promoter (including intron a with 4miRNA precursors of miRNA) driving expression of GFP but not CAR.

cCB L and CD3z among the 27 transcripts targeted in this example 4miRNA sequences against cCB L are SEQ ID NO 540 of P1, 541 of P2, 542 of P3, and 543 of P4 4miRNA sequences against CD3z are SEQ ID NO 544 of P1, 545 of P2, 546 of P3, and 547 of P4.

Note that since their specific sequences (miRNA for CD3 z) were designed, they would only target the RNA encoding endogenous CD3z and would not target the RNA encoding the CD3z domain within the CAR transgene. This is achieved by designing the miRNA to target a sequence within the 3' UTR of endogenous CD3z that is not present in the CAR transgene. Alternatively, the miRNA can be targeted to an mRNA sequence encoding the CD3z endodomain of both the endogenous CD3z and the CAR transgene, with the proviso that the codon usage for the CAR transgene is sufficiently different from that of endogenous CD3z so that the endogenous CD3z, but not the CAR transgene, mRNA is targeted for cleavage.

Lentiviral particle production

Library 315 and library 314 were used alone to produce lentiviral particles in 30ml suspension culture of 293T cells. Lentiviral particles were harvested and concentrated using PEG precipitation. Additional details regarding lentiviral particle generation are provided in example 17 herein.

Transduction of

On day 0, PBMCs were isolated from ACD peripheral blood and treated with intact OpTsizer 018 supplemented with 100IU/ml I L-2 (Novoprotein, GMP-CD66), 10ng/ml I L-7 (Novoprotein, GMP-CD47) and 50ng/ml anti-CD 3 antibody (Novoprotein, GMP-A)^TMCTS^TMT cell expansion SFM 5.0 × 10⁷Each surviving PBMC was seeded at 100ml into each of two 1L G-Rex devices to activate PBMCs including T cells and NK cells for virus transduction.Lentiviral particles were added directly to activated PBMCs in 1G-Rex of Bank 315 and the other G-Rex of Bank 314 at an MOI of 5, and incubated overnight at 37 ℃ and 5% CO₂Next, the G-Rex devices were incubated in a standard humidified tissue incubator with 100IU/ml recombinant human I L-2 and 10ng/ml recombinant human I L-7 solutions added every 48 hours and the cultures expanded until day 12, at which time the cells were predominantly T cells other details regarding PBMC isolation, transduction, and ex vivo expansion are provided in example 16 herein.

Tumor culture and administration of transduced cells.

Xenograft models using NOD Scid Gamma (NSG) mice were selected to probe the ability of human PBMCs transduced with lentiviral particles of pool 315 or pool 314 to survive and/or proliferate in vivo, with or without the tumor expressing the antigen recognized by the CARs encoded in the genomes of these lentiviral particles. Mice were treated according to protocols approved by the institute for Biochemical and cell laboratory Animal administration (Institutional Animal Care and Use Committee) of the Chinese academy of sciences. Subcutaneous (sc) tumor xenografts were established in 12-week-old female NOD-Prkdc^scidIl2rg^tm1Briefly, cultured CHO cells transfected to stably express either human ROR2(CHO-ROR2) or human AX L (CHO-AX L), individually washed in DPBS (thermo Fisher), counted, resuspended in cold DPBS, and plated on ice at 0.47 × 10⁶Concentration of individual cells/200. mu.lAnimals were prepared for injection using standard approved anesthesia with hair (Nair) removed prior to injection, 200 μ l of any one of the cell suspensions of ECM were injected subcutaneously into the posterior side of CHO cells (n ═ 2), CHO-ROR2 cells (n ═ 1), and CHO-AX L cells (n ═ 1), respectively.

5 days after tumor inoculation, 1 mouse bearing the CHO tumor and 1 mouse bearing the CHO-ROR2 tumor were administered Intravenously (IV) by tail vein injection with 1 × 10 containing lentiviral particles transduced with the library 315⁷200 μ l DPBS of PBMCs 5 days after tumor inoculation, 1 mouse bearing a CHO tumor and 1 mouse bearing a CHO-Axl tumor were administered Intravenously (IV) by tail vein injection with 1 × 10 transduced with lentiviral particles from pool 314⁷200. mu.l DPBS of each PBMC.

Tumor Collection and DNA sequencing

On day 20 after administration of transduced PBMCs, tumors were excised DNA was extracted from half of each tumor, and 4ug from each tumor was used as a template in a PCR reaction for 25 cycles to amplify the EF-1 α intron, amplicons were cloned into sequencing vectors, converted to bacteria and streaked onto disks, 18 total colonies (about 5 per mouse) were selected, and DNA was prepared and analyzed using Sanger sequencing to determine the sequence of a sample of miRNA constructs present in the tumors.

Results

A mouse xenograft model was used to determine whether mirnas targeting cCB L or CD3z increased proliferation and/or survival in vivo of transduced PBMCs expressing CARs, with xenografts being tumors with or without expression of the targeted antigen of the CAR for this analysis, a library of miRNA constructs consisting of 4 positions for 4 individual mirnas, as shown in figure 26B and examples 17 and 18, was generated consisting of mirnas against cCB L, endogenous CD3z or 25 other target mirnas was generated for this analysis tumor DNA was analyzed by sequencing the EF-1 α intron to identify which miRNA constructs were present 20 days after injection of transduced PBMCs and thus which miRNA constructs increased proliferation and/or survival.

Among the 18 EF-1 α introns sequenced, 13 contained miRNA constructs, where all 4 mirnas in the construct were directed against one target, although the target differed among the constructs, notably 2 EF-1 α introns contained miRNA constructs with all 4 mirnas directed against cCB L (SEQ ID NO:540 to 543), and 1 EF-1 α intron contained miRNA constructs with all 4 mirnas directed against CD3z (SEQ ID NO:544 to 547), 4 consecutive mirnas directed against cCB L were found in CHO-ROR2 tumors from mice receiving PBMC transduced with library 315, and CHO tumors from mice receiving PBMC transduced with library 314. 4 consecutive mirnas directed against CD3z were found in CHO tumors from mice receiving PBMC transduced with library 315. this indicates that there are advantages of CHO and CD3 survival in CD3 and CD3 survival in mice receiving PBMC and that the knockout gene for this species confers an increased proliferation and/or increased tumor-cell-proliferation-specific ratio, and/or increased tumor-specific tumor-proliferation-expressing tumor-expressing species.

The disclosed embodiments, examples and experiments are not intended to limit the scope of the invention or to represent the experiments below as all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. It is understood that variations can be made to the method as described without changing the basic aspects that the experiment is intended to illustrate.

Many modifications and other embodiments may be devised by those skilled in the art which fall within the scope and spirit of the principles of this invention. Indeed, the described materials, methods, figures, experiments, examples and embodiments may be varied by those skilled in the art without changing the basic aspects of the invention. Any of the disclosed embodiments can be used in combination with other disclosed embodiments.

In some cases, some concepts are described with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the present disclosure as set forth in the claims below. Accordingly, the specification and figures are 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 7 partial P1-P2, P1, P2, P3 and P4 codes, names and amino acid sequences

TABLE 8 optimal constructs for library 1A.

TABLE 9 optimal constructs for Bank 2B.

TABLE 10 optimal constructs for library 1.1A.

TABLE 11 optimal constructs for library 1.1B.

TABLE 12 optimal constructs for Bank 2.1B.

TABLE 13 optimal constructs for library 3A.

TABLE 14 optimal constructs for library 3B.

TABLE 15 optimal constructs for library 3.1A.

TABLE 16 optimal constructs for library 3.1B.

TABLE 17 optimal constructs for library 4B.

TABLE 18 optimal constructs for library 4.1B.

TABLE 19L ib 3P 4_ STOP.

TABLE 20 constructs with particularly noteworthy enrichment in pools 1A and 1.1A.

TABLE 21 constructs with particularly noteworthy enrichment in pools 2B and 2.1B.

Table 22 constructs with particularly noteworthy enrichment in pools 3A and 3.1A.

Table 23 constructs from pools 3B and 3.1B with particularly noteworthy enrichment.

Table 24 constructs from pools 4B and 4.1B with particularly noteworthy enrichment.

Table 25 constructs of the first 100 hits present in at least one replicate of pool 3.1A or 3.1B.

Sequence listing

<110> F1 tumor Co., Ltd

<120> methods and compositions for genetically modifying and expanding lymphocytes and modulating their activity

<130>F1.001.WO.04

<150>PCT/US2018/020818

<151>2018-03-03

<150>US 62/560,176

<151>2017-09-18

<150>US 62/564,253

<151>2017-09-27

<150>US 62/564,991

<151>2017-09-28

<150>US 62/728,056

<151>2018-09-06

<160>548

<170>PatentIn version 3.5

<210>1

<211>42

<212>PRT

<213> Intelligent people

<400>1

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>2

<211>41

<212>PRT

<213> Intelligent people

<400>2

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>3

<211>41

<212>PRT

<213> Intelligent people

<400>3

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>4

<211>35

<212>PRT

<213> Intelligent people

<400>4

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>5

<211>37

<212>PRT

<213> Intelligent people

<400>5

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>6

<211>49

<212>PRT

<213> Intelligent people

<400>6

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>7

<211>114

<212>PRT

<213> Intelligent people

<400>7

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>8

<211>187

<212>PRT

<213> Intelligent people

<400>8

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 GluThr 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>9

<211>54

<212>PRT

<213> Intelligent people

<400>9

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>10

<211>60

<212>PRT

<213> Intelligent people

<400>10

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>11

<211>163

<212>PRT

<213> Intelligent people

<400>11

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>12

<211>164

<212>PRT

<213> Intelligent people

<400>12

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 AspGly 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>13

<211>112

<212>PRT

<213> Intelligent people

<400>13

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>14

<211>21

<212>PRT

<213> Intelligent people

<400>14

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>15

<211>22

<212>PRT

<213> Intelligent people

<400>15

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>16

<211>21

<212>PRT

<213> Intelligent people

<400>16

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>17

<211>171

<212>PRT

<213> Intelligent people

<400>17

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>18

<211>127

<212>PRT

<213> Intelligent people

<400>18

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>19

<211>21

<212>PRT

<213> Intelligent people

<400>19

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>20

<211>206

<212>PRT

<213> Intelligent people

<400>20

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>21

<211>21

<212>PRT

<213> Intelligent people

<400>21

Asn ProAsp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser

1 5 10 15

Gly Leu Asn Gln Arg

20

<210>22

<211>182

<212>PRT

<213> Intelligent people

<400>22

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 ThrIle 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>23

<211>21

<212>PRT

<213> Intelligent people

<400>23

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>24

<211>226

<212>PRT

<213> Intelligent people

<400>24

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>25

<211>188

<212>PRT

<213> Intelligent people

<400>25

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 ProPro 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>26

<211>21

<212>PRT

<213> Intelligent people

<400>26

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>27

<211>113

<212>PRT

<213> Intelligent people

<400>27

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>28

<211>107

<212>PRT

<213> Intelligent people

<400>28

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro LeuLeu

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>29

<211>102

<212>PRT

<213> Intelligent people

<400>29

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 ValMet 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>30

<211>101

<212>PRT

<213> Intelligent people

<400>30

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 ArgIle 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>31

<211>21

<212>PRT

<213> Intelligent people

<400>31

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>32

<211>86

<212>PRT

<213> Intelligent people

<400>32

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 ArgLys 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>33

<211>21

<212>PRT

<213> Intelligent people

<400>33

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>34

<211>20

<212>PRT

<213> Intelligent people

<400>34

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>35

<211>68

<212>PRT

<213> Intelligent people

<400>35

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>36

<211>619

<212>PRT

<213> Intelligent people

<400>36

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 GlyGly 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>37

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic HA Epitope

<400>37

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

<210>38

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic F L AG Epitope

<400>38

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210>39

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic c-myc Epitope

<400>39

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210>40

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic His5 Affinity

<400>40

His His His His His

1 5

<210>41

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic HisX6 Affinity

<400>41

His His His His His His

1 5

<210>42

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Strep Tag Affinity

<400>42

Trp Ser His Pro Gln Phe Glu Lys

1 5

<210>43

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic HisX6 Affinity

<400>43

Arg Tyr Ile Arg Ser

1 5

<210>44

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic affinity

<400>44

Phe His His Thr

1

<210>45

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic affinity

<400>45

Trp Glu Ala Ala Ala Arg Glu Ala Cys Cys Arg Glu Cys Cys Ala Arg

1 5 10 15

Ala

<210>46

<211>24

<212>PRT

<213> Intelligent people

<400>46

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>47

<211>23

<212>PRT

<213> Intelligent people

<400>47

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>48

<211>25

<212>PRT

<213> Intelligent people

<400>48

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>49

<211>23

<212>PRT

<213> Intelligent people

<400>49

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>50

<211>27

<212>PRT

<213> Intelligent people

<400>50

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>51

<211>26

<212>PRT

<213> Intelligent people

<400>51

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>52

<211>24

<212>PRT

<213> Intelligent people

<400>52

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>53

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 1

<400>53

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>54

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 2

<400>54

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>55

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 3

<400>55

Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210>56

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 4

<400>56

Gly Gly Ser Gly

1

<210>57

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 5

<400>57

Gly Gly Ser Gly Gly

1 5

<210>58

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 6

<400>58

Gly Ser Gly Ser Gly

1 5

<210>59

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 7

<400>59

Gly Ser Gly Gly Gly

1 5

<210>60

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 8

<400>60

Gly Gly Gly Ser Gly

1 5

<210>61

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic linker 9

<400>61

Gly Ser Ser Ser Gly

1 5

<210>62

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 1

<400>62

Cys Pro Pro Cys

1

<210>63

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 2

<400>63

Asp Lys Thr His Thr

1 5

<210>64

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 3

<400>64

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

1 5 10 15

<210>65

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 4

<400>65

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr

1 5 10

<210>66

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 5

<400>66

Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10

<210>67

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 6

<400>67

Lys Cys Cys Val Asp Cys Pro

1 5

<210>68

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 7

<400>68

Lys Tyr Gly Pro Pro Cys Pro

1 5

<210>69

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 8

<400>69

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210>70

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 9

<400>70

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210>71

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> resultant hinge 10

<400>71

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro

<210>72

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 11

<400>72

Ser Pro Asn Met Val Pro His Ala His His Ala Gln

1 5 10

<210>73

<211>45

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hinge 12

<400>73

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>74

<211>21

<212>PRT

<213> Intelligent people

<400>74

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>75

<211>69

<212>PRT

<213> Intelligent people

<400>75

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 1015

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>76

<211>66

<212>PRT

<213> Intelligent people

<400>76

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>77

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic 2A-1 cleavage Signal

<400>77

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>78

<211>357

<212>PRT

<213> Intelligent people

<400>78

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>79

<211>45

<212>PRT

<213> Intelligent people

<400>79

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>80

<211>39

<212>PRT

<213> Intelligent people

<400>80

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

35

<210>81

<211>113

<212>PRT

<213> Intelligent people

<400>81

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

ProGln 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>82

<211>463

<212>PRT

<213> Intelligent people

<400>82

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 Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Pro Pro Cys Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser

245 250 255

Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile

260 265 270

Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu

275 280 285

His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro

290 295 300

Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala

305 310 315 320

Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu

325 330 335

Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn

340 345 350

Cys Pro Ser Glu Asp Val Val Val Thr Pro Glu Ser Phe Gly Arg Asp

355 360 365

Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro

370 375 380

Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn

385 390 395 400

Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn

405 410 415

Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu

420 425 430

Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn

435 440 445

Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

450 455 460

<210>83

<211>13673

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic Dox-rapamycin inducible riboswitched lentivirus genome

