CN117295506A - Compositions and methods for reducing therapeutic T cytotoxicity - Google Patents

Compositions and methods for reducing therapeutic T cytotoxicity Download PDF

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CN117295506A
CN117295506A CN202280034086.2A CN202280034086A CN117295506A CN 117295506 A CN117295506 A CN 117295506A CN 202280034086 A CN202280034086 A CN 202280034086A CN 117295506 A CN117295506 A CN 117295506A
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thr
leu
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M·L·达维拉
G·李
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H Li Moffett Cancer Center And Institute Co ltd
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H Li Moffett Cancer Center And Institute Co ltd
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Priority claimed from PCT/US2022/071070 external-priority patent/WO2022192895A1/en
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Abstract

Disclosed are ready-made immune effector cells engineered to express an anti-CD 3 antibody disclosed herein, the anti-CD 3 antibody configured to automatically activate the immune effector cells, thereby reducing expression of T cell receptors (e.g., TCR αβ) that may cause GVHD. Also disclosed are methods for modifying donor immune effector cells to render them suitable for ready treatment of an allogeneic subject. These methods involve engineering cells to express anti-CD 3 antibodies configured to activate the cells. In some embodiments, the antibody is a bispecific antibody that binds to a CD3 complex on immune effector cells. In other embodiments, the antibody is a membrane-bound anti-CD 3 antibody that automatically activates immune effector cells.

Description

Compositions and methods for reducing therapeutic T cytotoxicity
Cross reference to related applications
The present application claims the benefit of U.S. provisional application No. 63/159,222 filed on 3/10 of 2021, 63/209,094 filed on 6/10 of 2021, and 63/225,715 filed on 7/26 of 2021, which are hereby incorporated by reference in their entireties.
Sequence listing
The present application contains an ascii. Txt sequence listing file filed in electronic form, entitled "320803-2800 Sequence Listing_ST25", created at 2022, 3, 8, and having 226,162 bytes. The contents of the sequence listing are incorporated herein in their entirety.
Background
Autologous T cells, such as Chimeric Antigen Receptor (CAR) T cells, have altered the therapeutic promise of hematological malignancies. However, the use of allogeneic T cells from donors has many potential advantages over autologous methods, such as immediate availability of cryopreserved batches for patient treatment, possible standardization of T cell products, multiple cell modifications, re-administration or combination of T cells to different targets, and reduced costs using an industrial process. However, allogeneic T cells may cause life-threatening graft versus host disease and may be rapidly eliminated by the host immune system. Thus, there is a need in the art for methods for modifying allogeneic T cells to reduce the likelihood of graft versus host disease.
Disclosure of Invention
Disclosed are ready-made immune effector cells engineered to express an anti-CD 3 antibody disclosed herein, the anti-CD 3 antibody configured to automatically activate the immune effector cells, thereby reducing expression of T cell receptors (e.g., TCR αβ) that may cause GVHD.
Also disclosed are methods of modifying donor immune effector cells to render them suitable for ready treatment of an allogeneic subject. These methods involve engineering cells to express anti-CD 3 antibodies configured to activate the cells. In some embodiments, the immune effector cells are further engineered to express a Chimeric Antigen Receptor (CAR). In other embodiments, the immune effector cells do not express the CAR, but instead rely on antibodies for targeting.
In some embodiments, the antibody is a bispecific antibody that crosslinks a CD3 complex on an immune effector cell to another cell. In other embodiments, the antibody is a membrane-bound anti-CD 3 antibody that automatically activates immune effector cells. In other embodiments, the antibody is a monospecific antibody, such as an scFv.
Thus, also disclosed are methods of enhancing CAR-T cells for allogeneic cell transfer, which involve engineering CAR-T cells to secrete monospecific anti-CD 3 antibodies.
In some embodiments, the immune effector cells are obtained from an allogeneic donor. Thus, also disclosed are methods for treating cancer in a subject, the methods involving obtaining immune effector cells from an allogeneic donor; engineering an immune effector cell to express an anti-CD 3 multispecific antibody, wherein the antibody is configured to bind to the CD3 complex on the immune effector cell and a second antigen on another cell in a manner sufficient to activate the CD3 complex; and administering the engineered immune effector cells to the subject in an amount effective to treat the cancer.
In some embodiments, immune effector cells include any CD3 expressing lymphocyte that is involved in protecting the body against infectious diseases and foreign substances.
Thus, bispecific molecules capable of cross-linking a CD3 complex on a ready-made immune effector cell with an antigen on another cell are also disclosed. The bispecific molecule may be an engineered self-fusion polypeptide comprising: 1) A variable domain of an antibody that specifically binds to a target cell surface receptor, and 2) a variable domain of an antibody that specifically binds to CD 3. Either or both components of the bispecific molecule can also be engineered from non-antibody scaffolds, including but not limited to nanobodies, monomers, cyclic peptides, small molecules, and engineered ankyrin repeat proteins (darkins).
In some embodiments, the bispecific molecule is a bispecific T cell engagement (BiTE) antibody (fusion polypeptide) having, for example, the formula:
SP–V L R–V H R-/-V L 3–V H 3、
SP–V H R–V L R-/-V H 3–V L 3、
SP–V L R–V H R-/-V H 3–V L 3、
SP–V H R–V L R-/-V L 3–V H 3、
SP–V L 3–V H 3-/-V L R–V H R、
SP–V H 3–V L 3-/-V H R–V L R、
SP–V L 3–V H 3-/-V H R–V L r, or
SP–V H 3–V L 3-/-V L R–V H R，
Wherein "SP" represents an optional signal peptide,
wherein "V L R "is a light chain variable domain specific for a target cell surface receptor;
wherein "V H 3 "is a heavy chain variable domain specific for CD 3;
wherein "V L 3 "is a light chain variable domain specific for CD 3;
wherein "V H R "is a heavy chain variable domain specific for a target cell surface receptor;
wherein "-" consists of a peptide linker or peptide bond; and
wherein "-/-" consists of peptide hinge sequences.