<400>83

gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata 60

acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca 120

ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac 180

gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg 240

ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc 300

tttctagggt taagacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca360

atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 420

gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 480

atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 540

acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 600

catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 660

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 720

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 780

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 840

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 900

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 960

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 1020

ttgttttgga accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 1080

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc 1140

agtgatagag atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt 1200

cagatcgcct ggagacgccc tcgaagccgc ggtgcgggtg ccagggcgtg ccctgggtct 1260

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 1320

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 1380

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1440

gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1500

ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1560

ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1620

ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1680

aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1740

tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1800

caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1860

aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1920

aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1980

agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 2040

gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 2100

ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 2160

acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 2220

ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 2280

ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 2340

tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 2400

aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2460

aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2520

aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2580

acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2640

agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2700

tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2760

gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 2820

cgattagact gtagcccagg aatatggcag ctagattgta cacatttaga aggaaaagtt 2880

atcttggtag cagttcatgt agccagtgga tatatagaag cagaagtaat tccagcagag 2940

acagggcaag aaacagcata cttcctctta aaattagcag gaagatggcc agtaaaaaca 3000

gtacatacag acaatggcag caatttcacc agtactacag ttaaggccgc ctgttggtgg 3060

gcggggatca agcaggaatt tggcattccc tacaatcccc aaagtcaagg agtaatagaa 3120

tctatgaata aagaattaaa gaaaattata ggacaggtaa gagatcaggc tgaacatctt 3180

aagacagcag tacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg 3240

gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 3300

ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat 3360

ccagtttggc tgcattgatc acgtgagggg ctctagactc tagacacaca aaaaaccaac 3420

acacagatct aatgaaaata aagatctttt attgagaaac ttatacaggg tagcataatg 3480

ggctactgac cccgccttca aacctatttg gagactataa gtgaaaatta tcactggttt 3540

tggtagaagc tggacatggt gacatatgct tcttcttgat ttgatcccag ggaagtaaga 3600

atgggctgac cctgagcaac tgggttcaat gtcaggattc cagattggag agaaaatgga 3660

gggggcagcg tgctgtttgt agtcccaagg ctaagcagga ggtcctggta cacatgaggc 3720

ccattcttgc cactctccct gcagtctagg gacctggaag aggagagaat aggggcgtca 3780

catgcactga cattcccagc caggcatgtg agggatgaat ctcttccaaa gctttctgga 3840

gtgacgacta catcctcaga tgggcagttg gggctctgca catcccctcc aagcctctgc 3900

ttctcagatt cttctagttg ctgaggaaac gtatcttgca gaaaaccttc cacttcatct 3960

ctagcttgaa tgtcatccac cctatgaatc tggcagtcca ggaaactttc aggattgaaa 4020

ctcacattta aattttttct tggtttctta caaagatgtt ccagagtctt cttatgatcg 4080

gggagactgg gccatacgat aggcttaatc ctttttttcc ataacacaca ggccaagatg 4140

accaacagag cgacagagaa aaaactcaaa atgctgatgg tcaggcatgg tggtagtaag 4200

ataggatcca tctcccctga gctattattg atctctggag ttctgaagta ataacttgga 4260

ctccattcac tccagaagcc tttaaaatag tgatcaggga tggatcgaac tttaatctca 4320

tacattgctg ccggttggag ctttctctgc aggagtgtca gctttgtgct ggataaattc 4380

acatgcgtcc atttgttttc atccttttcc tggcggtaag ctacatcatg cattaaaact 4440

tttacatact tcttttgcaa gtgtgatgta ttaaatgtca ccacaaagtc attggctcct 4500

tcccgataga tgacactcag gtcaaaagga gcctcaggtt taactatagt ggttaggtct 4560

atttttttgc aggttagact cttttctcca accttcacac atatattgct ctttccaatc 4620

agtaagaatt tctttgtctc gatgaaatat atctcttgta gtttcctgaa attcaggcac 4680

tttacctcca cgagggcccc acatatttca aattccagat tggtggtgtt gacatctggg 4740

tcctcaaaag cacatgtcag tgaatgctgc gatccattca cttccaactg gctatagcat 4800

gagaatgagt agtcatccag ttctgcatct tccaagtctc cattttgagc atagccactt 4860

tctccagaaa cgacttgaag taaagaaaaa accatgccaa aagttgtacc tagaattgtc 4920

attgggccgg gattttcctc cacgtccccg catgttagta gacttcccct gccctcgccg 4980

gagcgagggg gcagggcctg catgtgaagg gcgtcgtagg tgtccttggt ggctgtactg 5040

agaccctggt aaaggccatc gtgccccttg cccctccggc gctcgccttt catcccaatc 5100

tcactgtagg cctccgccat cttatctttc tgcagttcat tgtacaggcc ttcctgaggg 5160

ttcttccttc tcggctttcc ccccatctca gggtcccggc cacgtctctt gtccaaaaca 5220

tcgtactcct ctcttcgtcc tagattgagc tcgttataga gctggttctg gccctgcttg 5280

tacgcggggg cgtctgcgct cctgctgaac ttcactctca gttcacatcc tccttcttct 5340

tcttctggaa atcggcagct acagccatct tcctcttgag tagtttgtac tggtctcata 5400

aatggttgtt tgaatatata caggagtttc tttctgcccc gtttgcagta aagggtgata 5460

accagtgaca ggagaaggac cccacaagtc ccggccaagg gcgcccagat gtagatatca 5520

caggcgaagt ccagccccct cgtgtgcact gcgccccccg ccgctggccg gcacgcctct 5580

gggcgcaggg acaggggctg cgacgcgatg gtgggcgccg gtgttggtgg tcgcggcgct 5640

ggcgtcgtgg ttgaggagac ggtgactgag gttccttggc cccagtagtc catagcatag 5700

ctaccaccgt agtaataatg tttggcacag tagtaaatgg ctgtgtcatc agtttgcaga 5760

ctgttcattt ttaagaaaac ttggctcttg gagttgtcct tgatgatggt cagtctggat 5820

ttgagagctg aattatagta tgtggtttca ctaccccata ttactcccag ccactccaga 5880

ccctttcgtg gaggctggcg aatccagctt acaccatagt cgggtaatga gacccctgag 5940

acagtgcatg tgacggacag gctctgtgag ggcgccacca ggccaggtcc tgactcctgc 6000

agtttcacct cagatccgcc gccacccgac ccaccaccgc ccgagccacc gccacctgtg 6060

atctccagct tggtcccccc tccgaacgtg tacggaagcg tattaccctg ttggcgaagt 6120

aggtggcaat atcttcttgc tccaggttgc taattgtcag agaataatct gttccagacc 6180

cactgccact gaaccttgat gggactcctg agtgtaatct tgatgtatgg tagatcagga 6240

gtttaacagt tccatctggt ttctgctgat accaatttaa atatttacta atgtcctgac 6300

ttgccctgca actgatggtg actctgtctc ccagagaggc agacagggag gatgtagtct 6360

gtgtcatctg gatgtccggc ctggcggcgt ggagcagcaa ggccagcggc aggagcaagg 6420

cggtcactgg taaggccatg gatcctctag atcacgacac ctgaaatgga agaaaaaaac 6480

tttgaaccac tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt 6540

ccaaggagaa ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt 6600

ggggaaactc catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc 6660

tcgagaacta atcgaggtgc ctggacggcg cccggtactc agtggagtca catgaagcga 6720

cggctgagga cggaaaggcc cttttccttt gtgtgggaga aacttataca gggtagcata 6780

atgggctact gaccccgcct tcaaacctat ttggagacta taagtgaaaa tgactcaccc 6840

gcccgctctc ccggcacctt catcttgtcc tttccctcag aaagaggctg ggaggcagag 6900

gctgaggcag cggtggccgg gacggttagg agaaaaggag tctctgctgg ttttattctg 6960

cagctacctc cccaggaagt ggaggactgt ggggcctttg agaagcacct gccgacaggg 7020

ccaagaaatt cgcactcccc ctttcggttc acaggcagga agccctggag gtttgagggt 7080

ttggggtgtg tgtatgtatc tgtctgtctg aattttgctt tttctctcat ttgaccattg 7140

ttttaatgct ccttttttta aaaaaaataa ttcttatcta attcctatct tgattggtaa 7200

agtccatctc taggcaaata caagttctcg atggaaaaca ataagtaatg taaaatacag 7260

catagcaaaa ctttaacctc caaatcaagc ctctacttga atccttttct gagggatgaa 7320

taaggcatag gcatcagggg ctgttgccaa tgtgcattag ctgtttgcag cctcaccttc 7380

tttcatggag tttaagatat agtgtatttt cccaaggttt gaactagctc ttcatttctt 7440

tatgttttaa atgcactgac ctcccacatt ccctttttag taaaatattc agaaataatt 7500

taaatacatc attgcaatga aaataaatgt tttttattag gcagaatcca gatgctcaag 7560

gcccttcata atatccccca gtttagtagt tggacttagg gaacaaagga acctttaata 7620

gaaattggac agcaagaaag cgagcttagt gatacttgtg atcctctaga tcacgacacc 7680

tgaaatggaa gaaaaaaact gcaccttcat cttgtccttt ccctcagaaa gaggctggga 7740

ggcagaggct gaggcagcgg tggccgggac ggttaggaga aaaggagtct ctgctggttt 7800

tattctgcag ctacctcccc aggaagtgga ggactgtggg gcctttgaga agcacctgcc 7860

gacagggcca agaaattcgc actccccctt tcggttcaca ggcaggaagc cctggaggtt 7920

tgagggtttg gggtgtgtgt atgtatctgt ctgtctgaat tttgcttttt ctctcatttg 7980

accattgttt taatgctcct ttttttaaaa aaaataattc ttatctaatt cctatcttga 8040

ttggtaaagt ccatctctag gcaaatacaa gttctcgatg gaaaacaata agtaatgtaa 8100

aatacagcat agcaaaactt taacctccaa atcaagcctc tacttgaatc cttttctgag 8160

ggatgaataa ggcataggca tcaggggctg ttgccaatgt gcattagctg tttgcagcct 8220

caccttcttt catggagttt aagatatagt gtattttccc aaggtttgaa ctagctcttc 8280

atttctttat gttttaaatg cactgacctc ccacattccc tttttagtaa aatattcaga 8340

aataatttaa atacatcatt gcaatgaaaa taaatgtttt ttattaggca gaatccagat 8400

gctcaaggcc cttcataata tcccccagtt tagtagttgg acttagggaa caaaggaacc 8460

tttaatagaa attggacagc aagaaagcga gcttagtgat acttgtaaaa agagacgcgt 8520

ctctaaaagt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 8580

tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 8640

ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 8700

tgggccgcct ccccgcctgg aattcgagct cggtaccttt aagaccaatg acttacaagg 8760

cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 8820

cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct 8880

gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 8940

cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 9000

tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta 9060

ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg 9120

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 9180

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc 9240

tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 9300

ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 9360

ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtatggcca 9420

caacctgggc tccccgggcg cgtactccac ctcacccatc atccacgctg ttttatgagt 9480

aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt 9540

aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt 9600

acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact 9660

actttctctt atggtgttca atgcttttca agatacccag atcatatgaa acggcatgac 9720

tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat 9780

gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga 9840

atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggacacaaattggaa 9900

tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa tggaatcaaa 9960

gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc agaccattat 10020

caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcc 10080

acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag 10140

tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata ggacctccat 10200

agaagacacc gggaccgatc caataacttc gtatagcata cattatacga agttatgcct 10260

ccggactcta gcgtttaaac ttaagcttgg gaagttccta ttccgaagtt cctattctct 10320

agaaagtata ggaacttcta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 10380

agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca 10440

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 10500

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 10560

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 10620

gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 10680

gcggaaagtt aaccctagaa agataatcat attgtgacgt acgttaaaga taatcatgcg 10740

taaaattgac gcatgtgttt tatcggtctg tatatcgagg tttatttatt aatttgaata 10800

gatattaagt tttattatat ttacacttac atactaataa taaattcaac aaacaattta 10860

tttatgttta tttatttatt aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac 10920

aaaaaccagc tggggctcga agttcctata ctttctagag aataggaact tctatagtga 10980

gtcgaataag ggcgacacaa aatttattct aaatgcataa taaatactga taacatctta 11040

tagtttgtat tatattttgt attatcgttg acatgtataa ttttgatatc aaaaactgat 11100

tttcccttta ttattttcga gatttatttt cttaattctc tttaacaaac tagaaatatt 11160

gtatatacaa aaaatcataa ataatagatg aatagtttaa ttataggtgt tcatcaatcg 11220

aaaaagcaac gtatcttatt taaagtgcgt tgcttttttc tcatttataa ggttaaataa 11280

ttctcatata tcaagcaaag tgacaggcgc ccttaaatat tctgacaaat gctctttccc 11340

taaactcccc ccataaaaaa acccgccgaa gcgggttttt acgttatttg cggattaacg 11400

attactcgtt atcagaaccg cccagggggc ccgagcttaa gactggccgt cgttttacaa 11460

cacagaaaga gtttgtagaa acgcaaaaag gccatccgtc aggggccttc tgcttagttt 11520

gatgcctggc agttccctac tctcgccttc cgcttcctcg ctcactgact cgctgcgctc 11580

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 11640

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 11700

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 11760

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 11820

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 11880

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 11940

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 12000

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 12060

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 12120

tgctacagag ttcttgaagt ggtgggctaa ctacggctac actagaagaa cagtatttgg 12180

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 12240

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 12300

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 12360

cgacgcgcgc gtaactcacg ttaagggatt ttggtcatga gcttgcgccg tcccgtcaag 12420

tcagcgtaat gctctgcttt tagaaaaact catcgagcat caaatgaaac tgcaatttat 12480

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 12540

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 12600

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 12660

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 12720

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 12780

cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac 12840

aattacaaac aggaatcgag tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 12900

tttcacctga atcaggatat tcttctaata cctggaacgc tgtttttccg gggatcgcag 12960

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagtggca 13020

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 13080

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaag cgatagattg 13140

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 13200

tgttggaatt taatcgcggc ctcgacgttt cccgttgaat atggctcata ttcttccttt 13260

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 13320

gtatttagaa aaataaacaa ataggggtca gtgttacaac caattaacca attctgaaca 13380

ttatcgcgag cccatttata cctgaatatg gctcataaca ccccttgttt gcctggcggc 13440

agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc 13500

gatggtagtg tggggactcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg 13560

aaaggctcag tcgaaagact gggcctttcg cccgggctaa ttagggggtg tcgcccttat 13620

tcgactctat agtgaagttc ctattctcta gaaagtatag gaacttctga agt 13673

<210>84

<211>15025

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic Dox-rapamycin inducible GAG PO L ENV

<400>84

gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata 60

acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca 120

ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac 180

gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg 240

ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc 300

tttctagggt taagacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca 360

atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 420

gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 480

atgaagaatc tgcttagggt taggcgtttt gaagttccta tactttctag agaataggaa 540

cttcggaata ggaacttcgg atgcaatttc ctcattttat taggaaagga cagtgggagt 600

ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact 660

agaaggcaca gcgttagtga tacttgtggg ccagggcatt agccacacca gccaccactt 720

tctgataggc agcctgcact ggtggggtga attccgcgga agcttgtgta attgttaatt 780

tctctgtccc actccatcca ggtcgtgtga ttccaaatct gttccagaga tttattactc 840

caactagcat tccaaggcac agcagtggtg caaatgagtt ttccagagca accccaaatc 900

cccaggagct gttgatcctt taggtatctt tccacagcca ggattcttgc ctggagctgc 960

ttgatgcccc agactgtgag ttgcaacaga tgctgttgcg cctcaatagc cctcagcaaa 1020

ttgttctgct gctgcactat accagacaat aattgtctgg cctgtaccgt cagcgtcatt 1080

gacgctgcgc ccatagtgct tcctgctgct cccaagaacc caaggaacaa agctcctgcg 1140

gccgctccgg aattccatgt gttaatcctc atcctgtcta cttgccacac aatcatcacc 1200

tgccatctgt tttccataat ccctgatgat ctttgctttt cttcttggca ctacttttat 1260

gtcactatta tcttgtatta ctactgcccc ttcacctttc cagaggagct ttgctggtcc 1320

tttccaaact ggatctctgc tgtccctgta ataaacccga aaattttgaa tttttgtaat 1380

ttgtttttgt aattctttag tttgtatgtc tgttgctatt atgtctacta ttctttcccc 1440

tgcactgtac cccccaatcc ccccttttct tttaaaattg tggatgaata ctgccatttg 1500

tactgctgtc ttaagatgtt cagcctgatc tcttacctgt cctataattt tctttaattc 1560

tttattcata gattctatta ctccttgact ttggggattg tagggaatgc caaattcctg 1620

cttgatcccc gcccaccaac aggcggcctt aactgtagta ctggtgaaat tgctgccatt 1680

gtctgtatgt actgttttta ctggccatct tcctgctaat tttaagagga agtatgctgt 1740

ttcttgccct gtctctgctg gaattacttc tgcttctata tatccactgg ctacatgaac 1800

tgctaccaag ataacttttc cttctaaatg tgtacaatct agctgccata ttcctgggct 1860

acagtctact tgtccatgca tggcttcccc ttttagctga catttatcac agctggctac 1920

tatttctttt gctactacag gtggtaggtt aaaatcacta gccattgctc tccaattact 1980

gtgatatttc tcatgttctt cttgggcctt atctattcca tctaaaaata gtactttcct 2040

gattccagca ctgaccaatt tatctacttg ttcatttcct ccaattcctt tgtgtgctgg 2100

tacccatgcc aggtagactt tttccttttt tattaactgc tctattattt gactgactaa 2160

ctctgattca ctcttatctg gttgtgcttg aatgattccc aatgcatatt gtgagtctgt 2220

cactatgttt acttctaatc ccgaatcctg caaagctaga tgaattgctt gtaactcagt 2280

cttctgattt gttgtgtccg ttagggggac aactttttgt cttcctctgt cagttacata 2340

tcctgctttt cctaatttag tttccctatt ggctgcccca tctacataga aagtttctgc 2400

tcctattatg ggttctttct ctaactggta ccataacttc actaagggag gggtattgac 2460

aaactcccac tcaggaatcc aggtggcttg ccaatactct gtccaccatg cttcccatgt 2520

ttccttttgt atgggtaatt taaatttagg agtctttccc catattacta tgctttctgt 2580

ggctattttt tgtactgcct ctgttaattg tttcacatca ttagtgtggg cacccttcat 2640

tcttgcatac tttcctgttt tcagattttt aaatggctct tgataaattt gatatgtcca 2700

ttggccttgc ccctgcttct gtatttctgc tattaagtct tttgatgggt cataatacac 2760

tccatgtacc ggttctttta gaatctccct gttttctgcc agttctagct ctgcttcttc 2820

tgttagtggt actacttctg ttagtgcttt ggttccccta agaagtttac ataattgcct 2880

tactttaatc cctgcataaa tctgacttgc ccaattcaat tttcccacta atttctgtat 2940

gtcattgaca gtccagctgt ccttttctgg cagcactata ggctgtactg tccatttatc 3000

aggatggagt tcataaccca tccaaaggaa tggaggttct ttctgatgtt ttttgtctgg 3060

tgtggtaaat ccccacctca acagatgttg tctcagttcc tctatttttg ttctatgctg 3120

ccctatttct aagtcagatc ctacatacaa atcatccatg tattgataga tgactatgtc 3180

tggattttgt tttctaaaag gctctaagat ttttgtcatg ctacactgga atattgctgg 3240

tgatcctttc catccctgtg gaagcacatt gtactgatat ctaatccctg gtgtctcatt 3300

gtttatacta ggtatggtaa atgcagtata cttcctgaag tctttatcta agggaactga 3360

aaaatatgca tcgcccacat ccagtactgt tactgatttt ttctgtttta accctgcagg 3420

atgtggtatt cctaattgaa cttcccagaa atcttgagtt ctcttattaa gttctctgaa 3480

atctactaat tttctccatt tagtactgtc ttttttcttt atggcaaata ctggagtatt 3540

gtatggattt tcaggcccaa tttttgaaat ttttccttcc ttttccattt ctgtacaaat 3600

ttctactaat gcttttattt tttcttctgt caatggccat tgtttaactt ttgggccatc 3660

cattcctggc tttaatttta ctggtacagt ctcaatagga ctaatgggaa aatttaaagt 3720

gcagccaatc tgagtcaaca gatttcttcc aattatgttg acaggtgtag gtcctactaa 3780

tactgtacct atagctttat gtccgcagat ttctatgagt atctgatcat actgtcttac 3840

tttgataaaa cctccaattc cccctatcat ttttggtttc catcttcctg gcaaattcat 3900

ttcttctaat actgtatcat ctgctcctgt atctaataga gcttccttta attgcccccc 3960

tatctttatt gtgacgaggg gtcgctgcca aagagtgatc tgagggaagc taaaggatac 4020

agttccttgt ctatcggctc ctgcttctga gagggagttg ttgtctcttc cccaaacctg 4080

aagctctctt ctggtggggc tgttggctct ggtctgctct gaagaaaatt ccctggcctt 4140

cccttgtggg aaggccagat cttccctaaa aaattagcct gtctctcagt acaatctttc 4200

atttggtgtc cttcctttcc acatttccaa cagccctttt tcctaggggc cctgcaattt 4260

ttggctatgt gcccttcttt gccacaattg aaacacttaa cagtctttct ttggttccta 4320

aaattgcctt tctgtatcat tatggtagct ggatttgtta cttggctcat tgcttcagcc 4380

aaaactcttg ctttatggcc gggtcccccc actccctgac atgctgtcat catttcttct 4440

agtgtcgctc ctggtcccaa tgcttttaaa atagtcttac aatctgggtt cgcattttgg 4500

accaacaagg tttctgtcat ccaatttttt acctcttgtg aagcttgctc ggctcttaga 4560

gttttataga atcggtctac atagtctcta aagggttcct ttggtccttg tcttatgtcc 4620

agaatgctgg tagggctata cattcttact attttattta atcccaggat tatccatctt 4680

ttatagattt ctcctactgg gataggtgga ttatgtgtca tccatcctat ttgttcctga 4740

agggtactag tagttcctgc tatgtcactt ccccttggtt ctctcatctg gcctggtgca 4800

ataggccctg catgcactgg atgcactcta tcccattctg cagcttcctc attgatggtc 4860

tcttttaaca tttgcatggc tgcttgatgt ccccccactg tgtttagcat ggtgtttaaa 4920

tcttgtgggg tggctccttc tgataatgct gaaaacatgg gtatcacttc tgggctgaaa 4980

gccttctctt ctactacttt tacccatgca tttaaagttc taggtgatat ggcctgatgt 5040

accatttgcc cctggatgtt ctgcactata gggtaatttt ggctgacctg attgctgtgt 5100

cctgtgtcag ctgctgcttg ctgtgctttt ttcttacttt tgttttgctc ttcctctatc 5160

ttgtctaaag cttccttggt gtcttttatc tctatccttt gatgcacaca atagagggtt 5220

gctactgtat tatataatga tctaagttct tctgatcctg tctgaaggga tggttgtagc 5280

tgtcccagta tttgtctaca gccttctgat gtttctaaca ggccaggatt aactgcgaat 5340

cgttctagct ccctgcttgc ccatactata tgttttaatt tatatttttt ctttccccct 5400

ggccttaacc gaattttttc ccatcgatct aattctcccc cgcttaatac tgacgctctc 5460

gcacccatgg cggcggcaga tctcgaattc agatctcacgtgctttgcca aagtgatggg 5520

ccagcacaca gaccagcacg ttgcccagga gctgtgggag gaagataaga ggtatgaaca 5580

tgattagcaa aagggcctag cttggactca gaataatcca gccttatccc aaccataaaa 5640

taaaagcaga atggtagctg gattgtagct gctattagca atatgaaacc tcttacatca 5700

gttacaattt atatgcagaa atatttatat gcagaaatat tgctattgcc ttaacccaga 5760

aattatcact gttattcttt agaatggtgc aaagaggcat gatacattgt atcattattg 5820

ccctgaaaga aagagattag ggaaagtatt agaaataaga taaacaaaaa agtatattaa 5880

aagaagaaag cattttttaa aattacaaat gcaaaattac cctgatttgg tcaatatgtg 5940

taccctgtta cttctcccct tcctatgaca tgaacttaac catagaaaag aaggggaaag 6000

aaaacatcaa gggtcccata gactcaccct gaagttctca ggatccgagc tcggtaccac 6060

atgtaagctt cgaggggagg ctggatcggt cccggtgtct tctatggagg tcaaaacagc 6120

gtggatggcg tctccaggcg atctgacggt tcactaaacg ctgcttcgcg atgtacgggc 6180

cagatatacg cgttgcgatc tgacggttca ctaaacgagc tctgcttata taggcctccc 6240

accgtacacg ccacctcgac atactcgagt ttactcccta tcagtgatag agaacgtatg 6300

aagagtttac tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat 6360

agagaacgta taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc 6420

cctatcagtg atagagaacg tatctacagt ttactcccta tcagtgatag agaacgtata 6480

tccagtttac tccctatcag tgatagagaa cgtataagct ttaggcgtgt acggtgggcg 6540

cctataaaag cagagctcgt ttagtgaacc gtcagatcgc ctggagcaat tccacaacac 6600

ttttgtctta taccaacttt ccgtaccact tcctaccctc gtaaatcgtc gacgagctcg 6660

tttagtgaac cgtcagatcg cctggagacg ccctcgaagc cgcggtgcgg gtgccagggc 6720

gtgcccttgg gctccatgtc catcatgggt ctcaaggtga acgtctctgc catattcatg 6780

gcagtactgt taactctcca aacacccacc ggtcaaatcc attggggcaa tctctctaag 6840

ataggggtgg taggaatagg aagtgcaagc tacaaagtta tgactcgttc cagccatcaa 6900

tcattagtca taaaattaat gcccaatata actctcctca ataactgcac gagggtagag 6960

attgcagaat acaggagact actgagaaca gttttggaac caattagaga tgcacttaat 7020

gcaatgaccc agaatataag accggttcag agtgtagctt caagtaggag acacaagaga 7080

tttgcgggag tagtcctggc aggtgcggcc ctaggcgttg ccacagctgc tcagataaca 7140

gccggcattg cacttcacca gtccatgctg aactctcaag ccatcgacaa tctgagagcg 7200

agcctggaaa ctactaatca ggcaattgag gcaatcagac aagcagggca ggagatgata 7260

ttggctgttc agggtgtcca agactacatc aataatgagc tgataccgtc tatgaaccaa 7320

ctatcttgtg atttaatcgg ccagaagctc gggctcaaat tgctcagata ctatacagaa 7380

atcctgtcat tatttggccc cagcttacgg gaccccatat ctgcggagat atctatccag 7440

gctttgagct atgcgcttgg aggagacatc aataaggtgt tagaaaagct cggatacagt 7500

ggaggtgatt tactgggcat cttagagagc agaggaataa aggcccggat aactcacgtc 7560

gacacagagt cctacttcat tgtcctcagt atagcctatc cgacgctgtc cgagattaag 7620

ggggtgattg tccaccggct agagggggtc tcgtacaaca taggctctca agagtggtat 7680

accactgtgc ccaagtatgt tgcaacccaa gggtacctta tctcgaattt tgatgagtca 7740

tcgtgtactt tcatgccaga ggggactgtg tgcagccaaa atgccttgta cccgatgagt 7800

cctctgctcc aagaatgcct ccgggggtcc accaagtcct gtgctcgtac actcgtatcc 7860

gggtcttttg ggaaccggtt cattttatca caagggaacc taatagccaa ttgtgcatca 7920

atcctttgca agtgttacac aacaggaacg atcattaatc aagaccctga caagatccta 7980

acatacattg ctgccgatca ctgcccggta gtcgaggtga acggcgtgac catccaagtc 8040

gggagcagga ggtatccaga cgctgtgtac ttgcacagaa ttgacctcgg tcctcccata 8100

tcattggaga ggttggacgt agggacaaat ctggggaatg caattgctaa gttggaggat 8160

gccaaggaat tgttggagtc atcggaccag atattgagga gtatgaaagg tttatcgagc 8220

actagcatag tctacatcct gattgcagtg tgtcttggag ggttgatagg gatccccgct 8280

ttaatatgtt gctgcagggg gcgttgaccc ctctccctcc ccccccccta acgttactgg 8340

ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 8400

gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 8460

taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 8520

agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 8580

gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 8640

tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 8700

atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 8760

tatgggatct gatctggggc ctcggtacac atgctttaca tgtgtttagt cgaggttaaa 8820

aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata 8880

atatggccac aaccatggga agtaggatag tcattaacag agaacatctt atgattgata 8940

gaccttatgt tttgctggct gttctgtttg tcatgtttct gagcttgatc gggttgctag 9000

ccattgcagg cattagactt catcgggcag ccatctacac cgcagagatc cataaaagcc 9060

tcagcaccaa tctagatgta actaactcaa tcgagcatca ggtcaaggac gtgctgacac 9120

cactcttcaa aatcatcggt gatgaagtgg gcctgaggac acctcagaga ttcactgacc 9180

tagtgaaatt catctctgac aagattaaat tccttaatcc ggatagggag tacgacttca 9240

gagatctcac ttggtgtatc aacccgccag agagaatcaa attggattat gatcaatact 9300

gtgcagatgt ggctgctgaa gagctcatga atgcattggt gaactcaact ctactggaga 9360

ccagaacaac caatcagttc ctagctgtct caaagggaaa ctgctcaggg cccactacaa 9420

tcagaggtca attctcaaac atgtcgctgt ccctgttaga cttgtattta ggtcgaggtt 9480

acaatgtgtc atctatagtc actatgacat cccagggaat gtatggggga acttacctag 9540

tggaaaagcc taatctgagc agcaaaaggt cagagttgtc acaactgagc atgtaccgag 9600

tgtttgaagt aggtgttatc agaaatccgg gtttgggggc tccggtgttc catatgacaa 9660

actatcttga gcaaccagtc agtaatgatc tcagcaactg tatggtggct ttgggggagc 9720

tcaaactcgc agccctttgt cacggggaag attctatcac aattccctat cagggatcag 9780

ggaaaggtgt cagcttccag ctcgtcaagc taggtgtctg gaaatcccca accgacatgc 9840

aatcctgggt ccccttatca acggatgatc cagtgataga caggctttac ctctcatctc 9900

acagaggtgt tatcgctgac aaycaagcaa aatgggctgt cccgacaaca cgaacagatg 9960

acaagttgcg aatggagaca tgcttccaac aggcgtgtaa gggtaaaatc caagcactct 10020

gcgagaatcc cgagtgggca ccattgaagg ataacaggat tccttcatac ggggtcttgt 10080

ctgttgatct gagtctgaca gttgagctta aaatcaaaat tgcttcggga ttcgggccat 10140

tgatcacaca cggttcaggg atggacctat acaaatccaa ccacaacaat gtgtattggc 10200

tgactatccc rccaatgaag aacctagcct taggtgtaat caacacattg gagtggatac 10260

cgagattcaa ggttagtccc tacctcttca mtgtcccaat taaggaagca ggcgaagact 10320

gccatgcccc aacataccta cctgcggagg tggatggtga tgtcaaactc agttccaatc 10380

tggtgattct acctggtcaa gatctccaat atgttttggc aacctacgat acttccaggg 10440

ttgaacatgc tgtggtttat tacgtttaca gcccaagccg ctcattttct tacttttatc 10500

cttttaggtt gcctataaag ggggtcccca tcgaattaca agtggaatgc ttcacatggg 10560

accaaaaact ctggtgccgt cacttctgtg tgcttgcgga ctcagaatct ggtggacata 10620

tcactcactc tgggatggtg ggcatgggag tcagctgcac agtcacccgg gaagatggaa 10680

ccaatcgcag atagggctgc tagtgaacya atcwcatgat gtcacccaga catcaggcat 10740

acccactagt gtgaaataga catcagaatt aagaaaaatg ggctccccgg gcgcgtactc 10800

cacctcaccc atcatccacg ctcggcaata aaaagacaga ataaaacgca cgggtgttgg 10860

gtcgtttgtt cgccgggcgc gtactccacc tcacccatca tccacgctgt tttatggata 10920

gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt gcacatggag ggctccgtga 10980

acggccacga gttcgagatc gagggcgagg gcgagggcaa gccctacgag ggcacccaga 11040

ccgccaagct gcaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc 11100

cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca 11160

agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg 11220

gcgtggtgac cgtgacccag gactcctccc tgcaggacgg caccttcatc taccacgtga 11280

agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actctgggct 11340

gggagccctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca 11400

aggcgctgaa gctgaagggc ggcggccact acctggtgga gttcaagtca atctacatgg 11460

ccaagaagcc cgtgaagctg cccggctact actacgtgga ctccaagctg gacatcacct 11520

cccacaacga ggactacacc gtggtggagc agtacgagcg cgccgaggcc cgccaccacc 11580

tgttccagta ggacctccat agaagacacc gggaccgatc caataacttc gtatagcata 11640

cattatacga agttatgcct ccggactcta gcgtttaaac ttaagcttgg taccgagctc 11700

ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg 11760

agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 11820

catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 11880

tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 11940

tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 12000

ctggggatgc ggtgggctct atggcttctg aggcggaaag ttaaccctag aaagataatc 12060

atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc 12120

tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt 12180

acatactaat aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa 12240

acaaaaactc aaaatttctt ctataaagta acaaaaacca gctggggctc gaagttccta 12300

tactttctag agaataggaa cttctatagt gagtcgaata agggcgacac aaaatttatt 12360

ctaaatgcat aataaatact gataacatct tatagtttgt attatatttt gtattatcgt 12420

tgacatgtat aattttgata tcaaaaactg attttccctt tattattttc gagatttatt 12480

ttcttaattc tctttaacaa actagaaata ttgtatatac aaaaaatcat aaataataga 12540

tgaatagttt aattataggt gttcatcaat cgaaaaagca acgtatctta tttaaagtgc 12600

gttgcttttt tctcatttat aaggttaaat aattctcata tatcaagcaa agtgacaggc 12660

gcccttaaat attctgacaa atgctctttc cctaaactcc ccccataaaa aaacccgccg 12720

aagcgggttt ttacgttatt tgcggattaa cgattactcg ttatcagaac cgcccagggg 12780

gcccgagctt aagactggcc gtcgttttac aacacagaaa gagtttgtag aaacgcaaaa 12840

aggccatccg tcaggggcct tctgcttagt ttgatgcctg gcagttccct actctcgcct 12900

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 12960

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 13020

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 13080

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 13140

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 13200

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 13260

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 13320

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 13380

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 13440

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtgggct 13500

aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 13560

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 13620

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 13680

atcttttcta cggggtctga cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga 13740

ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta atgctctgct tttagaaaaa 13800

ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt 13860

ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc 13920

aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt 13980

cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg 14040

tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg 14100

ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc 14160

gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg agtgcaaccg 14220

gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa 14280

tacctggaac gctgtttttc cggggatcgc agtggtgagt aaccatgcat catcaggagt 14340

acggataaaa tgcttgatgg tcggaagtgg cataaattcc gtcagccagt ttagtctgac 14400

catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg 14460

cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg 14520

agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgacgt 14580

ttcccgttga atatggctca tattcttcct ttttcaatat tattgaagca tttatcaggg 14640

ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 14700

cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta tacctgaata 14760

tggctcataa caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca 14820

tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga 14880

gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 14940

cgcccgggct aattaggggg tgtcgccctt attcgactct atagtgaagt tcctattctc 15000

tagaaagtat aggaacttct gaagt15025

<210>85

<211>6584

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic rapamycin inducible TET activators

<400>85

gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata 60

acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca 120

ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac 180

gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg 240

ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc 300

tttctagggt taagacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca 360

atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 420

gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 480

atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 540

acgcgttctc catagaagac accgaataaa atatctttat tttcattaca tctgtgtgtt 600

ggttttttgt gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa 660

aacaaactag caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctgg 720

accgatccaa taacttcgta tagcatacat tatacgaagt tatgcctccg gactctagcg 780

ttttagttat tactagcgct accggactca gatctcgagc tcaagcttcg aattctgcag 840

tcgacggtac cgcggcttac gcgtgctagc taatgatggg cgctcgagta atgatgggcg 900

gtcgactaat gatgggcgct cgagtaatga tgggcgtcta gctaatgatg ggcgctcgag 960

taatgatggg cggtcgacta atgatgggcg ctcgagtaat gatgggcgtc tagctaatga 1020

tgggcgctcg agtaatgatg ggcggtcgac taatgatggg cgctcgagta atgatgggcg 1080

tctagaacgc gaattaattc aacattttga cacccccata atatttttcc agaattaaca 1140

gtataaattg catctcttgt tcaagagttc cctatcactc tctttaatca ctactcacag 1200

taacctcaac tcctgccaca agcttgccct gcagcgggaa ttccaaactt aagcttggta 1260

ccgagctcgg atccactagt ccagtgtggt ggaattctgc agatatccag cacagtggcg 1320

gccgctcgag tctagagggc ccgtttaaac ccgctgatca atgtctagac tggacaagag 1380

caaagtcata aactctgctc tggaattact caatggagtc ggtatcgaag gcctgacgac 1440

aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa 1500

caagcgggcc ctgctcgatg ccctgccaat cgagatgctg gacaggcatc atacccactc 1560

ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcataccg 1620

ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga 1680

gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct 1740

ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga 1800

ggaacaggag catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc 1860

cccacttctg aaacaagcaa ttgagctgtt cgaccggcag ggagccgaac ctgccttcct 1920

tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg 1980

gccgaccgac gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga 2040

ctttgacctt gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc 2100

cgggtaagtc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 2160

tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 2220

cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 2280

ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 2340

caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 2400

ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 2460

cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 2520

aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 2580

tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg 2640

gggacgtggt tttcctttga aaaacacgat gataaatgga tagcactgag aacgtcatca 2700

agcccttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga 2760

tcgagggcga gggcgagggc aagccctacg agggcaccca gaccgccaag ctgcaggtga 2820

ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct 2880

ccaaggtgta cgtgaagcac cccgccgacatccccgacta caagaagctg tccttccccg 2940

agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc 3000

aggactcctc cctgcaggac ggcaccttca tctaccacgt gaagttcatc ggcgtgaact 3060

tcccctccga cggccccgta atgcagaaga agactctggg ctgggagccc tccaccgagc 3120

gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca caaggcgctg aagctgaagg 3180

gcggcggcca ctacctggtg gagttcaagt caatctacat ggccaagaag cccgtgaagc 3240

tgcccggcta ctactacgtg gactccaagc tggacatcac ctcccacaac gaggactaca 3300

ccgtggtgga gcagtacgag cgcgccgagg cccgccacca cctgttccag tagctcgact 3360

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3420

gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3480

agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3540

gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaagt 3600

taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc gtaaaattga 3660

cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag 3720

ttttattata tttacactta catactaata ataaattcaa caaacaattt atttatgttt 3780

atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaaaccag 3840

ctggggctcg aagttcctat actttctaga gaataggaac ttctatagtg agtcgaataa 3900

gggcgacaca aaatttattc taaatgcata ataaatactg ataacatctt atagtttgta 3960

ttatattttg tattatcgtt gacatgtata attttgatat caaaaactga ttttcccttt 4020

attattttcg agatttattt tcttaattct ctttaacaaa ctagaaatat tgtatataca 4080

aaaaatcata aataatagat gaatagttta attataggtg ttcatcaatc gaaaaagcaa 4140

cgtatcttat ttaaagtgcg ttgctttttt ctcatttata aggttaaata attctcatat 4200

atcaagcaaa gtgacaggcg cccttaaata ttctgacaaa tgctctttcc ctaaactccc 4260

cccataaaaa aacccgccga agcgggtttt tacgttattt gcggattaac gattactcgt 4320

tatcagaacc gcccaggggg cccgagctta agactggccg tcgttttaca acacagaaag 4380

agtttgtaga aacgcaaaaa ggccatccgt caggggcctt ctgcttagtt tgatgcctgg 4440

cagttcccta ctctcgcctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4500

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4560

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4620

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4680

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4740

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4800

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4860

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4920

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4980

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga5040

gttcttgaag tggtgggcta actacggcta cactagaaga acagtatttg gtatctgcgc 5100

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5160

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5220

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgcg 5280

cgtaactcac gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa 5340

tgctctgctt ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag 5400

gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga 5460

ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat 5520

caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat 5580

gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt 5640

caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca 5700

ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga caattacaaa 5760

caggaatcga gtgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg 5820

aatcaggata ttcttctaat acctggaacg ctgtttttcc ggggatcgca gtggtgagta 5880

accatgcatc atcaggagta cggataaaat gcttgatggt cggaagtggc ataaattccg 5940

tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat 6000

gtttcagaaa caactctggc gcatcgggct tcccatacaa gcgatagatt gtcgcacctg 6060

attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat 6120

ttaatcgcgg cctcgacgtt tcccgttgaa tatggctcat attcttcctt tttcaatatt 6180

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 6240

aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga 6300

gcccatttat acctgaatat ggctcataac accccttgtt tgcctggcgg cagtagcgcg 6360

gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt 6420

gtggggactc cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca 6480

gtcgaaagac tgggcctttc gcccgggcta attagggggt gtcgccctta ttcgactcta 6540

tagtgaagtt cctattctct agaaagtata ggaacttctg aagt 6584

<210>86

<211>11528

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic rapamycin inducible REV srcVpx