In some embodiments, the antibody is a bispecific antibody containing intact heavy and light chain regions. In this embodiment, antibodies can be produced by methods described such as the "knob and hole" format (published in Ridgway JB, et al, protein eng.1996 9 (7): 617-21).
In some cases, the target cell surface receptor may be any other cell surface receptor, channel, or transporter expressed by the cell. In some embodiments, the receptor is a Tumor Associated Antigen (TAA). Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell mediated immune response. The additional antigen binding domain may be a natural ligand for an antibody or tumor antigen. The choice of additional antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, CSPG4, FAP, B7H3, kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, lectin-reactive AFP, fos-associated antigen 1, ADRB3, thyroglobulin, ephA2, RAGE-1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, globoH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid, PLAC1, RU1 RU2 (AS), enterocarboxylesterase, lewisY, sLe, LY K, mut hsp70-2, M-CSF, MYCN, rhoC, TRP-2, CYPIBI, BORIS, prostase, prostate Specific Antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53 mutant, ras mutant, gp100, prostaprotein, OR51E2, PANX3, PSMA, PSCA, her/neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin (survivin) and telomerase, legumain (legumain), HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-2, melanA/MART 1, XAGE1, ELF2M, ERG (PRSS 2), fusion genes 17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin Growth Factor (IGF) -I, IGFII, IGF-I receptor, GD2, ortho acetyl-GD 2, GD3, GM3, GPRC5D, GPR, CXORF61, folate receptor (FRa), folate receptor β, ROR1, flt3, TAG72, TN Ag, tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof. Non-limiting examples of tumor antigens include the following: differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated oncogenes such as p53, ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocation; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and Human Papilloma Virus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, P185erbB2, P180erbB-3, C-MET, nm-23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, nuMa, K-ras, β -catenin, CDK4, mum-1, P15, P16, 43-9F, 5T4, 791Tgp72, alpha fetoprotein, β -HCG, BCA225, BTA, CA 125, CA 15-3\CA 27.29\BCA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, ga 733\CAM, HTgp 175, MG 344, MA-50, MA 7-Ag 18, NB K, NY, NB/70-C1, CO 16, CA 16, TAC 6\6\6, TAC 1, TAG 1, CAL 6\6, TAG 1 and related proteins.
Also disclosed are isolated nucleic acids encoding the disclosed fusion polypeptides, as well as nucleic acid vectors containing the isolated nucleic acids operably linked to expression control sequences. Also disclosed are cells transfected with these vectors and the use of these cells for producing the disclosed fusion polypeptides.
Bispecific antigen binding molecules can be formed by dimerization of heavy and light chains. In these embodiments, V L R and V H R dimerizes to form an antigen binding site for a target cell surface receptor and V H 3 and V L 3 dimerize to form an antigen binding site for CD3.
Bispecific antibodies are also disclosed that are single polypeptide chains comprising bispecific antibodies having a first antigen binding region and a second antigen binding region. In some cases, the first antigen binding region is capable of specifically binding to a target receptor on a cell; and the second antigen binding region is capable of specifically binding CD3 on immune effector cells.
Each of the first portion and the second portion may comprise 1, 2, 3 or more antibody variable domains. In a specific embodiment, each of the first portion and the second portion comprises two variable domains: variable heavy chain (V) H ) Domain and variable light chain (V L ) Domain.
In some cases, the bispecific antibody has a binding to target receptor and CD3 of corresponding to about 10 -7 M、10 -8 M、10 -9 M or less K D Is a compound of formula (I).
Each of the first and second portions may be derived from a natural antibody, such as a monoclonal antibody. In some cases, the antibody is human. In some cases, bispecific antibodies have undergone alterations that result in reduced immunogenicity when administered to humans. For example, the alteration includes one or more techniques selected from the group consisting of: chimeric, humanized, CDR-grafted, deimmunized, and mutated framework amino acids to correspond to the closest human germline sequence.
Also disclosed are pharmaceutical compositions comprising the molecules disclosed herein in a pharmaceutically acceptable carrier. Also disclosed are methods for targeted ubiquitination of a target receptor in a subject, involving administering to the subject a therapeutically effective amount of the disclosed pharmaceutical compositions. Kits comprising the bispecific antibodies disclosed herein are also disclosed.
Also disclosed are expression vectors comprising an isolated nucleic acid encoding a bispecific antibody disclosed herein operably linked to an expression control sequence. Also disclosed are cells comprising the disclosed expression vectors. The cells may be primary cells, transformed cells, cell lines, and the like. In some cases, the cell is a mammalian cell line. In some cases, the cell is a non-mammalian cell line. For example, the cells may be bacterial or insect cell lines.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Drawings
FIGS. 1A-1C show in vitro CD3/TCRαβ expression after treatment with: CD19 BiTE or Her2 BiTE (fig. 1A); her2 bite+αher2BB, her2 car+αcd3εil15RA, or Her2 car+αcd3εscfv (fig. 1B); and CD19 bite+41BBLCD80, CD19 tripe 1 (aCD 28) or CD19 tripe 2 (41 BBL) (fig. 1C).