<400>86

gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata 60

acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca 120

ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac 180

gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg 240

ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc 300

tttctagggt taagacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca 360

atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 420

gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 480

atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 540

acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 600

catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 660

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 720

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 780

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 840

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 900

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 960

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 1020

ttgttttgga accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 1080

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcggcattga 1140

ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg 1200

gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 1260

cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 1320

tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat1380

catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 1440

gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc 1500

gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac 1560

tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 1620

aatcaacggg actttccaaa atgtcgtaac aactgcgatc gcccgccccg ttgacgcaaa 1680

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 1740

agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg 1800

cttctgacac aacagtctcg aacttaagct gcagtgactc tcttaaggta gccttgcaga 1860

agttggtcgt gaggcactgg gcaggtaagt atcaaggtta caagacaggt ttaaggagac 1920

caatagaaac tgggcttgtc gagacagaga agactcttgc gtttctgata ggcacctatt 1980

ggtcttactg acatccactt tgcctttctc tccacaggtg tccactccca gttcaattac 2040

agctcttaag gctagagtac ttaatacgac tcactatagg ctagcctcga gaattcatgg 2100

cttctagaat cctctggcat gagatgtggc atgaaggcct ggaagaggca tctcgtttgt 2160

actttgggga aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg catgctatga 2220

tggaacgggg cccccagact ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt 2280

taatggaggc ccaagagtgg tgcaggaagt acatgaaatc agggaatgtc aaggacctcc 2340

tccaagcctg ggacctctat tatcatgtgt tccgacgaat ctcaaagact agagatgagt 2400

ttcccaccat ggtgtttcct tctgggcaga tcagccaggc ctcggccttg gccccggccc 2460

ctccccaagt cctgccccag gctccagccc ctgcccctgc tccagccatg gtatcagctc 2520

tggcccaggc cccagcccct gtcccagtcc tagccccagg ccctcctcag gctgtggccc 2580

cacctgcccc caagcccacc caggctgggg aaggaacgct gtcagaggcc ctgctgcagc 2640

tgcagtttga tgatgaagac ctgggggcct tgcttggcaa cagcacagac ccagctgtgt 2700

tcacagacct ggcatccgtc gacaactccg agtttcagca gctgctgaac cagggcatac 2760

ctgtggcccc ccacacaact gagcccatgc tgatggagta ccctgaggct ataactcgcc 2820

tagtgacagg ggcccagagg ccccccgacc cagctcctgc tccactgggg gccccggggc 2880

tccccaatgg cctcctttca ggagatgaag acttctcctc cattgcggac atggacttct 2940

cagccctgct gagtcagatc agctccacta gttattaaga attcacgcgt cgagcatgca 3000

tctagggcgg ccaattccgc ccctctcccc cccccccctc tccctccccc ccccctaacg 3060

ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca 3120

ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga 3180

gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga 3240

aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca 3300

ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag 3360

atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa 3420

gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac 3480

cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga 3540

ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac 3600

gatgataagc ttgccacaac ccgggatcct ctagagtcga catggactat cctgctgcca 3660

agagggtcaa gttggactct agagaacgcc catatgcttg ccctgtcgag tcctgcgatc 3720

gccgcttttc tcgctcggat gagcttaccc gccatatccg catccacaca ggccagaagc 3780

ccttccagtg tcgaatctgc atgcgtaact tcagtcgtag tgaccacctt accacccaca 3840

tccgcaccca cacaggcggc ggccgcagga ggaagaaacg caccagcata gagaccaaca 3900

tccgtgtggc cttagagaag agtttcttgg agaatcaaaa gcctacctcg gaagagatca 3960

ctatgattgc tgatcagctc aatatggaaa aagaggtgat tcgtgtttgg ttctgtaacc 4020

gccgccagaa agaaaaaaga atcaacacta gaggagtgca ggtggaaacc atctccccgg 4080

gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac accgggatgc 4140

ttgaagatgg aaagaaattt gattcctccc gggacagaaa caagcccttt aagtttatgc 4200

taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg agtgtgggtc 4260

agagagccaa actgactata tctccagatt atgcctatgg tgccactggg cacccaggca 4320

tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg gaagtcgagg 4380

gcgtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag cgcggccaga 4440

cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaatttgat tcctcccggg 4500

acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga ggctgggaag 4560

aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct ccagattatg 4620

cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc gtcttcgatg 4680

tggagcttct aaaactggaa actagaggag tgcaggtgga aaccatctcc ccaggagacg 4740

ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg atgcttgaag 4800

atggaaagaa atttgattcc tcccgggaca gaaacaagcc ctttaagttt atgctaggca 4860

agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg ggtcagagag 4920

ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca ggcatcatcc 4980

caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaaact agttattaag 5040

tcgacccggg cggccgcttc cctttagtga gggttaatgc ttcgagcaga catgataaga 5100

tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 5160

gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 5220

ctccatagaa gacaccggga ccgatccaat aacttcgtat agcatacatt atacgaagtt 5280

atggctgcta gtgaacyaat cwcatgatgt cacccagaca tcaggcatac ccactagtgt 5340

gaaatagaca tcagaattaa gaaaaatggg ctccccgggc gcgtactcca cctcacccat 5400

catccacgct cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttcg 5460

ttttatggat agcactgaga acgtcatcaa gcccttcatg cgcttcaagg tgcacatgga 5520

gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggca agccctacga 5580

gggcacccag accgccaagctgcaggtgac caagggcggc cccctgccct tcgcctggga 5640

catcctgtcc ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat 5700

ccccgactac aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt 5760

cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcaccttcat 5820

ctaccacgtg aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa 5880

gactctgggc tgggagccct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg 5940

cgagatccac aaggcgctga agctgaaggg cggcggccac tacctggtgg agttcaagtc 6000

aatctacatg gccaagaagc ccgtgaagct gcccggctac tactacgtgg actccaagct 6060

ggacatcacc tcccacaacg aggactacac cgtggtggag cagtacgagc gcgccgaggc 6120

ccgccaccac ctgttccagt aggacctcca tagaagacac cgaataaaat atctttattt 6180

tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat 6240

caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt 6300

gccagaacat ttctctggac cgatccaata acttcgtata gcatacatta tacgaagtta 6360

tgcctccgga ctctagcgtt ttagttatta ctagcgctac cggactcaga tctcgagctc 6420

aagcttcgaa ttctgcagtc gacggtaccg cggcttacgc gtgctagcta atgatgggcg 6480

ctcgagtaat gatgggcggt cgactaatga tgggcgctcg agtaatgatg ggcgtctagc 6540

taatgatggg cgctcgagta atgatgggcg gtcgactaat gatgggcgct cgagtaatga 6600

tgggcgtcta gctaatgatg ggcgctcgag taatgatggg cggtcgacta atgatgggcg 6660

ctcgagtaat gatgggcgtc tagaacgcga attaattcaa cattttgaca cccccataat 6720

atttttccag aattaacagt ataaattgca tctcttgttc aagagttccc tatcactctc 6780

tttaatcact actcacagta acctcaactc ctgccacaag cttgccctgc agcgggaatt 6840

ccaaacttaa gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag 6900

atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcaga 6960

tggcaggaag aagcggagac agcgacgaag acctcctcaa ggcagtcaga ctcatcaagt 7020

ttctctatca aagcaaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 7080

gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcctta 7140

gcacttattt gggacgatct gcggacgctg tgcctcttca gctaccaccg cttgagagac 7200

ttactcttga ttgtgacgag gattgtggaa cttctgggac gcagggggtg ggaagccctc 7260

aaatattggt ggaatctcct acaatattgg agtcaggagc taaagaatag tccctccccc 7320

ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 7380

ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 7440

ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 7500

aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 7560

accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 7620

cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 7680

gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 7740

cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 7800

gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt 7860

gaaaaacacg atgataatgg ggaggaggaa gaggaagccg aaggatccga aggcgagggt 7920

gttggcggag gcggattaca aggacgacga tgacaagatg tcagatccca gggagagaat 7980

cccacctgga aacagtggag aagagacaat aggagaggcc ttcgaatggc taaacagaac 8040

agtagaggag ataaacagag aggcagtaaa ccacctacca agggagctga ttttccaggt 8100

ttggcaaagg tcttgggaat actggcatga tgaacaaggg atgtcacaaa gctatgtaaa 8160

atacagatac ttgtgtttaa tgcaaaaggc tttatttatg cattgcaaga aaggctgtag 8220

atgtctaggg gaaggacacg gggcaggagg atggagacca ggacctcctc ctcctccccc 8280

tccaggacta gcataacctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 8340

tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 8400

gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 8460

caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 8520

tctatggctt ctgaggcgga aagttaaccc tagaaagata atcatattgt gacgtacgtt 8580

aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg gtctgtatat cgaggtttat 8640

ttattaattt gaatagatat taagttttat tatatttaca cttacatact aataataaat 8700

tcaacaaaca atttatttat gtttatttat ttattaaaaa aaaacaaaaa ctcaaaattt 8760

cttctataaa gtaacaaaaa ccagctgggg ctcgaagttc ctatactttc tagagaatag 8820

gaacttctat agtgagtcga ataagggcga cacaaaattt attctaaatg cataataaat 8880

actgataaca tcttatagtt tgtattatat tttgtattat cgttgacatg tataattttg 8940

atatcaaaaa ctgattttcc ctttattatt ttcgagattt attttcttaa ttctctttaa 9000

caaactagaa atattgtata tacaaaaaat cataaataat agatgaatag tttaattata 9060

ggtgttcatc aatcgaaaaa gcaacgtatc ttatttaaag tgcgttgctt ttttctcatt 9120

tataaggtta aataattctc atatatcaag caaagtgaca ggcgccctta aatattctga 9180

caaatgctct ttccctaaac tccccccata aaaaaacccg ccgaagcggg tttttacgtt 9240

atttgcggat taacgattac tcgttatcag aaccgcccag ggggcccgag cttaagactg 9300

gccgtcgttt tacaacacag aaagagtttg tagaaacgca aaaaggccat ccgtcagggg 9360

ccttctgctt agtttgatgc ctggcagttc cctactctcg ccttccgctt cctcgctcac 9420

tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 9480

aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 9540

gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 9600

ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 9660

ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 9720

gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 9780

ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 9840

cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 9900

cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 9960

gaggtatgta ggcggtgcta cagagttctt gaagtggtgg gctaactacg gctacactag 10020

aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 10080

tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 10140

gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 10200

tgacgctcag tggaacgacg cgcgcgtaac tcacgttaag ggattttggt catgagcttg 10260

cgccgtcccg tcaagtcagc gtaatgctct gcttttagaa aaactcatcg agcatcaaat 10320

gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct 10380

gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt 10440

ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa 10500

ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt 10560

tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac 10620

tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgaggcga aatacgcgat 10680

cgctgttaaa aggacaatta caaacaggaa tcgagtgcaa ccggcgcagg aacactgcca 10740

gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aacgctgttt 10800

ttccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga 10860

tggtcggaag tggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat 10920

cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat 10980

acaagcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat 11040

ataaatcagc atccatgttg gaatttaatc gcggcctcga cgtttcccgt tgaatatggc 11100

tcatattctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 11160

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcagtgtt acaaccaatt 11220

aaccaattct gaacattatc gcgagcccat ttatacctga atatggctca taacacccct 11280

tgtttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg 11340

aaacgccgta gcgccgatgg tagtgtgggg actccccatg cgagagtagg gaactgccag 11400

gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgcccgg gctaattagg 11460

gggtgtcgcc cttattcgac tctatagtga agttcctatt ctctagaaag tataggaact 11520

tctgaagt 11528

<210>87

<400>87

000

<210>88

<400>88

000

<210>89

<400>89

000

<210>90

<400>90

000

<210>91

<400>91

000

<210>92

<400>92

000

<210>93

<400>93

000

<210>94

<400>94

000

<210>95

<400>95

000

<210>96

<400>96

000

<210>97

<400>97

000

<210>98

<400>98

000

<210>99

<400>99

000

<210>100

<400>100

000

<210>101

<211>461

<212>PRT

<213> Intelligent people

<400>101

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 Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Thr Ile Ser Lys Cys His Leu Ser Phe Phe Ser Val Ala

245 250 255

Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro

260 265 270

Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu

275 280 285

Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser

290 295 300

Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp

305 310 315 320

Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu

325 330 335

Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro

340 345 350

Ser Glu Asp Val Val Val Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser

355 360 365

Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu

370 375 380

Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro

385 390 395 400

His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr

405 410 415

Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro

420 425 430

Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu

435 440 445

Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

450 455 460

<210>102

<211>463

<212>PRT

<213> Intelligent people

<400>102

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 510 15

Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Phe Ser Cys Gly Pro Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser

245 250 255

Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile

260 265 270

Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu

275 280 285

His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro

290 295 300

Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala

305 310 315 320

Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu

325 330335

Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn

340 345 350

Cys Pro Ser Glu Asp Val Val Val Thr Pro Glu Ser Phe Gly Arg Asp

355 360 365

Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro

370 375 380

Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn

385 390 395 400

Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn

405 410 415

Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu

420 425 430

Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn

435 440 445

Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

450 455 460

<210>103

<211>462

<212>PRT

<213> Intelligent people

<400>103

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 Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Thr Cys His Leu Ile Ser Ile Leu Ser Phe Phe Ser Val

245 250 255

Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys

260 265 270

Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His

275 280 285

Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu

290 295 300

Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg

305 310 315 320

Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu

325 330 335

Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys

340 345 350

Pro Ser Glu Asp Val Val Val Thr Pro Glu Ser Phe Gly Arg Asp Ser

355 360 365

Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile

370 375 380

Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly

385 390 395 400

Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser

405 410 415

Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn

420 425 430

Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln

435 440 445

Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

450 455 460

<210>104

<211>466

<212>PRT

<213> Intelligent people

<400>104

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 Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Thr Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu LysAsp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Thr Pro Pro Val Cys Ser Val Thr Ile Ser Ile Leu Ser

245 250 255

Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys

260 265 270

Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys

275 280 285

Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser

290 295 300

Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp

305 310 315 320

Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr PhePro

325 330 335

Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln

340 345 350

Ser Pro Asn Cys Pro Ser Glu Asp Val Val Val Thr Pro Glu Ser Phe

355 360 365

Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys

370 375 380

Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser

385 390 395 400

Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly

405 410 415

Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile

420 425 430

Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu

435 440 445

Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln

450 455 460

Asn Gln

465

<210>105

<211>523

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Fc-delta-30

<400>105

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>106

<211>599

<212>PRT

<213> Artificial sequence

<220>

<223> synthesized Hc-delta-18

<400>106

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 HisGln 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>107

<211>529

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-I L7-DAF fusion

<400>107

Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu Pro Leu Leu Gly

1 5 10 15

Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys Leu Pro Ala Asp

20 25 30

Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu Met

35 40 45

Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser Asn

50 55 60

Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp Ala

65 70 75 80

Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln

85 90 95

Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu Lys

100 105 110

Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val Lys

115 120 125

Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu

130 135 140

Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys

145 150 155 160

Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile

165 170 175

Leu Met Gly Thr Lys Glu His Cys Gly Leu Pro Pro Asp Val Pro Asn

180 185 190

Ala Gln Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu Asp Thr Val

195 200 205

Ile Thr Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile Pro Gly Glu Lys

210 215 220

Asp Ser Val Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu

225 230 235 240

Phe Cys Asn Arg Ser Cys Glu Val Pro Thr Arg Leu Asn Ser Ala Ser

245 250 255

Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly Thr Val

260 265 270

Val Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser

275 280 285

Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala Val Glu

290 295 300

Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly

305 310 315 320

Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe

325 330 335

Ser Cys Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys

340 345 350

Leu Ile Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu Pro Glu Cys

355 360 365

Arg Glu Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile

370 375 380

Gln Gly Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala

385390 395 400

Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr Cys Thr

405 410 415

Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg

420 425 430

Gly Lys Ser Leu Thr Ser Lys Val Pro Pro Thr Val Gln Lys Pro Thr

435 440 445

Thr Val Asn Val Pro Thr Thr Glu Val Ser Pro Thr Ser Gln Lys Thr

450 455 460

Thr Thr Lys Thr Thr Thr Pro Asn Ala Gln Ala Thr Arg Ser Thr Pro

465 470 475 480

Val Ser Arg Thr Thr Lys His Phe His Glu Thr Thr Pro Asn Lys Gly

485 490 495

Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr Cys

500 505 510

Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu Leu

515 520 525

Thr

<210>108

<400>108

000

<210>109

<400>109

000

<210>110

<400>110

000

<210>111

<400>111

000

<210>112

<400>112

000

<210>113

<400>113

000

<210>114

<400>114

000

<210>115

<400>115

000

<210>116

<400>116

000

<210>117

<400>117

000

<210>118

<400>118

000

<210>119

<400>119

000

<210>120

<400>120

000

<210>121

<400>121

000

<210>122

<400>122

000

<210>123

<400>123

000

<210>124

<400>124

000

<210>125

<400>125

000

<210>126

<400>126

000

<210>127

<400>127

000

<210>128

<400>128

000

<210>129

<400>129

000

<210>130

<400>130

000

<210>131

<400>131

000

<210>132

<400>132

000

<210>133

<400>133

000

<210>134

<400>134

000

<210>135

<400>135

000

<210>136

<400>136

000

<210>137

<400>137

000

<210>138

<400>138

000

<210>139

<400>139

000

<210>140

<400>140

000

<210>141

<400>141

000

<210>142

<400>142

000

<210>143

<400>143

000

<210>144

<400>144

000

<210>145

<400>145

000

<210>146

<400>146

000

<210>147

<400>147

000

<210>148

<400>148

000

<210>149

<400>149

000

<210>150

<400>150

000

<210>151

<400>151

000

<210>152

<400>152

000

<210>153

<400>153

000

<210>154

<400>154

000

<210>155

<400>155

000

<210>156

<400>156

000

<210>157

<400>157

000

<210>158

<400>158

000

<210>159

<400>159

000

<210>160

<400>160

000

<210>161

<400>161

000

<210>162

<400>162

000

<210>163

<400>163

000

<210>164

<400>164

000

<210>165

<400>165

000

<210>166

<400>166

000

<210>167

<400>167

000

<210>168

<400>168

000

<210>169

<400>169

000

<210>170

<400>170

000

<210>171

<400>171

000

<210>172

<400>172

000

<210>173

<400>173

000

<210>174

<400>174

000

<210>175

<400>175

000

<210>176

<400>176

000

<210>177

<400>177

000

<210>178

<400>178

000

<210>179

<400>179

000

<210>180

<400>180

000

<210>181

<400>181

000

<210>182

<400>182

000

<210>183

<400>183

000

<210>184

<400>184

000

<210>185

<400>185

000

<210>186

<400>186

000

<210>187

<400>187

000

<210>188

<400>188

000

<210>189

<400>189

000

<210>190

<400>190

000

<210>191

<400>191

000

<210>192

<400>192

000

<210>193

<400>193

000

<210>194

<400>194

000

<210>195

<400>195

000

<210>196

<400>196

000

<210>197

<400>197

000

<210>198

<400>198

000

<210>199

<400>199

000

<210>200

<400>200

000

<210>201

<400>201

000

<210>202

<400>202

000

<210>203

<400>203

000

<210>204

<400>204

000

<210>205

<400>205

000

<210>206

<400>206

000

<210>207

<400>207

000

<210>208

<400>208

000

<210>209

<400>209

000

<210>210

<400>210

000

<210>211

<400>211

000

<210>212

<400>212

000

<210>213

<400>213

000

<210>214

<400>214

000

<210>215

<400>215

000

<210>216

<400>216

000

<210>217

<400>217

000

<210>218

<400>218

000

<210>219

<400>219

000

<210>220

<400>220

000

<210>221

<400>221

000

<210>222

<400>222

000

<210>223

<400>223

000

<210>224

<400>224

000

<210>225

<400>225

000

<210>226

<400>226

000

<210>227

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Src-Flag-Vpx membrane targeting domain

<400>227

Met Gly Ser Ser Lys Ser Lys Pro Lys Asp Pro

1 5 10

<210>228

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic viral protease cleavage Domain

<400>228

Lys Ala Arg Val Leu Ala Glu Ala

1 5

<210>229

<211>458

<212>PRT

<213> Intelligent people

<400>229

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 Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu

245 250 255

Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val

260 265 270

Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys

275 280 285

Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu

290 295 300

Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val

305 310 315 320

Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu

325 330 335

Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu

340 345 350

Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr

355 360 365

Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser

370 375 380

Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val

385 390 395 400

Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro

405 410 415

Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala

420 425 430

Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala

435 440 445

Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn

450 455

<210>230

<211>723

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic GFP-linker-P2A-I L7 Ra IncPPC L codon (except P2A) and splice optimized

<400>230

Met Ser Gly Gly Glu Glu Leu Phe Ala Gly Ile Val Pro Val Leu Ile

1 5 10 15

Glu Leu Asp Gly Asp Val His Gly His Lys Phe Ser Val Arg Gly Glu

20 25 30

Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu Ile Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Cys Tyr Gly Ile Gln Cys Phe Ala Arg Tyr Pro Glu His Met Lys Met

65 70 75 80

Asn Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg

85 90 95

Thr Ile Gln Phe Gln Asp Asp Gly Lys Tyr Lys Thr Arg Gly Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Lys

115 120125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Ser

130 135 140

Phe Asn Ser His Asn Val Tyr Ile Arg Pro Asp Lys Ala Asn Asn Gly

145 150 155 160

Leu Glu Ala Asn Phe Lys Thr Arg His Asn Ile Glu Gly Gly Gly Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro Leu Gly Asp Gly Pro

180 185 190

Val Leu Ile Pro Ile Asn His Tyr Leu Ser Thr Gln Thr Lys Ile Ser

195 200 205

Lys Asp Arg Asn Glu Ala Arg Asp His Met Val Leu Leu Glu Ser Phe

210 215 220

Ser Ala Cys Cys His Thr His Gly Met Asp Glu Leu Tyr Arg Gly Ser

225 230 235 240

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

245 250 255

Asn Pro Gly Pro Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe

260 265 270

Ser Leu Leu Gln Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly

275 280 285

Asp Leu Glu Asp Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser

290 295 300

Gln Leu Glu Val Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu

305 310 315 320

Asp Pro Asp Val Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala

325 330 335

Leu Val Glu Val Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr

340 345 350

Phe Ile Glu Thr Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys

355 360 365

Val Lys Val Gly Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr

370 375 380

Thr Ile Val Lys Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg

385 390 395 400

Glu Gly Ala Asn Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln

405 410 415

Lys Lys Tyr Val Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu

420 425 430

Lys Asp Glu Asn Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu

435 440 445

Thr Leu Leu Gln Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys

450 455 460

Val Arg Ser Ile Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp

465 470 475 480

Ser Pro Ser Tyr Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly

485 490 495

Glu Met Asp Pro Ile Leu Leu Pro Pro Cys Leu Thr Ile Ser Ile Leu

500 505 510

Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp

515 520 525

Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys

530 535 540

Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val

545 550 555 560

Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp

565 570 575

Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe

580 585 590

Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val

595 600 605

Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser

610 615 620

Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala

625 630 635 640

Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu

645 650 655

Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu

660 665 670

Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly

675 680 685

Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser

690 695 700

Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr

705 710 715 720

Gln Asn Gln

<210>231

<211>733

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic GFP-linker-P2A-Myc Tag-I L7 Ra IncPPC L

Codon (except P2A) and splice-optimized

<400>231

Met Ser Gly Gly Glu Glu Leu Phe Ala Gly Ile Val Pro Val Leu Ile

1 5 10 15

Glu Leu Asp Gly Asp Val His Gly His Lys Phe Ser Val Arg Gly Glu

20 25 30

Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu Ile Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Cys Tyr Gly Ile Gln Cys Phe Ala Arg Tyr Pro Glu His Met Lys Met

65 70 75 80

Asn Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg

85 90 95

Thr Ile Gln Phe Gln Asp Asp Gly Lys Tyr Lys Thr Arg Gly Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Lys

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Ser

130 135 140

Phe Asn Ser His Asn Val Tyr Ile Arg Pro Asp Lys Ala Asn Asn Gly

145 150 155 160

Leu Glu Ala Asn Phe Lys Thr Arg His Asn Ile Glu Gly Gly Gly Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro Leu Gly Asp Gly Pro

180 185 190

Val Leu Ile Pro Ile Asn His Tyr Leu Ser Thr Gln Thr Lys Ile Ser

195 200 205

Lys Asp Arg Asn Glu Ala Arg Asp His Met Val Leu Leu Glu Ser Phe

210 215 220

Ser Ala Cys Cys His Thr His Gly Met Asp Glu Leu Tyr Arg Gly Ser

225 230 235 240

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

245 250 255

Asn Pro Gly Pro Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe

260 265 270

Ser Leu Leu Gln Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu

275 280 285

Asp Leu Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu

290 295 300

Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly

305 310 315 320

Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Ile

325 330 335

Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys

340 345 350

Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys

355 360 365

Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys

370 375 380

Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu

385 390 395 400

Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn Asp Phe

405 410 415

Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val

420 425 430

Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp

435 440 445

Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys

450 455 460

Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp

465 470 475 480

His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe

485 490 495

Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro Ile Leu

500 505 510

Leu Pro Pro Cys Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala

515 520 525

Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro

530 535 540

Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu

545 550 555 560

Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser

565 570 575

Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp

580 585 590

Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu

595 600 605

Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro

610 615 620

Ser Glu Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser

625 630 635 640

Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu

645 650 655

Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro

660 665 670

His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr

675 680 685

Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro

690 695 700

Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu

705 710 715 720

Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

725 730

<210>232

<211>572

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic GFP-linker-P2A-I L7 RaSP-Myc Tag-I L7 RaIncPPC L

C-terminal truncated codon (except for P2A) and splicing

<400>232

Met Ser Gly Gly Glu Glu Leu Phe Ala Gly Ile Val Pro Val Leu Ile

1 5 10 15

Glu Leu Asp Gly Asp Val His Gly His Lys Phe Ser Val Arg Gly Glu

20 25 30

Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu Ile Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Cys Tyr Gly Ile Gln Cys Phe Ala Arg Tyr Pro Glu His Met Lys Met

65 70 75 80

Asn Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg

85 90 95

Thr Ile Gln Phe Gln Asp Asp Gly Lys Tyr Lys Thr Arg Gly Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Lys

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Ser

130 135 140

Phe Asn Ser His Asn Val Tyr Ile Arg Pro Asp Lys Ala Asn Asn Gly

145 150 155 160

Leu Glu Ala Asn Phe Lys Thr Arg His Asn Ile Glu Gly Gly Gly Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro Leu Gly Asp Gly Pro

180 185 190

Val Leu Ile Pro Ile Asn His Tyr Leu Ser Thr Gln Thr Lys Ile Ser

195 200 205

Lys Asp Arg Asn Glu Ala Arg Asp His Met Val Leu Leu Glu Ser Phe

210 215 220

Ser Ala Cys Cys His Thr His Gly Met Asp Glu Leu Tyr Arg Gly Ser

225 230 235 240

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

245 250 255

Asn Pro Gly Pro Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe

260 265 270

Ser Leu Leu Gln Val Val Ser Gly Glu Gln Lys Leu Ile Ser Glu Glu

275 280 285

Asp Leu Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu

290 295 300

Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly

305 310 315 320

Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Ile

325 330 335

Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys

340 345 350

Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys

355 360 365

Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys

370 375 380

Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu

385 390 395 400

Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn Asp Phe

405 410 415

Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val

420 425 430

Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp

435 440 445

Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys

450 455 460

Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp

465 470 475 480

His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe

485 490 495

Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro Ile Leu

500505 510

Leu Pro Pro Cys Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala

515 520 525

Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro

530 535 540

Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu

545 550 555 560

Cys Lys Lys Pro Arg Lys Val Ser Val Phe Gly Ala

565 570

<210>233

<211>562

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic GFP-linker-P2A-I L7 RaSP-I L7 RaIncPPC L

C-terminal truncated codon (except P2A) and splice-optimized

<400>233

Met Ser Gly Gly Glu Glu Leu Phe Ala Gly Ile Val Pro Val Leu Ile

1 5 10 15

Glu Leu Asp Gly Asp Val His Gly His Lys Phe Ser Val Arg Gly Glu

20 25 30

Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu Ile Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Cys Tyr Gly Ile Gln Cys Phe Ala Arg Tyr Pro Glu His Met Lys Met

65 70 75 80

Asn Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg

85 90 95

Thr Ile Gln Phe Gln Asp Asp Gly Lys Tyr Lys Thr Arg Gly Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Lys

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Ser

130 135 140

Phe Asn Ser His Asn Val Tyr Ile Arg Pro Asp Lys Ala Asn Asn Gly

145 150 155 160

Leu Glu Ala Asn Phe Lys Thr Arg His Asn Ile Glu Gly Gly Gly Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro Leu Gly Asp Gly Pro

180 185 190

Val Leu Ile Pro Ile Asn His Tyr Leu Ser Thr Gln Thr Lys Ile Ser

195 200 205

Lys Asp Arg AsnGlu Ala Arg Asp His Met Val Leu Leu Glu Ser Phe

210 215 220

Ser Ala Cys Cys His Thr His Gly Met Asp Glu Leu Tyr Arg Gly Ser

225 230 235 240

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

245 250 255

Asn Pro Gly Pro Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe

260 265 270

Ser Leu Leu Gln Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly

275 280 285

Asp Leu Glu Asp Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser

290 295 300

Gln Leu Glu Val Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu

305 310 315 320

Asp Pro Asp Val Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala

325 330 335

Leu Val Glu Val Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr

340 345 350

Phe Ile Glu Thr Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys

355 360 365

Val Lys Val Gly Glu LysSer Leu Thr Cys Lys Lys Ile Asp Leu Thr

370 375 380

Thr Ile Val Lys Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg

385 390 395 400

Glu Gly Ala Asn Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln

405 410 415

Lys Lys Tyr Val Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu

420 425 430

Lys Asp Glu Asn Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu

435 440 445

Thr Leu Leu Gln Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys

450 455 460

Val Arg Ser Ile Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp

465 470 475 480

Ser Pro Ser Tyr Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly

485 490 495

Glu Met Asp Pro Ile Leu Leu Pro Pro Cys Leu Thr Ile Ser Ile Leu

500 505 510

Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp

515 520 525

Lys Lys Arg Ile Lys Pro Ile ValTrp Pro Ser Leu Pro Asp His Lys

530 535 540

Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Val Ser Val Phe

545 550 555 560

Gly Ala

<210>234

<211>824

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic GFP-linker-P2A-eTag-I L7 RaIncPPC L

N-terminally deleted codons (except P2A) and splice-optimized

<400>234

Met Ser Gly Gly Glu Glu Leu Phe Ala Gly Ile Val Pro Val Leu Ile

1 5 10 15

Glu Leu Asp Gly Asp Val His Gly His Lys Phe Ser Val Arg Gly Glu

20 25 30

Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu Ile Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Cys Tyr Gly Ile Gln Cys Phe Ala Arg Tyr Pro Glu His Met Lys Met

65 70 75 80

Asn Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg

85 90 95

Thr Ile Gln Phe Gln Asp Asp Gly Lys Tyr Lys Thr Arg Gly Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Lys

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Ser

130 135 140

Phe Asn Ser His Asn Val Tyr Ile Arg Pro Asp Lys Ala Asn Asn Gly

145 150 155 160

Leu Glu Ala Asn Phe Lys Thr Arg His Asn Ile Glu Gly Gly Gly Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro Leu Gly Asp Gly Pro

180 185 190

Val Leu Ile Pro Ile Asn His Tyr Leu Ser Thr Gln Thr Lys Ile Ser

195 200 205

Lys Asp Arg Asn Glu Ala Arg Asp His Met Val Leu Leu Glu Ser Phe

210 215 220

Ser Ala Cys Cys His Thr His Gly Met Asp Glu Leu Tyr Arg Gly Ser

225 230 235 240

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

245 250 255

Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu

260 265 270

Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly

275 280 285

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

290 295 300

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

305 310 315 320

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

325 330 335

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

340 345 350

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

355 360 365

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

370 375 380

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

385 390 395 400

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

405 410 415

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

420 425 430

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

435 440 445

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

450 455 460

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

465 470 475 480

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

485 490 495

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

500 505 510

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

515 520 525

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

530 535 540

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

545 550 555 560

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

565 570 575

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Glu Ile

580 585 590

Asn Asn Ser Ser Gly Glu Met Asp Pro Ile Leu Leu Pro Pro Cys Leu

595 600 605

Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu

610 615 620

Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser

625 630 635 640

Leu Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg

645 650 655

Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln

660 665 670

Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe

675 680 685

Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg

690 695 700

Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val

705 710 715 720

Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala

725 730 735

Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser

740 745 750

Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp

755 760 765

Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe

770 775 780

Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln

785 790 795 800

Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr

805 810 815

Met Ser Ser Phe Tyr Gln Asn Gln

820

<210>235

<211>593

<212>PRT

<213> Artificial sequence

<220>

<223> synthesized MV (ed) -HD24

<400>235

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

180185 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

340345 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 505510

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>236

<400>236

000

<210>237

<400>237

000

<210>238

<400>238

000

<210>239

<400>239

000

<210>240

<400>240

000

<210>241

<400>241

000

<210>242

<400>242

000

<210>243

<400>243

000

<210>244

<400>244

000

<210>245

<400>245

000

<210>246

<400>246

000

<210>247

<400>247

000

<210>248

<400>248

000

<210>249

<400>249

000

<210>250

<400>250

000

<210>251

<400>251

000

<210>252

<211>560

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-aCD3scFv (UCHT1) IgG1 Fc-CD14GPI

<400>252

Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu Pro Leu Leu Gly

1 5 10 15

Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys Leu Pro 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

145150 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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