Fig. 2A and 2B show GvDH model tests. FIG. 2A shows NSG mice treated with whole body irradiation and 1e7 Her2 BiTE-T or CD19 BiTE-T cells. FIG. 2B shows NSG mice treated with chemotherapy and 1e7CTX+CAR-T or CTX+BiTE-T cells.
FIG. 3 shows in vivo CD 3/TCR. Alpha. Beta. Expression in the absence of antigen. NSG mice received irradiation and then received T cells. Donor T cells in peripheral blood were evaluated at certain time points. D12, day 12 after T cell injection.
FIG. 4 shows in vivo CD 3/TCR. Alpha. Beta. Expression in the presence of antigen. NSG mice were injected with tumor cells and then received T cell therapy. Donor T cells in peripheral blood or tumors were evaluated at certain time points.
Figures 5A-5L show that BiTE-T cells produce less cytokine and are comparable or better in efficacy to CAR-T in the presence of host T cells. Cytotoxicity (FIG. 5A) and cytokine production (FIG. 5B) of CD19 BiTE-T or CAR-T cells against 3T3.HCD19 cells. Her2 BiTE-T or CAR-T cells were cytotoxic against a375.her2 cells (fig. 5C) and cytokine production (fig. 5D). FIGS. 5E-5H show comparative studies of CD19 BiTE-T and CAR-T cells in vivo in the presence of host T cells. Fig. 5E shows the study design. Male NSG mice received 1.2Gy whole body irradiation and then were implanted with 700 ten thousand HLA-A2+ activated T cells after one day. Nalm6GL cells were administered on day 0. Mice were treated with HLA-A2-CD19 BiTE-T or CAR-T cells on day 3. Survival (fig. 5F), donor T cells in peripheral blood (fig. 5G), and recipient T cells (fig. 6H) were monitored over time. FIGS. 5I-5L show comparative studies of Her2 BiTE-T and CAR-T cells in vivo in the presence of host T cells. Fig. 5I shows the study design. Male NSG mice received 1.2Gy whole body irradiation and then were implanted with 500 ten thousand HLA-A2+ activated T cells after one day. A375.her2 cells were given on day 0. Mice were treated with HLA-A2-Her2 BiTE-T or CAR-T cells on day 8. Tumor growth (fig. 5J), donor T cells in peripheral blood (fig. 5K), and recipient T cells (fig. 5L) were monitored over time. DTCs, donor T cells; RTC, recipient T cells.
Figures 6A-6O show that co-stimulation or cytokines enhance BiTE-T cell efficacy to match or outperform CAR-T cells. Figures 6A to 6C show a continuous killing assay of a375.Her2 cells. Fig. 6A shows T cell proliferation over time. Fig. 6B shows tcrαβ expression over time. Fig. 6C shows CD3 expression over time. Figures 6D to 6H show efficacy comparisons of co-stimulated enhanced Her2 BiTE-T and CAR-T cells. Fig. 6D shows the study design. Fig. 6E shows tumor growth. Fig. 6F shows the percentage of tumor-infiltrating T cells. Fig. 6G shows donor T cells in peripheral blood over time. Fig. 7H shows the weight change over time. Figures 6I through 6M show data from comparative studies of co-stimulated enhanced CD19 BiTE-T and CAR-T cells. Fig. 6I shows the study design. Figure 6J shows survival. Small arrows indicate death events unrelated to leukemia or GvHD. Fig. 6K shows the weight change. Fig. 6L shows donor T persistence in peripheral blood. FIG. 6M shows CD 3/TCR. Alpha. Beta. Expression on WK 4 blood T cells. FIG. 6N (Her 2) and FIG. 6O (CD 19) show the evaluation of BiTE-T cells overexpressing IL7 and IL15 in an in vitro continuous killing assay.
Figures 7A-7I show that CD3 engagement induced TCR αβ/CD3 down-regulation and BiTE engineered T cells have reduced alloreactivity. FIG. 7A shows mouse TCR/CD3 expression on mouse T cells after 24 hours of treatment with plate-coated mCD3 epsilon antibodies. Figure 7B shows a schematic of BiTE and CAR constructs used in the study. Figure 7C shows tcrαβ evaluation of UT, biTE-T, or CAR-T cells using two different clones of tcrαβ antibodies. FIG. 7D shows TCRαβ/CD3 expression on CAR-T cells engineered with membrane-bound or soluble secreted CD3 εscFv. Figure 7E shows cytotoxicity of CAR-T cells engineered with membrane-bound or soluble secreted CD3 epsilon scFv. FIG. 7F shows the immunophenotype of BiTE-T and CAR-T cells. Fig. 7G-7I show GvHD risk studies using chemotherapy. Mice were treated with 250mg/kg Cyclophosphamide (CTX) and then given 1000 ten thousand transduced T cells or non-transduced T cells (UTs). Fig. 7G shows survival. Fig. 7H shows weight change. FIG. 7I shows long term leukemia protection by BiTE-T cells. In the same study, surviving mice were challenged with NALM6GL cells 4.5 months after BiTE-T cells. Leukemia cells in peripheral blood were evaluated 3 weeks after challenge.
FIG. 8A shows a representative flow chart for CD19 BiTE-T cells inducing CD3/TCRαβ down-regulation on HLA-mismatched donor T cells. FIG. 8B shows that Her2 BiTE-T cells reduce CD 3/TCR. Alpha. Beta. Expression on allogeneic T cells. HLA-A2+Her2BiTE-T cells were co-cultured with HLA-A2-PBMC in the absence of cytokines for 5 days. Flow analysis was performed on total cells.