275 280 285

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

290 295 300

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

305310 315 320

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

325 330 335

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

340 345 350

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

355 360 365

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

370 375 380

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

385 390 395 400

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

405 410 415

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

420 425 430

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

435 440 445

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

450 455 460

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

465 470475 480

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

485 490 495

Gln Lys Ser Leu Ser Leu Ser Pro Gly Val Asp Asn Leu Thr Leu Asp

500 505 510

Gly Asn Pro Phe Leu Val Pro Gly Thr Ala Leu Pro His Glu Gly Ser

515 520 525

Met Asn Ser Gly Val Val Pro Ala Cys Ala Arg Ser Thr Leu Ser Val

530 535 540

Gly Val Ser Gly Thr Leu Val Leu Leu Gln Gly Ala Arg Gly Phe Ala

545 550 555 560

<210>253

<211>276

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-CD80ECD-CD16GPI

<400>253

Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu Pro Leu Leu Gly

1 5 10 15

Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys Leu Pro Val Ile

20 25 30

His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly His

35 40 45

Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys

50 55 60

Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp

65 70 75 80

Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser

85 90 95

Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys

100 105 110

Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala

115 120 125

Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser

130 135 140

Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr

145 150 155 160

Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu

165 170 175

Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu

180 185 190

Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His

195 200 205

Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr

210 215 220

Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn Val Ser

225 230 235 240

Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln Val Ser Phe Cys Leu

245 250 255

Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val

260 265 270

Lys Thr Asn Ile

275

<210>254

<211>533

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-I L7-DAF

<400>254

Met Ala Thr Thr Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu

1 5 10 15

Pro Leu Leu Gly Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys

20 25 30

Leu Pro Ala Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu

35 40 45

Ser Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu

50 55 60

Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His

65 70 75 80

Ile Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg

85 90 95

Lys Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu

100 105 110

His Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr

115 120 125

Gly Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro

130 135 140

Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu

145 150 155 160

Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys

165 170 175

Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His Cys Gly Leu Pro Pro

180 185 190

Asp Val Pro Asn Ala Gln Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro

195 200 205

Glu Asp Thr Val Ile Thr Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile

210 215 220

Pro Gly Glu Lys Asp Ser Val Ile Cys Leu Lys Gly Ser Gln Trp Ser

225 230 235 240

Asp Ile Glu Glu Phe Cys Asn Arg Ser Cys Glu Val Pro Thr Arg Leu

245 250 255

Asn Ser Ala Ser Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro

260 265 270

Val Gly Thr Val Val Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu

275 280 285

Pro Ser Leu Ser Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser

290 295 300

Thr Ala Val Glu Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu

305 310 315 320

Ile Arg Asn Gly Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly Ala

325 330 335

Thr Ile Ser Phe Ser Cys Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr

340 345 350

Ser Ser Phe Cys Leu Ile Ser Gly Ser Ser Val Gln Trp Ser Asp Pro

355 360 365

Leu Pro Glu Cys Arg Glu Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp

370 375 380

Asn Gly Ile Ile Gln Gly Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser

385 390 395 400

Val Thr Tyr Ala Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser

405 410 415

Ile Tyr Cys Thr Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro

420 425 430

Pro Glu Cys Arg Gly Lys Ser Leu Thr Ser Lys Val Pro Pro Thr Val

435 440 445

Gln Lys Pro Thr Thr Val Asn Val Pro Thr Thr Glu Val Ser Pro Thr

450 455 460

Ser Gln Lys Thr Thr Thr Lys Thr Thr Thr Pro Asn Ala Gln Ala Thr

465 470 475 480

Arg Ser Thr Pro Val Ser Arg Thr Thr Lys His Phe His Glu Thr Thr

485 490 495

Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser

500 505 510

Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr

515 520 525

Met Gly Leu Leu Thr

530

<210>255

<211>1823

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic EF-1a promoter and having miRs

<400>255

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

tagttcctgg aggcttgctg aaggctgtat gctgacatgg tacagttcaa tggtggtttt 1020

ggccactgac tgaccaccat tgctgtacca tgtcaggaca caaggcctgt tactagcact 1080

cacatggaac aaatggccca cattggtgcc ggatgaagct cttatgttgc acggtcatct 1140

ggaggcttgc tgaaggctgt atgctgtcag tctgttcatc ttctggcgtt ttggccactg 1200

actgacgcca gaaggaacag actgacagga cacaaggcct gttactagca ctcacatgga 1260

acaaatggcc gttgccggag tcttggcagc gagagatcac tatcaactaa ctggaggctt 1320

gctgaaggct gtatgctgaa gcgtgaagtg aatcaacggg ttttggccac tgactgaccc 1380

gttgatactt cacgcttcag gacacaaggc ctgttactag cactcacatg gaacaaatgg 1440

ccgtgttaat tgtccatgta gcgaggcatc cttatggcgt ggctggaggc ttgctgaagg 1500

ctgtatgctg gcagtatcct agtacattga cgttttggcc actgactgac gtcaatgtta 1560

ggatactgcc aggacacaag gcctgttact agcactcaca tggaacaaat ggccgctttt 1620

ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg 1680

agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg 1740

ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt 1800

ttcttccatt tcaggtgtcg tga1823

<210>256

<211>28

<212>DNA

<213> mouse

<400>256

ctggaggctt gctgaaggct gtatgctg 28

<210>257

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>257

acatggtaca gttcaatggt g 21

<210>258

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA coding Loop

<400>258

gttttggcca ctgactgac 19

<210>259

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA-encoding stem

<400>259

caccattgct gtaccatgt19

<210>260

<211>45

<212>DNA

<213> mouse

<400>260

caggacacaa ggcctgttac tagcactcac atggaacaaa tggcc 45

<210>261

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>261

tcagtctgtt catcttctgg c 21

<210>262

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>262

gccagaagga acagactga 19

<210>263

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>263

aagcgtgaag tgaatcaacg g 21

<210>264

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>264

ccgttgatac ttcacgctt 19

<210>265

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>265

gcagtatcct agtacattga c 21

<210>266

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>266

gtcaatgtta ggatactgc 19

<210>267

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>267

atatgtactt ggctggacag c 21

<210>268

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>268

gctgtccaca agtacatat 19

<210>269

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA-encoding linker

<400>269

cacattggtg ccggatgaag ctcttatgtt gccggtcat 39

<210>270

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>270

ctgttaatgc taatcgtgat a 21

<210>271

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>271

tatcacgatt attaacag 18

<210>272

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA-encoding linker

<400>272

gttgccggag tcttggcagc gagagatcac tatcaactaa 40

<210>273

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>273

taccagttta gcacgaagct c 21

<210>274

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>274

gagcttcgct aaactggta19

<210>275

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA-encoding linker

<400>275

gtgttaattg tccatgtagc gaggcatcct tatggcgtgg 40

<210>276

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>276

tgccgctgaa atccaaggca a 21

<210>277

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic DNA encoding MiRNA Stem

<400>277

ttgccttgtt tcagcggca 19

<210>278

<211>534

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic UCHT1scFvFc-GPI

<400>278

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

145 150 155 160

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

165 170 175

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

180 185 190

Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

195 200 205

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 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 Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp

260 265 270

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

305 310 315 320

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Thr Pro Ile Glu

370 375 380

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

385 390 395 400

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

405 410 415

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

420 425 430

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

435 440 445

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

450 455 460

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

485 490 495

Gly Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu

500 505 510

Ser Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val

515 520 525

Thr Met Gly Leu Leu Thr

530

<210>279

<211>530

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic OKT3scFvFc-GPI

<400>279

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Ser Gly Ala Arg Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

50 55 60

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser

145 150 155 160

Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr

165 170 175

Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

180 185 190

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

195 200 205

Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met

210 215 220

Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

225 230 235 240

Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr

245 250 255

Thr Leu Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr

260 265 270

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

275 280 285

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

290 295 300

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

305 310 315 320

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

325 330 335

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

340 345 350

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

355 360 365

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

370 375 380

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

385 390 395 400

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

405 410 415

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

420 425 430

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

435 440 445

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Pro Asn Lys

485 490 495

Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr

500 505 510

Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu

515 520 525

Leu Thr

530

<210>280

<211>280

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic hCD80-CD16 GPI

<400>280

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr

1 5 10 15

Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys

20 25 30

Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu

35 40 45

Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

50 55 60

Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

65 70 75 80

Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

85 90 95

Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly

100 105 110

Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

115 120 125

Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

130 135 140

Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

145 150 155 160

Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu

165 170 175

Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp

180 185 190

Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met

195 200 205

Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg

210 215 220

Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro

225 230 235 240

Asp Asn Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln Val

245 250 255

Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu

260 265 270

Tyr Phe Ser Val Lys Thr Asn Ile

275 280

<210>281

<400>281

000

<210>282

<400>282

000

<210>283

<400>283

000

<210>284

<211>357

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic C1 control polypeptide comprising the signal sequence of GMCSF alpha chain (amino acids 1-22) and eTAG (amino acids 23-357) nucleotides

<400>284

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 9095

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 250255

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>285

<211>240

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic OKT3ScFv

<400>285

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr MetThr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

115 120 125

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

130 135 140

Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

145 150 155 160

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

165 170 175

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

180 185 190

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

195 200 205

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

210 215 220

Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

225 230 235 240

<210>286

<211>532

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-I L7-DAF fusion

<400>286

Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu Pro Leu Leu Gly

1 5 10 15

Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys Leu Pro Ala Val

20 25 30

Trp Gly Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser

35 40 45

Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile

50 55 60

Gly Ser Asn Cys Leu AsnAsn Glu Phe Asn Phe Phe Lys Arg His Ile

65 70 75 80

Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys

85 90 95

Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His

100 105 110

Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly

115 120 125

Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr

130 135 140

Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn

145 150 155 160

Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp

165 170 175

Asn Lys Ile Leu Met Gly Thr Lys Glu His Cys Gly Leu Pro Pro Asp

180 185 190

Val Pro Asn Ala Gln Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu

195 200 205

Asp Thr Val Ile Thr Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile Pro

210 215 220

Gly Glu Lys Asp Ser Val Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp

225 230 235 240

Ile Glu Glu Phe Cys Asn Arg Ser Cys Glu Val Pro Thr Arg Leu Asn

245 250 255

Ser Ala Ser Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val

260 265 270

Gly Thr Val Val Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro

275 280 285

Ser Leu Ser Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr

290 295 300

Ala Val Glu Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile

305 310 315 320

Arg Asn Gly Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly Ala Thr

325 330 335

Ile Ser Phe Ser Cys Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser

340 345 350

Ser Phe Cys Leu Ile Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu

355 360 365

Pro Glu Cys Arg Glu Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn

370 375 380

Gly Ile Ile Gln Gly Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val

385 390 395 400

Thr Tyr Ala Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile

405 410 415

Tyr Cys Thr Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro

420 425 430

Glu Cys Arg Gly Lys Ser Leu Thr Ser Lys Val Pro Pro Thr Val Gln

435 440 445

Lys Pro Thr Thr Val Asn Val Pro Thr Thr Glu Val Ser Pro Thr Ser

450 455 460

Gln Lys Thr Thr Thr Lys Thr Thr Thr Pro Asn Ala Gln Ala Thr Arg

465 470 475 480

Ser Thr Pro Val Ser Arg Thr Thr Lys His Phe His Glu Thr Thr Pro

485 490 495

Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly

500 505 510

His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr Met

515 520 525

Gly Leu Leu Thr

530

<210>287

<211>564

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-aCD3scFv (UCHT1) IgG1 Fc-CD14GPI

<400>287

Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu Pro Leu Leu Gly

1 5 10 15

Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys Leu Pro Ala Val

20 25 30

Trp Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

35 40 45

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg

50 55 60

Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

65 70 75 80

Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe

85 90 95

Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu

100 105 110

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu

115 120 125

Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

145 150 155 160

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

165 170 175

Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

180 185 190

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn

195 200 205

Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe

210 215 220

Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

225 230 235 240

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly

245 250 255

Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr

260 265 270

Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr

275 280 285

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

290 295 300

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

305 310 315 320

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

325 330 335

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

340 345 350

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

355 360 365

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

370 375 380

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

385 390 395 400

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

405 410 415

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

420 425 430

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

435 440 445

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

450 455 460

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

465 470 475 480

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

485 490 495

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Val Asp Asn

500 505 510

Leu Thr Leu Asp Gly Asn Pro Phe Leu Val Pro Gly Thr Ala Leu Pro

515 520 525

His Glu Gly Ser Met Asn Ser Gly Val Val Pro Ala Cys Ala Arg Ser

530 535 540

Thr Leu Ser Val Gly Val Ser Gly Thr Leu Val Leu Leu Gln Gly Ala

545 550 555 560

Arg Gly Phe Ala

<210>288

<211>280

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic DAFss-CD80ECD-CD16GPI

<400>288

Met Thr Val Ala Arg Pro Ser Val Pro Ala Ala Leu Pro Leu Leu Gly

1 5 10 15

Glu Leu Pro Arg Leu Leu Leu Leu Val Leu Leu Cys Leu Pro Ala Val

20 25 30

Trp Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu

35 40 45

Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

50 55 60

Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

65 70 75 80

Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

85 90 95

Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly

100 105 110

Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

115 120 125

Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

130 135 140

Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

145 150 155 160

Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu

165 170 175

Glu Asn Gly Glu Glu Leu Asn Ala IleAsn Thr Thr Val Ser Gln Asp

180 185 190

Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met

195 200 205

Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg

210 215 220

Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro

225 230 235 240

Asp Asn Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln Val

245 250 255

Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu

260 265 270

Tyr Phe Ser Val Lys Thr Asn Ile

275 280

<210>289

<400>289

000

<210>290

<400>290

000

<210>291

<400>291

000

<210>292

<211>368

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M001

<400>292

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 AspGly 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 CysHis 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>293

<211>232

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M002

<400>293

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 ArgGly 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>294

<211>194

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M007

<400>294

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>295

<211>174

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M008

<400>295

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>296

<211>184

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M009

<400>296

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 ThrLeu Leu Val Asp Leu Leu Trp Leu Leu Leu

165 170 175

Phe Leu Ala Ile Leu Ile Trp Met

180

<210>297

<211>162

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M010

<400>297

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>298

<211>363

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M012

<400>298

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 GlyGlu 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>299

<211>227

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M013

<400>299

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>300

<211>354

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M018

<400>300

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>301

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M019

<400>301

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

130135 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>302

<211>360

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M024

<400>302

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 4045

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>303

<211>224

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M025

<400>303

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>304

<211>359

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M030

<400>304

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>305

<211>223

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M031

<400>305

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>306

<211>368

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M036

<400>306

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>307

<211>232

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M037

<400>307

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>308

<211>360

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M042

<400>308

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 155160

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>309

<211>224

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M043

<400>309

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>310

<211>359

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M048

<400>310

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>311

<211>223

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide M049

<400>311

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>312

<211>368

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E006

<400>312

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>313

<211>369

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E007

<400>313

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>314

<211>370

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E008

<400>314

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

370

<210>315

<211>371

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E009

<400>315

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

3540 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 200205

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 360365

Ala Ala Ala

370

<210>316

<211>372

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E010

<400>316

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 GlnHis 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 ThrCys 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>317

<211>69

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E011

<400>317

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

2025 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>318

<211>70

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E012

<400>318

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>319

<211>71

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E013

<400>319

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>320

<211>72

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E014

<400>320

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>321

<211>73

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide E015

<400>321

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>322

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T001

<400>322

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>323

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T002

<400>323

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>324

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T003

<400>324

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>325

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T004

<400>325

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>326

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T005

<400>326

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>327

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T006

<400>327

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>328

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T007

<400>328

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>329

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T008

<400>329

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>330

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T009

<400>330

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>331

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T010

<400>331

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>332

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T011

<400>332

Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu

1 5 1015

Leu Val Leu Val Phe Ile

20

<210>333

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T012

<400>333

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>334

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T013

<400>334

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>335

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T014

<400>335

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>336

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T015

<400>336

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>337

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T016

<400>337

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>338

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T017

<400>338

Met Trp Val Leu Ala Leu Ile Val Ile Phe Leu Thr Ile Ala Val Leu

1 5 10 15

Leu Ala Leu

<210>339

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T018

<400>339

Met Trp Val Leu Ala Leu Ile Glu Ile Phe Leu Thr Ile Ala Val Leu

1 5 10 15

Leu Ala Leu

<210>340

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T019

<400>340

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>341

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T020

<400>341

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>342

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T021

<400>342

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>343

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T022

<400>343

Cys Cys Leu Thr Leu Ser Leu Ile Leu Val Val Ile Leu ValLeu Leu

1 5 10 15

Thr Val Leu Ala Leu Leu Ser

20

<210>344

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T023

<400>344

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>345

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T024

<400>345

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>346

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T025

<400>346

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>347

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T026

<400>347

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>348

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T027

<400>348

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>349

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T028

<400>349

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>350

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T029

<400>350

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>351

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T030

<400>351

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>352

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T031

<400>352

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>353

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T032

<400>353

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>354

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T033

<400>354

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>355

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T034

<400>355

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>356

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T035

<400>356

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>357

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T036

<400>357

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>358

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T037

<400>358

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>359

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T038

<400>359

Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu

1 5 1015

Ser Gly Leu

<210>360

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T039

<400>360

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>361

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T040

<400>361

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>362

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T041

<400>362

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>363

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T042

<400>363

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>364

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T043

<400>364

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>365

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T044

<400>365

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>366

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T045

<400>366

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>367

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T046

<400>367

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>368

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T047

<400>368

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>369

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T048

<400>369

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>370

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T049

<400>370

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>371

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T050

<400>371

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>372

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T051

<400>372

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>373

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T052

<400>373

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>374

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T053

<400>374

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>375

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T054

<400>375

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>376

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T055

<400>376

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>377

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T056

<400>377

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>378

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T057

<400>378

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>379

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T058

<400>379

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>380

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T059

<400>380

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>381

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T060

<400>381

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>382

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T061

<400>382

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>383

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T062

<400>383

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>384

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T063

<400>384

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>385

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T064

<400>385

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>386

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T065

<400>386

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>387

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T066

<400>387

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>388

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T067

<400>388

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>389

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T068

<400>389

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>390

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T069

<400>390

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>391

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T070

<400>391

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>392

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T071

<400>392

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>393

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T072

<400>393

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>394

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T073

<400>394

Val Gly Leu Ile Ile Ala Ile Leu Ile Pro Val Ala Val Ala Val Ile

1 510 15

Val Gly Val Val Thr Ser Ile Leu Cys

20 25

<210>395

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T074

<400>395

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>396

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T075

<400>396

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>397

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T076

<400>397

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>398

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T077

<400>398

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>399

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T078

<400>399

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>400

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T079

<400>400

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>401

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T080

<400>401

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>402

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T081

<400>402

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>403

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide T082

<400>403

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>404

<211>117

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S036

<400>404

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>405

<211>45

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S037

<400>405

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>406

<211>55

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S038

<400>406

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>407

<211>45

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S039

<400>407

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

3540 45

<210>408

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S042

<400>408

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>409

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S043

<400>409

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>410

<211>48

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S044

<400>410

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>411

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S045

<400>411

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>412

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S046

<400>412

Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Phe Tyr Lys

1 5 10 15

<210>413

<211>48

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S047

<400>413

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>414

<211>41

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S048

<400>414

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>415

<211>41

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S049

<400>415

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>416

<211>62

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S050

<400>416

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

2025 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>417

<211>66

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S051

<400>417

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>418

<211>61

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S052

<400>418

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>419

<211>49

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S053

<400>419

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>420

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S054

<400>420

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>421

<211>437

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S057

<400>421

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 LeuPro 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>422

<211>54

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S058

<400>422

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>423

<211>64

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S059

<400>423

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>424

<211>186

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S062

<400>424

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>425

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S063

<400>425

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>426

<211>133

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S064

<400>426

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

115120 125

Ile Thr Ser Val Leu

130

<210>427

<211>235

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S069

<400>427

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>428

<211>235

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S072

<400>428

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 CysPro 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>429

<211>42

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S074

<400>429

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>430

<211>77

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S075

<400>430

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>431

<211>77

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S076

<400>431

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>432

<211>350

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S077

<400>432

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 7075 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 230235 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>433

<211>38

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S080

<400>433

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 510 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>434

<211>100

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S081

<400>434

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>435

<211>251

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S082

<400>435

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>436

<211>67

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S083

<400>436

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>437

<211>223

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S084

<400>437

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>438

<211>69

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S085

<400>438

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>439

<211>271

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S086

<400>439

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>440

<211>242

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S087

<400>440

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>441

<211>179

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S098

<400>441

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>442

<211>210

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S099

<400>442

Lys Ile Asp Ile Val Leu Trp Tyr Arg Asp Ser Cys Tyr Asp Phe Leu

1 5 1015

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>443

<211>182

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S100

<400>443

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>444

<211>299

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S101

<400>444

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

180185 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>445

<211>207

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S102

<400>445

Leu Lys Met Phe Trp Ile Glu Ala Thr Leu Leu Trp Arg Asp Ile Ala

1 5 1015

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>446

<211>219

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S103

<400>446

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>447

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S104

<400>447

Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile

1 5 10

<210>448

<211>286

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S105

<400>448

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 GlnVal 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 GluLeu Gln Gly Gln Asp Pro Thr His Leu Val

275 280 285

<210>449

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S106

<400>449

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>450

<211>53

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S109

<400>450

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>451

<211>569

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S110

<400>451

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>452

<211>569

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S113

<400>452

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 ValVal 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 AlaArg 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 ProGly 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 Phe 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>453

<211>58

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S115

<400>453

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>454

<211>82

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S116

<400>454

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

3540 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>455

<211>277

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S117

<400>455

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>456

<211>196

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S120

<400>456

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 9095

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>457

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S121

<400>457

Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His

1 5 10 15

Lys Lys Thr Leu GluHis Leu Cys Lys Lys Pro Arg Lys Val Ser Val

20 25 30

Phe Gly Ala

35

<210>458

<211>230

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S126

<400>458

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>459

<211>322

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S129

<400>459

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>460

<211>83

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S130

<400>460

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>461

<211>31

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S135

<400>461

Arg Leu Arg Arg Gly Gly Lys Asp Gly Ser Pro Lys ProGly 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>462

<211>92

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S136

<400>462

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>463

<211>90

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S137

<400>463

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>464

<211>219

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S138

<400>464

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>465

<211>60

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S141

<400>465

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>466

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S142

<400>466

Arg Lys Pro Asn Thr Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp

1 5 10 15

Thr

<210>467

<211>39

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S143

<400>467

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>468

<211>525

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S144

<400>468

Cys Met Thr Trp Arg Leu Ala Gly Pro Gly Ser Glu Lys Tyr Ser Asp

15 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

165170 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

325330 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 490495

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>469

<211>189

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S145

<400>469

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 9095

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>470

<211>232

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S146

<400>470

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 2530

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>471

<211>219

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S147

<400>471

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 CysSer 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>472

<211>419

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S148

<400>472

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>473

<211>192

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S149

<400>473

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 LeuGlu Arg Glu Ala Ala Arg Leu Ala Asp Leu Gly

180 185 190

<210>474

<211>191

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S154

<400>474

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>475

<211>222

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S155

<400>475

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

3540 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>476

<211>282

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S156

<400>476

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 GluThr Tyr Leu Met Gln Phe Met Glu

260 265 270

Glu Trp Gly Leu Tyr Val Gln Met Glu Asn

275 280

<210>477

<211>57

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S157

<400>477

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>478

<211>285

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S158

<400>478

Ser Leu Lys Thr His Pro Leu Trp Arg Leu Trp LysLys 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>479

<211>325

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S161

<400>479

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 155160

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

305310 315 320

Val Gln Trp Glu Ser

325

<210>480

<211>253

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S165

<400>480

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

100105 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>481

<211>99

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S168

<400>481

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>482

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S169

<400>482

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>483

<211>189

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S170

<400>483

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>484

<211>106

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S171

<400>484

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>485

<211>303

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S174

<400>485

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 135140

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

290295 300

<210>486

<211>96

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S175

<400>486

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>487

<211>34

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S176

<400>487

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>488

<211>44

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S177

<400>488

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>489

<211>239

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S180

<400>489

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

145150 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>490

<211>202

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S183

<400>490

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>491

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S186

<400>491

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 TrpGln Gln Pro

115 120

<210>492

<211>304

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S189

<400>492

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>493

<211>296

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S190

<400>493

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

8590 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>494

<211>251

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S191

<400>494

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

6570 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>495

<211>191

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S192

<400>495

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 GluAsp

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>496

<211>146

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S193

<400>496

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>497

<211>172

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S194

<400>497

Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val SerSer

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>498

<211>127

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S195

<400>498

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

ProGlu Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile

115 120 125

<210>499

<211>304

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S196

<400>499

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 105110

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

260265 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>500

<211>296

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S197

<400>500

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>501

<211>251

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S198

<400>501

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 GlnGly

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>502

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S199

<400>502

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>503

<211>364

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S202

<400>503

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 310315 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>504

<211>42

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S211

<400>504

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>505

<211>188

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S212

<400>505

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>506

<211>42

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S213

<400>506

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>507

<211>60

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S214

<400>507

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>508

<211>58

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S215

<400>508

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>509

<211>51

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide S216

<400>509

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>510

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide X001

<400>510

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>511

<211>152

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide I L7

<400>511

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210>512

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide Flexible linker

<400>512

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210>513

<211>244

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide I L7 RA ECD and TM

<400>513

Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp

1 5 10 15

Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln

20 25 30

His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Ile Thr Asn

35 40 45

Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Asn

50 5560

Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu

65 70 75 80

Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys Ser Leu

85 90 95

Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro

100 105 110

Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn Asp Phe Val Val

115 120 125

Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met

130 135 140

His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp Thr His

145 150 155 160

Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln

165 170 175

Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr

180 185 190

Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr

195 200 205

Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro Ile Leu Leu Thr

210 215 220

Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala

225 230 235 240

Cys Val Leu Trp

<210>514

<211>286

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide I L2 RB ICD

<400>514

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 250255

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>515

<211>9

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide Kozak type sequence

<220>

<221> misc _ feature

<222>(7)..(7)

<223> n = T or U

<220>

<221> misc _ feature

<222>(9)..(9)

<223> n = G may or may not be present

<400>515

ccaccangn 9

<210>516

<211>9

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide Kozak type sequence 2

<220>

<221> misc _ feature

<222>(7)..(7)

<223> n = T or U

<220>

<221> misc _ feature

<222>(9)..(9)

<223> n = G may or may not be present

<400>516

ccgccangn 9

<210>517

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide Kozak type sequence 3

<220>

<221> misc _ feature

<222>(11)..(11)

<223> n = T or U

<220>

<221> misc _ feature

<222>(13)..(13)

<223> n = G may or may not be present

<400>517

gccgccgcca ngn 13

<210>518

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide Kozak type sequence 4

<220>

<221> misc _ feature

<222>(11)..(11)

<223> n = T or U

<220>

<221> misc _ feature

<222>(12)..(12)

<223> n = G may or may not be present

<400>518

gccgccacca nn 12

<210>519

<211>9

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide Kozak sequence

<400>519

gccgccacc 9

<210>520

<211>9

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide triple terminator sequence

<400>520

taatagtga 9

<210>521

<211>191

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide WPRE

<400>521

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>522

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic TRAF1, TRAF2 and TRAF3 consensus binding sequences

<220>

<221> misc _ feature

<222>(2)..(2)

<223> Xaa can be any natural amino acid

<220>

<221> misc _ feature

<222>(4)..(4)

<223> Xaa can be any natural amino acid

<400>522

Pro Xaa Gln Xaa Thr

1 5

<210>523

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic TRAF2 consensus binding sequence

<220>

<221> misc _ feature

<222>(2)..(3)

<223> Xaa can be any natural amino acid

<400>523

Ser Xaa Xaa Glu

1

<210>524

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic TRAF6 consensus binding sequence

<220>

<221> misc _ feature

<222>(2)..(2)

<223> Xaa can be any natural amino acid

<220>

<221> misc _ feature

<222>(4)..(4)

<223> Xaa can be any natural amino acid

<220>

<221> misc _ feature

<222>(6)..(6)

<223> Xaa can be any natural amino acid

<400>524

Gln Xaa Pro Xaa Glu Xaa

1 5

<210>525

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Box1 motifs

<220>

<221> misc _ feature

<222>(2)..(3)

<223> Xaa can be any natural amino acid

<400>525

Pro Xaa Xaa Pro

1

<210>526

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Shc phosphotyrosine binding motifs

<220>

<221> misc _ feature

<222>(2)..(3)

<223> Xaa can be any natural amino acid

<400>526

Asn Xaa Xaa Tyr

1

<210>527

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic STAT3 consensus binding sequence

<220>

<221> misc _ feature

<222>(2)..(3)

<223> Xaa can be any natural amino acid

<400>527

Tyr Xaa Xaa Gln

1

<210>528

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic STAT5 recruitment sequence

<400>528

Tyr Leu Pro Leu

1

<210>529

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic STAT5 consensus recruitment sequence

<220>

<221> misc _ feature

<222>(1)..(1)

<223> Xaa is phosphorylated tyrosine

<220>

<221> misc _ feature

<222>(3)..(3)

<223> Xaa can be any natural amino acid

<400>529

Xaa Leu Xaa Leu

1

<210>530

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic Kozak sequence

<220>

<221> misc _ feature

<222>(1)..(3)

<223> nnn, if present, GCC

<220>

<221> misc _ feature

<222>(10)..(10)

<223> n is A or G

<400>530

nnngccgccn ccatg 15

<210>531

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic Kozak sequence

<220>

<221> misc _ feature

<222>(11)..(11)

<223> n is T (DNA) or U (RNA)

<220>

<221> misc _ feature

<222>(12)..(12)

<223> n is G or absent

<400>531

gccgccgcca nn 12

<210>532

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic Kozak sequence

<220>

<221> misc _ feature

<222>(11)..(11)

<223> n is T (DNA) or U (RNA)

<220>

<221> misc _ feature

<222>(13)..(13)

<223> n is G or absent

<400>532

gccgccacca ngn 13

<210>533

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic translation initiation sequence

<400>533

agaggatcca tg12

<210>534

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic triple stop codon

<400>534

tagtctagac tag 13

<210>535

<211>563

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic baboon reverse transcription envelope glycoprotein

<400>535

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>536

<211>545

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Baboon reverse transcription envelope glycoprotein deltaR (HA)

<400>536

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>537

<211>546

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Baboon reverse transcription envelope glycoprotein deltaR (HAM)

<400>537

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 295300

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

450455 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>538

<211>654

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic Mu L V envelope protein

<400>538

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

180185 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>539

<211>905

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic fusion of anti-CD 3scFV to Mu L V envelope protein by UCHT1

<400>539

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 280285

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 ThrAsn 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>540

<211>223

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic cCB L miRNA at P1

<400>540

cggcacctgc gctgccgttg gatcggggat gaaagctggc gctggaggct tgctgaaggc 60

tgtatgctgt aataactccc aactcactgg gttttggcca ctgactgacc cagtgagggg 120

agttattaca ggacacaagg cctgttacta gcactcacat ggaacaaatg gcccacattg 180

gtgccggatg aagctcttat gttgcgtccc atcgcaggtg cct 223

<210>541

<211>204

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic cCB L miRNA at P2

<400>541

cctcacctgc ttgcgtccca ttctggaggc ttgctgaagg ctgtatgctg tttagtaatc 60

cgaaatgtgt cgttttggcc actgactgac gacacattgg attactaaac aggacacaag 120

gcctgttact agcactcaca tggaacaaat ggccgttgcc tgagtcttgg cagcgagaga 180

tcactaactg ctaagcaggt gctt 204

<210>542

<211>204

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic cCB L miRNA at P3

<400>542

tgtcacctgc actaactgct aactggaggc ttgctgaagg ctgtatgctg taatcattgc 60

aggtcagatc agttttggcc actgactgac tgatctgatg caatgattac aggacacaag 120

gcctgttact agcactcaca tggaacaaat ggccgtgtta attgtccatg tagcgaggca 180

tccttatggc gtgggcaggt gtcc 204

<210>543

<211>200

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic cCB L miRNA at P4

<400>543

ccttcacctg ccttatggcg tggctggagg cttgctgaag gctgtatgct gtttgtgaat 60

gaatttctgg aggttttggc cactgactga cctccagaat cattcacaaa caggacacaa 120

ggcctgttac tagcactcac atggaacaaa tggccggtgt ccgttatcgg ggaagaaggt 180

cgcgcacata gcaggtgtcc 200

<210>544

<211>223

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of CD3Z miRNA at P1

<400>544

cggcacctgc gctgccgttg gatcggggat gaaagctggc gctggaggct tgctgaaggc 60

tgtatgctga catggtacag ttcaatggtg gttttggcca ctgactgacc accattgctg 120

taccatgtca ggacacaagg cctgttacta gcactcacat ggaacaaatg gcccacattg 180

gtgccggatg aagctcttat gttgcgtccc atcgcaggtg cct 223

<210>545

<211>204

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of CD3Z miRNA at P2

<400>545

cctcacctgc ttgcgtccca ttctggaggc ttgctgaagg ctgtatgctg tcagtctgtt 60

catcttctgg cgttttggcc actgactgac gccagaagga acagactgac aggacacaag 120

gcctgttact agcactcaca tggaacaaat ggccgttgcc tgagtcttgg cagcgagaga 180

tcactaactg ctaagcaggt gctt 204

<210>546

<211>204

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of CD3Z miRNA at P3

<400>546

tgtcacctgc actaactgct aactggaggc ttgctgaagg ctgtatgctg aagcgtgaag 60

tgaatcaacg ggttttggcc actgactgac ccgttgatac ttcacgcttc aggacacaag 120

gcctgttact agcactcaca tggaacaaat ggccgtgtta attgtccatg tagcgaggca 180

tccttatggc gtgggcaggt gtcc 204

<210>547

<211>200

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of CD3Z miRNA at P4

<400>547

ccttcacctg ccttatggcg tggctggagg cttgctgaag gctgtatgct ggcagtatcc 60

tagtacattg acgttttggc cactgactga cgtcaatgtt aggatactgc caggacacaa 120

ggcctgttac tagcactcac atggaacaaa tggccggtgt ccgttatcgg ggaagaaggt 180

cgcgcacata gcaggtgtcc 200

<210>548

<211>511

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic VSV-G envelope protein

<400>548

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 185190

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

Claims (48)

1. Use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying a T cell or NK cell of an individual, wherein the use of the kit comprises:

contacting said T cell or said NK cell ex vivo with said replication deficient recombinant retroviral particle, wherein said replication deficient recombinant retroviral particle comprises on its surface a pseudotyped component, wherein said replication deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein said one or more transcription units encode a first polypeptide comprising a lymphoproliferative component (E) or a first polypeptide comprising E and a second polypeptide comprising a Chimeric Antigen Receptor (CAR), and wherein said contacting is performed for between 15 minutes and 18 hours to facilitate membrane fusion of said T cells or said NK cells with said replication deficient recombinant retroviral particle, wherein said 0E comprises an intracellular signaling domain from the group consisting of CD, CD8, CSF 8, CD79, CR 1F, RB2, CSF3, epogr 2, fcr 2, GHR, IFR 2R, IFR 21, nri 31, nri 51, nri 64, nri, PR 11, PR, sri, PR, sri, PR, sri, and nri 80, sri 80, PR 11, PR.

2. A genetically modified T cell or NK cell made by genetically modifying a T cell or NK cell according to a method comprising contacting the T cell or NK cell ex vivo with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises on its surface a pseudotyped component, wherein the replication deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first polypeptide comprising a lymphoid tissue proliferative component (E), or a first polypeptide comprising E and a second polypeptide comprising a Chimeric Antigen Receptor (CAR), and wherein the contacting is performed for between 15 minutes and 18 hours to facilitate membrane fusion of the T cell or the NK cell with the replication deficient recombinant retroviral particle, wherein the 0E comprises an intracellular signaling domain from CD, CD8, CD79, CR 1F, CSF2, epo 3, fcr 2R 417, fcr 2R 11, nrr 51, nrr 41, nra 31, nra 5, nra, 12, 11, ifi, and ifi 5, if.

3. A method for genetically modifying and/or transducing a T cell or NK cell, comprising contacting the T cell or the NK cell ex vivo with a replication deficient recombinant retroviral particle comprising a pseudotyped component on its surface, wherein the replication deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first polypeptide comprising a lymphoproliferative component (E), or a first polypeptide comprising E and a second polypeptide comprising a Chimeric Antigen Receptor (CAR), and wherein the contacting is performed for between 15 minutes and 18 hours to facilitate membrane fusion of the T cell or the NK cell by the replication deficient recombinant retroviral particle, wherein the 0E comprises an intracellular signaling domain from CD, CD8, CD79, CR 1F, CSF2, CSF3, EPOR, RB, FCGR2, fcrr 2, GHR 317, IFR, nar 17, nar 8, CD79, CR 2F, CSF3, RA2, rr 3, RB2, nrr 41, nrl 64, nrl.

4. The use, the genetically modified T cell or NK cell, or the method for genetically modifying and/or transducing a T cell or NK cell, according to any one of the preceding claims, wherein the L E is capable of promoting cell survival and/or cell proliferation of T cells during culture without the addition of I L-2.

5. Use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying a T cell or NK cell of an individual, wherein the use of the kit comprises:

contacting the T cell or the NK cell ex vivo with the replication-deficient recombinant retroviral particle, wherein the replication-deficient recombinant retroviral particle comprises a pseudotyping component on its surface, wherein the replication-deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in a T cell and/or an NK cell, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), a first polypeptide comprising a lymphoproliferative component (L E), or a first polypeptide comprising L E and a second polypeptide comprising a CAR, wherein the one or more transcription units further comprise one or more of a kowpzak-related sequence, a re component, and a triple termination sequence, wherein the L E is capable of promoting cell survival and/or cell proliferation of a T cell during culture without the addition of I L-2, and wherein the contacting is performed for between 15 minutes and 18 hours to facilitate membrane-mediated fusion of the T cell or the NK cell with the replication-deficient recombinant retroviral particle, thereby producing a genetically modified T cell or genetically fused T cell.