Figures 9A-9H show that BiTE-T cells are less durable than CAR-T cells. Figure 10A shows T cell expansion in a CD19 continuous killing assay. Fig. 9B shows NALM6GL cell killing in a continuous killing assay. Fig. 9C shows T cell expansion in an a375.her2 continuous killing assay. Fig. 9D shows a375.her2 cell killing in a continuous killing assay. Fig. 10E (survival) and fig. 9F (T cell persistence) show in vivo efficacy comparisons of CD19 BiTE-T and CAR-T cells. Fig. 9G (tumor growth) and fig. 9G (tumor growth) fig. 9H (T cell persistence) show in vivo efficacy comparisons of Her2 BiTE-T and CAR-T cells.
FIG. 10 shows bioluminescence data for co-stimulated enhanced CD19 BiTE-T cells compared to CD19 CAR-T cells.
Detailed Description
Before the present disclosure is described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where 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 stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and were set forth herein by reference to disclose and describe the methods and/or materials in connection with which the publications were cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any of the recited methods may be performed in the order of recited events or in any other order that is logically possible.
Unless otherwise indicated, embodiments of the present disclosure will employ chemical, biological, etc. techniques within the skill of the art.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods of and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in degrees celsius and pressure is at or near atmospheric. Standard temperature and standard pressure are defined as 20 ℃ and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that this disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, etc., as such may vary, unless otherwise specified. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps may be performed in a different order where logically possible.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
The term "antibody" refers to immunoglobulins, derivatives thereof which retain specific binding capacity, and proteins having binding domains which are homologous or largely homologous to immunoglobulin binding domains. These proteins may be derived from natural sources or may be partially or wholly synthetically produced. Antibodies may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class from any species, including any of the classes: igG, igM, igA, igD and IgE. In exemplary embodiments, the antibodies used with the methods and compositions described herein are derivatives of the IgG class.
The term "antibody fragment" refers to any derivative of an antibody that is less than full length. In exemplary embodiments, the antibody fragment retains at least a substantial portion of the specific binding capacity of a full length antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, scFv, fv, dsFv diabodies, fc, and Fd fragments. Antibody fragments may be produced by any means. For example, an antibody fragment may be produced enzymatically or chemically by fragmentation of an intact antibody, it may be produced recombinantly from a gene encoding a portion of the antibody sequence, or it may be produced wholly or partially synthetically. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple strands linked together, for example by disulfide bonds. The fragment may also optionally be a multi-molecular complex. Functional antibody fragments typically comprise at least about 50 amino acids, and more typically at least about 200 amino acids.
The term "antigen binding site" refers to a region of an antibody that specifically binds an epitope on an antigen.
The term "bispecific antibody" refers to an antibody having two different antigen binding regions defined by different antibody sequences. This is understood to mean that different targets bind, but also binding to different epitopes in one target is included.
The term "carrier" refers to a compound, composition, substance or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, availability, selectivity or any other feature of the compound or composition for its intended use or purpose. For example, the carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
The term "engineered antibody" refers to a recombinant molecule comprising at least an antibody fragment that comprises an antigen binding site derived from the antibody heavy and/or light chain variable domains, and may optionally comprise all or part of the variable and/or constant domains of antibodies from any Ig class (e.g., igA, igD, igE, igG, igM and IgY).
The term "epitope" refers to the region of an antigen to which an antibody preferentially and specifically binds. Monoclonal antibodies preferentially bind to a single specific epitope of a molecule that can be defined on the molecule. In the present invention, a multispecific antibody may recognize multiple epitopes.
"fusion protein" or "fusion polypeptide" refers to a hybrid polypeptide comprising polypeptide portions from at least two different polypeptides. These parts may be derived from proteins of the same organism, in which case the fusion proteins are referred to as "intraspecies", "intragenic", etc. In various embodiments, the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide sequence. Where more than one amino acid sequence is fused to a first polypeptide, the fusion sequence may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences. The first polypeptide may be fused to the N-terminus, the C-terminus, or both the N-and C-termini of the second polypeptide. In addition, the first polypeptide may be inserted within the sequence of the second polypeptide.
The term "Fab fragment" refers to an antibody fragment comprising an antigen binding site generated by cleavage of an antibody with papain that cleaves at the hinge region at the N-terminus of the disulfide bond between H chains and generates two Fab fragments from one antibody molecule.
The term "F (ab') 2 fragment" refers to an antibody fragment containing two antigen binding sites, which is produced by cleavage of an antibody molecule with pepsin, which cleaves at the hinge region of the C-terminal disulfide bond between H chains.
The term "Fc fragment" refers to an antibody fragment comprising its heavy chain constant domain.
The term "Fv fragment" refers to an antibody fragment comprising its heavy and light chain variable domains.
"genetic construct" refers to a nucleic acid, such as a vector, plasmid, viral genome, etc., which includes a "coding sequence" for a polypeptide or is otherwise transcribable into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), which can be transfected into a cell, e.g., into a mammalian cell in certain embodiments, and which can cause expression of the coding sequence in a cell transfected with the construct. The genetic construct may include one or more regulatory elements operably linked to the coding sequence, as well as intron sequences, polyadenylation sites, origins of replication, marker genes, and the like.