6. A genetically modified T cell or NK cell made by genetically modifying a T cell or NK cell according to a method comprising contacting the T cell or NK cell ex vivo with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises a pseudotyping component on its surface, wherein the replication deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), a first polypeptide comprising a lymphoproliferative component (L E), or a first polypeptide comprising L E and a second polypeptide comprising a CAR, wherein the one or more transcription units further comprise one or more of a Kozak-related sequence, a WPRE component and a triple termination sequence, wherein the L E is capable of promoting cell survival and/or cell proliferation of the T cell during culture without the addition of I L-2, and wherein the contacting is performed for between 15 minutes and 18 hours to facilitate the genetically modifying T cell to produce the genetically modified T cell or NK cell.

7. A method for genetically modifying and/or transducing a T cell or an NK cell, comprising contacting the T cell or the NK cell ex vivo with a replication-deficient recombinant retroviral particle comprising a pseudotyped component on its surface, wherein the replication-deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), a first polypeptide comprising a lymphoproliferative component (L E), or a first polypeptide comprising L E and a second polypeptide comprising a CAR, wherein the one or more transcription units further comprise one or more of a Kozak-related sequence, a WPRE component, and a triple termination sequence, wherein the L E is capable of promoting cell proliferation and/or cell proliferation of the T cell during culture without the addition of I L-2, and wherein the contacting is performed for between 15 minutes and 18 hours to facilitate survival of the T cell or the NK cell by the replication-deficient recombinant retroviral particle to produce a genetically modified T cell or NK cell.

8. The use according to claim 5, the genetically modified T cell or NK cell according to claim 6, or the method according to claim 7 for genetically modifying and/or transducing a T cell or NK cell according to claim 72, wherein the L E comprises an intracellular signaling domain from BT L A, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A, CD8B, CD B, a mutation B CD B, CD79B, CR B F B, CSF2 CSF B, CSF3 RB B, RB B/CD B, DAP B, EPOR, FCER 1B, FCGR2 GR B, FCGRA B, GHR B, ICOS, IFR B, IFR NARA B, IFRAP B, IFRA B, TFR B, TFR 3611, B, 3611, B, 3636363636363672, 3611, B, 36363636363672, B, 3636363636363636363672, 3611, 363672, 363636363611, 36363672, B, 363672, B, 36363672, B, 363636363636363636363636363672, 3636363672, B, 363672, 363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363672, 36.

9. The use according to claim 5, the genetically modified T-cell or NK-cell according to claim 6, or the method according to claim 7 for genetically modifying and/or transducing a T-cell or NK-cell, wherein the E comprises an intracellular signaling domain from CD, CD8, CD79, CR F, CSF2, CSF3, EPOR, FCGR2, GHR, IFNAR, IFNGR, IFN 0R, I11R, I21 RAP, I31R 41, I51R 62, I72 RA, I82 RB, I92 RG, I3 RA, I04 15RA, I26 ST, I49 510RA, I610 RB, I711 RA, I813 RA 913RA, I17 RA, I017 RB, I117 RC, I217 RD, I RE 317, I418 RAP, I418R, I49 RA, I720 RA, I620 RA, I518 RA, I711 RA, I31 MR 518, I MP R, I31 PR, MR 31R, MR.

10. The use, the genetically modified T cell or NK cell, or the method for genetically modifying and/or transducing a T cell or NK cell according to any one of the preceding claims, wherein the use or the method is performed without prior ex vivo stimulation.

11. The use, the genetically modified T cell or NK cell, or the method for genetically modifying and/or transducing a T cell or NK cell, according to any of the preceding claims, wherein the T cell is a resting T cell or the NK cell is a resting NK cell.

12. The use, the genetically modified T cell or NK cell, or the method for genetically modifying and/or transducing a T cell or NK cell, according to any of the preceding claims, wherein the replication deficient recombinant retroviral particle comprises on its surface a membrane bound T cell activation module.

13. The use, genetically modified T-cell or NK cell, or method for genetically modifying and/or transducing a T-cell or NK cell, of any one of the preceding claims, wherein the one or more transcriptional units encode the first polypeptide comprising the L E and the second polypeptide comprising the CAR, and wherein the genetically modified cell is a genetically modified T-cell.

14. The use, the genetically modified T cell or NK cell, or the method for genetically modifying and/or transducing a T cell or NK cell according to any of the preceding claims, wherein the use or the method further comprises:

collecting blood comprising the T cells or NK cells from an individual prior to contacting the T cells or NK cells with the replication deficient recombinant retroviral particles ex vivo; and

introducing the genetically modified T cells or NK cells into the individual, wherein the genetically modified T cells or NK cells are not expanded ex vivo after the contacting and prior to being introduced into the individual.

15. A replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises on its surface a pseudotyped component and a membrane bound T cell activating component, wherein the replication deficient recombinant retroviral particle 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 transcriptional units encode a first polypeptide comprising a lymphoproliferative component (E) and a second polypeptide comprising a Chimeric Antigen Receptor (CAR), and wherein the E comprises an intracellular signaling domain from CD, CD8, CD79, CR 0F, CSF2, CSF3, RAP, FCGR2, fcpr 2, GHR, IFNAR, IFNGR, IFN 1R, I21R, I31 RAP, I41R 51, I61R 72, I82 RA, I92 RB, I2 RG, I2 RB 03, I317 RA, I25 RA, I36 RA, ST 59, I42 RA, I42R, I811, I42R, I520 RA, I520, I53, I14R, I53, I14R, I14, RB, I53, I53, R, la, mr I14R, RB, PR 2R, PR 2, RB, PR 2R, RB, PR 2R, PR 2.

16. A replication-deficient recombinant retroviral particle, wherein the replication-deficient recombinant retroviral particle comprises on its surface a pseudotyping component and a membrane-bound T cell activation component, wherein the replication-deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a lymphoproliferative component and a second polypeptide comprising a Chimeric Antigen Receptor (CAR), wherein the L E is capable of promoting cell survival and/or cell proliferation of T cells during culture without the addition of I L-2, and wherein the one or more transcription units further comprise one or more of a Kozak-related sequence, a WPRE component, and a triple termination sequence.

17. The use, the genetically modified T cell or NK cell, the method for genetically modifying and/or transducing a T cell or NK cell, or the replication deficient recombinant retroviral particle of any one of claims 12, 15 and 16, wherein the activating module comprises anti-CD 3.

18. The use, the genetically modified T cell or NK cell, the method for genetically modifying and/or transducing a T cell or NK cell, or the replication deficient recombinant retroviral particle according to any one of the preceding claims, wherein the replication deficient recombinant retroviral particle is a lentiviral particle.

19. The use, the genetically modified T cell or NK cell, the method for genetically modifying and/or transducing a T cell or NK cell, or the replication-defective recombinant retroviral particle of any one of the preceding claims, wherein the L E is a chimeric L E comprising two or more intracellular signaling domains each from a different gene.

20. The use, genetically modified T cell or NK cell, method for genetically modifying and/or transducing a T cell or NK cell, isolated polypeptide, isolated polynucleotide or replication defective recombinant retroviral particle of any one of the preceding claims, wherein the L E is a chimeric L E comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain.

21. The use, genetically modified T cell or NK cell, method for genetically modifying and/or transducing a T cell or NK cell, isolated polypeptide, isolated polynucleotide or replication deficient recombinant retroviral particle of claim 20, wherein the extracellular domain of the chimeric L E comprises a dimerization motif.

22. The use, genetically modified T cell or NK cell, method for genetically modifying and/or transducing a T cell or NK cell, isolated polypeptide, isolated polynucleotide or replication defective recombinant retroviral particle of claim 20, wherein the extracellular domain of chimeric L E does not bind a ligand for an interleukin receptor.

23. The use, genetically modified T cell or NK cell, method for genetically modifying and/or transducing a T cell or NK cell, isolated polypeptide, isolated polynucleotide or replication defective recombinant retroviral particle of any one of the preceding claims, wherein the L E is constitutively active.

24. The use, the genetically modified T cell or NK cell, the method for genetically modifying and/or transducing a T cell or NK cell, the isolated polypeptide, the isolated polynucleotide or the replication deficient recombinant retroviral particle according to any one of the preceding claims,

wherein said L E possesses the following characteristics:

a) under the same conditions, between day 7 and day 21, day 28, day 35 and/or day 42 of in vitro culture in the absence of exogenously added interleukins, when transduced with a nucleic acid encoding an anti-CD 19CAR comprising a CD3zeta intracellular activation domain but no co-stimulatory domain, improved amplification of preactivated PBMCs transduced with retroviral particles comprising a nucleic acid encoding the L E, and/or compared to a control construct identical to the nucleic acid construct comprising the L E but not the L E, and/or the control construct

b) Preactivated PBMCs transduced with a nucleic acid construct encoding the L E are amplified at least 2-fold, 3-fold, 4-fold, 6-fold, 7-fold, or 8-fold, or between 3-fold and 25-fold, 5-fold and 20-fold, 5-fold and 15-fold, 7-fold and 15-fold, or 7-fold and 12-fold, between day 7 and 21, day 28, day 35, and/or day 42 of in vitro culture, in the absence of exogenously added interleukins, when transduced with a nucleic acid encoding an anti-CD 19CAR comprising a CD3 ζ intracellular activation domain but no co-stimulatory domain.

25. The use, the genetically modified T cell or NK cell, the method for genetically modifying and/or transducing a T cell or NK cell, the isolated polypeptide, the isolated polynucleotide or the replication deficient recombinant retroviral particle of claim 24, wherein the L E is capable of improved amplification according to element a), and wherein the improvement is determined using statistical methods and a cut-off p-value equal to or less than 0.1.

26. The use, genetically modified T cell or NK cell, method for genetically modifying and/or transducing a T cell or NK cell, isolated polypeptide, isolated polynucleotide or replication deficient recombinant retroviral particle according to any one of the preceding claims, wherein the L E comprises an intracellular signaling domain from CSF2RB, CSF3R, IFNGR1, I L2 RB, I L02 RG, I L6 ST, I L10 RA, I L17 RE, I L18R 1, I L22 RA1, I L31 RA, MP L, MyD88, OSMR or PR L R.

27. The use, genetically modified T-cell or NK-cell, method for genetically modifying and/or transducing a T-cell or NK-cell, isolated polypeptide, isolated polynucleotide or replication deficient recombinant retroviral particle according to any one of the preceding claims, wherein the L E comprises an intracellular signaling domain from CSF2RB, CSF3R, I L2 RB, I L2 RG, I L6 ST, I L31 RA, MP L and MyD 88.

28. The use, genetically modified T cell or NK cell, method for genetically modifying and/or transducing a T cell or NK cell, isolated polypeptide, isolated polynucleotide or replication deficient recombinant retroviral particle according to any one of the preceding claims, wherein the L E comprises an intracellular signaling domain from CD40, I L22 RA1, I L13 RA2, I L17 RA, I L17 RB, IFNGR2 and FCGR 2C.

29. The use according to any of the preceding claims, a genetically modified T cell or NK cell, a method for genetically modifying and/or transducing a T cell or NK cell, an isolated polypeptide, an isolated polynucleotide or a replication deficient recombinant retroviral particle, wherein said L E comprises a first and a second intracellular signaling domain selected from the group consisting of CSF2RA and TNFRSF4, CSF2RA and CD28, CSF2RA and TNFRSF8, CSF2RA and CD RA, CSFR RA and CD79 RA, IFNAR RA and TNFRSF RA, I RA RAP and CD79 RA, I3603 RA and CD RA, I RA RA and CD 3679, I36211 RA and FCGRA RA, I RA and CD RA, I36313 and TNFRSF RA, CD 36418 and CD3 RAP RA, CD RA and CD RA, CD RA and MyR RA, CD RA and CD RA, CD 36.

30. An isolated polypeptide comprising a membrane-associated motif and first and second intracellular signaling domains selected from the group consisting of CSF2 and TNFRSF, CSF2 and CD, CSF2 and TNFRSF, CSF2 and CD, CSFR and CD79, IFNAR and TNFRSF, I1 RAP and CD79, I3 RA and CD, I010 RA and CD79, I111 RA and FCGRA, I213 RA and TNFRSF, I318 RAP and CD3, I27 RA and FCGRA, EPR and CD3, IFR and TNFRSF, MP and CD79, MP and TNFRSF, MP and CD79, MyD and CD3, and MyD and CD79, or MyD and CD3, respectively.

31. The isolated polypeptide of claim 30, wherein the membrane associated motif is a transmembrane domain adjacent to the first intracellular signaling domain.

32. The isolated polypeptide of claim 31, further comprising an extracellular domain.

33. The isolated polypeptide of any one of claims 30-32, wherein the isolated polypeptide is capable of promoting cell survival and/or cell proliferation of T cells during culture without the addition of I L-2.

34. The isolated polypeptide of claim 33, wherein the isolated polypeptide is constitutively active.

35. An isolated polynucleotide encoding the isolated polypeptide of any one of claims 30 to 34.

36. The isolated polynucleotide of claim 35, further comprising a promoter active in T cells and/or NK cells.

37. An isolated replication-defective recombinant retroviral particle comprising the isolated polynucleotide of claim 36.

38. The isolated, replication-defective recombinant retroviral particle of claim 37, wherein the retroviral particle further encodes a chimeric antigen receptor.

39. Use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying a T cell or NK cell of an individual, wherein the use of the kit comprises:

contacting the T cell or the NK cell ex vivo with the replication-deficient recombinant retroviral particle, wherein the replication-deficient recombinant retroviral particle comprises a pseudotyping component on its surface, wherein the replication-deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in a T cell and/or an NK cell, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), a first polypeptide comprising a lymphoproliferative component (L E), or a first polypeptide comprising L E and a second polypeptide comprising a CAR, wherein the L E is capable of promoting cell survival and/or cell proliferation of a T cell during culture without the addition of I L-2, and wherein the contacting is performed for less than 15 minutes to facilitate fusion of the T cell or the NK cell with a membrane of the replication-deficient recombinant retroviral particle, thereby producing a genetically modified T cell or a genetically modified NK cell.

40. A genetically modified T cell or NK cell made by genetically modifying a T cell or NK cell according to a method comprising contacting the T cell or NK cell ex vivo with a replication deficient recombinant retroviral particle, wherein the replication deficient recombinant retroviral particle comprises a pseudotyped component on its surface, wherein the replication deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), a first polypeptide comprising a lymphoproliferative component (L E), or a first polypeptide comprising L E and a second polypeptide comprising a CAR, wherein the L E is capable of promoting cell survival and/or cell proliferation of the T cell during culture without the addition of I L-2, and wherein the contacting is performed for less than 15 minutes to facilitate fusion of the T cell or NK cell with a membrane of the replication deficient recombinant retroviral particle, thereby producing the genetically modified T cell or NK cell.

41. A method for genetically modifying and/or transducing a T cell or NK cell comprising contacting the T cell or the NK cell ex vivo with a replication deficient recombinant retroviral particle comprising a pseudotyping component on its surface, wherein the replication deficient recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in the T cell and/or NK cell, wherein the one or more transcription units encode a first polypeptide comprising a Chimeric Antigen Receptor (CAR), a first polypeptide comprising a lymphoproliferative component (L E), or a first polypeptide comprising L E and a second polypeptide comprising a CAR, wherein the L E is capable of promoting cell survival and/or cell proliferation of the T cell during culture without the addition of I L-2, and wherein the contacting is performed for less than 15 minutes to facilitate membrane fusion of the T cell or the NK cell by the replication deficient recombinant retroviral particle, thereby producing a genetically modified T cell or NK cell.

42. The use according to claim 39, a genetically modified T cell or NK cell according to claim 40, or a method according to claim 41 for genetically modifying and/or transducing a T cell or NK cell according to claim 72, wherein the L E comprises an intracellular signaling domain from BT L A, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A, CD8B, CD B, a mutation B CD B, CD79B, CR B F B, CSF2 CSF B, CSF3 RB B, RB B/CD B, DAP B, EPOR, FCER 1B, FCGR2 GR B, FCGRA B, GHR B, ICOS, IFR B, IFR NARA B, IFRAP B, IFRA B, TFR B, TFR B, TFR B, TFR 3611, B, 3611, B, 363636363611, B, 363636363636363611, B, 3636363672, B, 363636363636363636363672, 363636363611, 36363636363636363672, B, 36363672, B, 3636363672, 36363636363636363636363636363672, 3636363672, B, 36363672, 36363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636.

43. The use according to claim 39, the genetically modified T-cell or NK-cell according to claim 40, or the method according to claim 41 for genetically modifying and/or transducing a T-cell or NK-cell, wherein the E comprises an intracellular signaling domain from CD, CD8, CD79, CR F, CSF2, CSF3, EPOR, FCGR2, GHR, IFNAR, IFNGR, IFN 0R, I11R, I21 RAP, I31R 41, I51R 62, I72 RA, I82 RB, I92 RG, I3 RA, I04 15RA, I26 ST, I49 510RA, I610 RB, I711 RA, I813 RA 913, I RA, I17 RA, I017 RB, I117 RC, I217 RD, I RE 317, I418 RAP, I418 RA, I620 RA, I720 RA, I518 RA, I711 RA, I518, I31 PR 518, I31R, MR.

44. The use of claim 39, the genetically modified T cell or NK cell of claim 40, or the method of genetically modifying and/or transducing a T cell or NK cell of claim 41, wherein the L E comprises an intracellular signaling domain from CSF2RB, CSF3R, IFNGR1, I L2 RB, I L02 RG, I L6 ST, I L10 RA, I L17 RE, I L18R 1, I L22 RA1, I L31 RA, MP L, MyD88, OSMR, or OSPR L R.

45. The use of claim 39, the genetically modified T cell or NK cell of claim 40, or the method of claim 41 for genetically modifying and/or transducing a T cell or NK cell, wherein the use or the method is performed without prior ex vivo stimulation.

46. The use of claim 39, the genetically modified T cell or NK cell of claim 40, or the method of claim 41 for genetically modifying and/or transducing a T cell or NK cell, wherein the T cell is a resting T cell or the NK cell is a resting NK cell.

47. The use of claim 39, the genetically modified T cell or NK cell of claim 40, or the method of claim 41 for genetically modifying and/or transducing a T cell or NK cell, wherein the replication-defective recombinant retroviral particle comprises a membrane-bound T cell activation module on its surface.

48. The use of claim 39, the genetically modified T cell or NK cell of claim 40, or the method of claim 41 for genetically modifying and/or transducing a T cell or NK cell, wherein the one or more transcriptional units encode the first polypeptide comprising the L E and the second polypeptide comprising the CAR, and wherein the genetically modified cell is a genetically modified T cell.

Images