The term "isolated polypeptide" refers to a polypeptide, which may be prepared from recombinant DNA or RNA, or is of synthetic origin, some combination thereof, or which may be a naturally occurring polypeptide, which (1) is not associated with proteins with which it is normally associated in nature; (2) isolated from the cells in which it is normally present; (3) Substantially free of other proteins from the same cellular source; (4) expressed by cells from different species; or (5) not present in nature.
The term "isolated nucleic acid" refers to a polynucleotide of genomic, cDNA, synthetic, or natural origin, or some combination thereof, which (1) is not associated with cells that find "isolated nucleic acid" in nature; or (2) operably linked to a polynucleotide to which it is not linked in nature.
The term "linker" is art-recognized and refers to a molecule or group of molecules that connects two compounds, such as two polypeptides. The linker may comprise a single linker molecule or may comprise a linker molecule and a spacer molecule, intended to separate the linker molecule and the compound by a specific distance.
The term "multivalent antibody" refers to an antibody or engineered antibody that comprises more than one antigen recognition site. For example, a "bivalent" antibody has two antigen recognition sites, while a "tetravalent" antibody has four antigen recognition sites. The terms "monospecific", "bispecific", "trispecific", "tetraspecific", and the like, refer to the number of different antigen recognition sites specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody. For example, antigen recognition sites of "monospecific" antibodies all bind the same epitope. The "bispecific" antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites, all of which bind the same epitope. A "multivalent bispecific" antibody has a plurality of antigen recognition sites, some of which bind a first epitope and some of which bind a second epitope that is different from the first epitope.
The term "nucleic acid" refers to a polymeric form of nucleotides, ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide. These terms should also be understood to include, as equivalents, analogs of RNA or DNA made from nucleotide analogs, as well as single-stranded (such as sense or antisense) and double-stranded polynucleotides suitable for use in the described embodiments.
The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms thereof which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
As used herein, "peptide mimetic" refers to a mimetic of a peptide that includes some alteration of normal peptide chemistry. Peptide mimetics typically enhance some of the characteristics of the original peptide, such as increased stability, increased efficacy, enhanced delivery, increased half-life, and the like. Methods for preparing peptide mimetics based on known polypeptide sequences are described, for example, in U.S. patent No. 5,631,280;5,612,895; and 5,579,250. The use of peptidomimetics can involve the incorporation of a non-amino acid residue with a non-amide linkage at a given position. One embodiment of the invention is a peptidomimetic in which the compound has a bond, a peptide backbone, or an amino acid component replaced by a suitable mimetic. Some non-limiting examples of unnatural amino acids that can be suitable amino acid mimics include β -alanine, L- α -aminobutyric acid, L- γ -aminobutyric acid, L- α -aminoisobutyric acid, L- ε -aminocaproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid, N- ε -Boc-N- α -CBZ-L-lysine, N- ε -Boc-N- α -Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N- α -Boc-N- δ CBZ-L-ornithine, N- δ -Boc-N- α -CBZ-L-ornithine, boc-p-nitro-L-phenylalanine, boc-hydroxyproline, and Boc-L-thioproline.
When referring to a gene product, for example, when encoded by a coding sequence, the terms "protein" (if single-stranded), "polypeptide" and "peptide" are used interchangeably herein. When referring to "polypeptides" herein, one of skill in the art will recognize that proteins may be used instead unless the context clearly indicates otherwise. "protein" may also refer to the association of one or more polypeptides. "Gene product" refers to a molecule that results from transcription of a gene. Gene products include RNA molecules transcribed from the gene, as well as proteins translated from such transcripts.
The term "polypeptide fragment" or "fragment," when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally identical to the reference polypeptide. Such deletions may occur at the amino-or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments are typically at least about 5, 6, 8, or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40, or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500, or more amino acids long. Fragments may retain one or more biological activities of the reference polypeptide. In various embodiments, the fragment may comprise the enzymatic activity and/or interaction site of the reference polypeptide. In another embodiment, the fragment may have immunogenic properties.
The term "single chain variable fragment or scFv" refers to an Fv fragment in which the heavy and light chain domains are linked. One or more scFv fragments can be linked to other antibody fragments (such as constant domains of heavy or light chains) to form an antibody construct having one or more antigen recognition sites.
As used herein, the term "specific binding," when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological products. Thus, a particular ligand or antibody "specifically binds" to its particular "target" (e.g., antibody specifically binds to endothelial antigen) when it does not bind in significant amounts to other proteins present in the sample or other proteins to which the ligand or antibody may be exposed in an organism under the specified conditions (e.g., immunoassay conditions in the case of antibodies). Typically, a first molecule that "specifically binds" a second molecule has greater than about 10 to the second molecule 5 M -1 (e.g. 10 6 M -1 、10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 And 10 12 M -1 Or more) affinity constant (Ka).
As used herein, the term "specific delivery" refers to preferential association of a molecule with a cell or tissue carrying a particular target molecule or marker, rather than with a cell or tissue lacking the target molecule. Of course, it is recognized that some degree of non-specific interaction between the molecule and non-target cells or tissues may occur. However, specific delivery can be distinguished as being mediated by specific recognition of the target molecule. In general, specific delivery results in a much stronger association between the delivered molecule and the cell carrying the target molecule than between the delivered molecule and the cell lacking the target molecule.
The term "subject" refers to any individual as a target of administration or treatment. The subject may be a vertebrate, for example a mammal. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject being treated by a clinician (e.g., physician).
The term "therapeutically effective" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of the disease or disorder. This improvement need only be reduced or altered and not necessarily eliminated.
The term "treatment" refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active therapies, i.e. therapies directed specifically to ameliorating a disease, pathological condition or disorder, and also includes causal therapies, i.e. therapies directed to eliminating the cause of the associated disease, pathological condition or disorder. Furthermore, the term also includes palliative treatment, i.e. treatment intended to alleviate symptoms rather than cure a disease, pathological condition or disorder; prophylactic treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a related disease, pathological condition, or disorder; supportive treatment, i.e. treatment for supplementing another specific therapy aimed at ameliorating the associated disease, pathological condition or disorder.
Off-the-shelf immune effector cells
Ready-made immune effector cells engineered to be modified are disclosedThe anti-CD 3 antibodies disclosed herein are configured to automatically activate immune effector cells, thereby reducing the expression of T cell receptors (e.g., tcrαβ) that may cause GVHD. Thus, these cells are preferably obtained from a healthy donor subject. Immune effector cells may be obtained from a variety of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue, and tumors. The immune effector cells may use a variety of techniques known to those skilled in the art such as Ficoll TM Isolated, obtained from blood collected from a subject. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, the method comprises lysing the erythrocytes and depleting monocytes, e.g., by PERCOL TM Gradient centrifugation or elutriation by countercurrent centrifugation separates immune effector cells from peripheral blood lymphocytes. Specific subpopulations of immune effector cells may be further isolated by positive selection or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed against surface markers characteristic of the cells being selected, such as by incubating with antibody-conjugated beads for a time sufficient to positively select for the desired immune effector cells. Alternatively, enrichment of immune effector cell populations may be accomplished by negative selection using a combination of antibodies directed against surface markers specific for negatively selected cells.
In some embodiments, immune effector cells include any white blood cells that are involved in defending the body against infectious diseases and foreign substances. For example, the immune effector cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, the immune effector cells may include T lymphocytes, preferably Cytotoxic T Lymphocytes (CTLs).
T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a cell surface T Cell Receptor (TCR). They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with different functions.
T helper cell (T) H Cells) assist in the maturation of other leukocytes during immunization, including B cells into plasma cells and memory B cells, as well as activation of cytotoxic T cells and macrophages. These cells are also called cd4+ T cells because they express CD4 glycoproteins on their surface. When peptide antigens are presented to helper T cells by MHC class II molecules expressed on the surface of Antigen Presenting Cells (APCs), the helper T cells become activated. Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including T H 1、T H 2、T H 3、T H 17、T H 9 or T FH It secretes different cytokines to promote different types of immune responses.
Cytotoxic T cells (T C Cells or CTLs) destroy virus-infected cells and tumor cells, and are also associated with transplant rejection. These cells are also known as CD8 + T cells because they express CD8 glycoprotein on the surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. By means of IL-10, adenosine and other molecules secreted by regulatory T cells, CD8+ cells can be deactivated to non-responsive state, so as to prevent autoimmune diseases.
Memory T cells are a subset of antigen-specific T cells that persist for a long period of time after infection has subsided. When they are re-exposed to homologous antigens, they rapidly expand into a large number of effector T cells, providing the immune system with a "memory" against past infections. The memory cell may be CD4 + Or CD8 + . Memory T cells typically express the cell surface protein CD45RO.
Regulatory T cells (T reg Cells) were previously referred to as suppressor T cells, which are critical for maintaining immune tolerance. Their primary effects are to shut down T cell mediated immunity at the end of the immune response and to suppress autoreactive T cells that escape the thymic negative selection process. Two broad classes of CD4 have been described + T reg Cells-naturally occurring T reg Cells and adaptive T reg And (3) cells.
Natural Killer T (NKT) cells (not to be confused with Natural Killer (NK) cells) link the adaptive immune system with the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by Major Histocompatibility Complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by CD1d molecules.
In some embodiments, the T cells comprise a mixture of cd4+ cells. In other embodiments, T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T cells include cytotoxic CD8 + T lymphocytes. In some embodiments, the T cells comprise γδ T cells that possess a unique T Cell Receptor (TCR) with one γ chain and one δ chain instead of the α and β chains.
Natural Killer (NK) cells are CD56 + CD3 - Large granular lymphocytes, which kill virally infected and transformed cells, and constitute a critical subset of cells of the innate immune system (Godfrey J, et al Leuk Lymphoma 2012 53:1666-1676). With cytotoxic CD8 + Unlike T lymphocytes, NK cells initiate cytotoxicity against tumor cells without prior sensitization, and can also eradicate MHC-I negative cells (Narni-Mancinelli E, et al int Immunol 2011 23:427-431). NK cells are safer effector cells because they can avoid the following potentially fatal complications: cytokine storm (Morgan RA, et al mol Ther 2010:843-851), oncolytic syndrome (Porter DL, et al N Engl J Med 2011:725-733), and mid-target non-tumor effects. While NK cells have a well known role as cancer cell killers, and NK cell damage has been widely documented as critical to MM progression (Godfrey J, et al Leuk Lymphoma 2012 53:1666-1676;Fauriat C,et al.Leukemia 2006 20:732-733), prior to the disclosed CAR, means of enhancing NK cell mediated anti-MM activity have not been explored substantially.
Epstein-Barr virus (EBV) induced lymphoproliferative disease (EBV-LPD) and other EBV-associated cancers are important causes of morbidity and mortality in allogeneic Hematopoietic Cell Transplantation (HCT) or Solid Organ Transplantation (SOT) recipients, particularly those receiving certain T-cell reactive antibodies to prevent or treat GVHD. Prevention and treatment by adoptive transfer of autologous or allogeneic EBV-specific cytotoxic T cells, and subsequent long-term restoration of immunity to EBV-related lymphoproliferation, provides positive results in treating these consistent fatal complications of allogeneic tissue transfer. Thus, in some embodiments, the disclosed immune effector cells comprising one or more CAR polypeptides of the invention are allogeneic or autologous EBV-specific Cytotoxic T Lymphocytes (CTLs). For example, the generation of EBV-specific cytotoxic T cells may involve isolation of PBMCs from EBV seropositive autologous or allogeneic donors and enrichment by depletion of monocytes and NK cells. EBV-specific cytotoxic T cells can also be produced by contacting donor PBMCs or purified donor T cells with "stimulator" cells that express one or more EBV antigens and present the one or more EBV antigens to unstimulated T cells, thereby causing stimulation and expansion of the EBV-specific CTLs. EBV antigens include, for example, latent Membrane Proteins (LMP) and EBV nuclear antigen (EBNA) proteins, such as LMP-1, LMP-2A and LMP-2B, and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP. Cytotoxic T cells comprising one or more T cell receptors that recognize one or more EBV specific antigens are considered to have been "sensitized" to those EBV antigens, and are therefore referred to herein as "EBV sensitized cytotoxic T cells". Known methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations that may comprise one or more of the CAR polypeptides of the invention are described, for example, in Barker et al, blood 2010 116 (23): 5045-49; doubrovina, et al, blood 2012 119 (11): 2644-56; koehne, et al blood 2002 99 (5): 1730-40; and Smith et al cancer Res 2012 72 (5): 1116-25, which is incorporated by reference for these teachings.
Bispecific antibodies
Bispecific antibodies may contain heavy chains comprising one or more variable regions and/or light chains comprising one or more variable regions. The use of antibody variable domains alone can be used to construct a dtexA specific antibody. A fairly efficient and relatively simple way is to let V H And V L The linker sequences between the domains are so short that they cannot fold and bind to each other. Reducing the linker length to 3-12 residues prevents monomer configuration of the scFv molecule and facilitates intermolecular VH-VL pairing to form a 60kDa non-covalent scFv dimer "diabody". Diabody forms can also be used to generate recombinant bispecific antibodies obtained by non-covalent association of two single chain fusion products consisting of VH domains from one antibody linked to VL domains of the other antibody by short linkers. Further reduction of the linker length below three residues may result in the formation of trimers ("trisomy", about 90 kDa) or tetramers ("tetrasomy", about 120 kDa). For a review of engineered antibodies, particularly single domain fragments, see Holliger and Hudson,2005,Nature Biotechnology,23:1126-1136. All such engineered antibodies can be used in the fusion polypeptides provided herein.
Peptide linkers (-) suitable for the production of scFv antibodies are described in Kumaday, et al Biochemical Engineering journal.2007 (2): 158-165; albrecht H, et al J Immunol methods.2006 310 (1-2): 100-16; feng J, et al J Immunol methods 2003 282 (1-2): 33-43; griffiths AD, et al, curr Opin Biotechnol 1998 9 (1): 102-8; huston JS, et al methods enzymol.1991 203:46-88; bird RE, et al science.1988 242 (4877): 423-6; takkien K, et al protein Eng.1991 4 (7): 837-41; smallshaw JE, et al protein Eng.1999.12 (7): 623-30; argos P.J Mol biol 1990 211 (4): 943-58; and Whitlow M, et al protein Eng.1993 (8): 989-95, which is hereby incorporated by reference for the teachings of these linkers and methods of using various linkers to generate scFv antibodies directed against different targets.
Tetravalent typeCan be prepared essentially as described in WO1999/057150 A3 or US 2006/023787 for the preparation of +.>Molecular methodThe teachings are incorporated by reference.
The antigen recognition site or the entire variable region of the engineered antibody may be derived from one or more parent antibodies against any antigen of interest (e.g., target receptor ECD or TMUL ECD). The parent antibody may include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, antibodies constructed de novo using sequences of antibodies or antibody fragments known to have specificity for the antigen of interest. Sequences that may be derived from a parent antibody include heavy and/or light chain variable regions and/or CDRs, framework regions or other portions thereof.
In some cases, V L 3 comprises the amino acid sequence MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRA (SEQ ID NO: 1), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:1 that is capable of binding CD 3. In some cases, V H 3 comprises the amino acid sequence EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 2), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:2 that is capable of binding CD 3.
Other anti-CD 3 antibody sequences useful in the disclosed systems are known in the art, such as OKT3.
In some cases, the target cell surface receptor is Her2. Thus, in some cases, V H R comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEPKSCDKTHTCP (SEQ ID NO: 3), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:3 that is capable of binding Her2. In some cases, V L R comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 4), or it has at least 70% with SEQ ID NO:4A fragment or variant capable of binding Her2 that is 75%, 80%, 85%, 90%, 95% or 100% sequence identity.
In some cases, the target cell surface receptor is EGFR. In some cases, V H R comprises the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTEYPIHWVKQAPGKGFKWMGMIYTDIGKPTYAEEFGRFAFSLETSASTAYLQINNLKNEDTATYFCVRDRYDSLFDYWGQGTTLTVSS (SEQ ID NO: 5), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:5 that is capable of binding EGFR. In some cases, V L R comprises the amino acid sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK (SEQ ID NO: 6), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:6 that is capable of binding EGFR.
In some cases, the target cell surface receptor is CSPG4. In some cases, V H R comprises the amino acid sequence EVQLVESGAEVKKPGDSLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSETTYSPAFQGDVTISVDKSISTAYLQWNSLKASDTGIYYCARRRGNYYMDVWGNGTLVTVSSLKS (SEQ ID NO: 7), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:7 that is capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence RSTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRHVFGTGTQLTVLG (SEQ ID NO: 8), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:8 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is CD20. In some cases, V H R comprises the amino acid sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 9), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:9 that is capable of binding CD20. In some cases, V L R comprises the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 10), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:10 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is CD20. In some cases, V H R comprises the amino acid sequence EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS (SEQ ID NO: 11), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:11 that is capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO: 12), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:12 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is PSMA. In some cases, V H R comprises an amino acid sequence MARGSSSLDLNWYSLGLQXKLSCKSSKUtYTQFYINGWLRQRPEQGLEWUWGPQGGgnTNTNENFKGKATLTIDKSSTTAYIQLSrltsedSavYFCARDGNFPYYAMDSQGTSVTVSSAKTTPKL (SEQ ID NO: 13), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID No. 13 capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence DIVMTQIPLSLPVILGDQASISCRSSQSLVYSNGNTYLHWFLQKPGQSPKLLIYNVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPTFGGGTKLEIKRADAAAAGS (SEQ ID NO: 14), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:14 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is BCMA. In some cases, V H R comprises the amino acid sequence QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSA (SEQ ID NO: 15), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:15 capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence YVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVL (SEQ ID NO: 16), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:16 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is mesothelin. In some cases, V H R comprises the amino acid sequence QVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSS (SEQ ID NO: 17), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:17 that is capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence DIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK (SEQ ID NO: 18), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:18 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is GPC3. In some cases, V H R comprises the amino acid sequence QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWYAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLSPEDTALYFCARGWFVEPLSWGQGTLVTVSS (SEQ ID NO: 19), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:19 that is capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL (SEQ ID NO: 20), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:20 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is EpCAM. In some cases, V H R comprises the amino acid sequence QVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDPFLHYWGQGTLVT (SEQ ID NO: 21), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:21 that is capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence EIELTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTFTFGPGTKVEI (SEQ ID NO: 22), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:22 that is capable of binding CSPG 4.
In some cases, the target cell surface receptor is GD2. In some cases, V H R comprises the amino acid sequence QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSS (SEQ ID NO: 23), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:23 that is capable of binding CSPG 4. In some cases, V L R comprises the amino acid sequence EIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKR (SEQ ID NO: 24), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:24 that is capable of binding CSPG 4.
The specific length of the peptide linker (- -) used to link the scFv molecules together is important to determine the half-life, immunogenicity and activity of the whole construct. In some embodiments, the linker sequence (- -) is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length. In some embodiments, the linker sequence (- -) comprises GGGGS (SEQ ID NO: 25). In some cases, the linker comprises 2, 3, 4, 5 or more GGGGS (SEQ ID NO: 25) sequences.
In some embodiments, the hinge sequence (-/-)) Comprises EPKSCDKTHTCP (SEQ ID NO: 26). The linker is preferably long enough not to interfere with V H -V L The correct folding and association of the chains, but not so long as to result in increased immunogenicity.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEPKSCDKTHTCPEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 27), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:27 that is capable of binding CD3 and Her 2.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ ID NO: 28), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:28 capable of binding CD3 and Her 2.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QIQLVQSGPELKKPGETVKISCKASGYTFTEYPIHWVKQAPGKGFKWMGMIYTDIGKPTYAEEFGRFAFSLETSASTAYLQINNLKNEDTATYFCVRDRYDSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKEPKSCDKTHTCPEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 29), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:29 that is capable of binding CD3 and EGFR.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQIQLVQSGPELKKPGETVKISCKASGYTFTEYPIHWVKQAPGKGFKWMGMIYTDIGKPTYAEEFGRFAFSLETSASTAYLQINNLKNEDTATYFCVRDRYDSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKEPKSCDKTHTCP (SEQ ID NO: 30), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:30 that is capable of binding CD3 and EGFR.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: EVQLVESGAEVKKPGDSLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSETTYSPAFQGDVTISVDKSISTAYLQWNSLKASDTGIYYCARRRGNYYMDVWGNGTLVTVSSLKSGGGGSGGGGSGGGGSRSTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRHVFGTGTQLTVLGEPKSCDKTHTCPEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 31), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:31 that is capable of binding CD3 and CSPG 4.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPEVQLVESGAEVKKPGDSLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSETTYSPAFQGDVTISVDKSISTAYLQWNSLKASDTGIYYCARRRGNYYMDVWGNGTLVTVSSLKSGGGGSGGGGSGGGGSRSTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRHVFGTGTQLTVLGEPKSCDKTHTCP (SEQ ID NO: 32), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:32 that is capable of binding CD3 and CSPG 4.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 33), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:33 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 34), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:34 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 35), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:35 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO: 36), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:36 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MARKSSSSLQQQKQQKQSKQQQQQQQQFKQQQQFQQQFQQQQQQQFQQQQVQFQQQQQFQQQQQFQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQtiming Qtiming timing Qtiming four, is QQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ (SEQ ID NO: (SEQ ID NO: 37), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID No. 37 capable of binding CD3 and PSMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQQQQQQSSLSLSASLGDRVTDRDRDRVSSASSEDSSEDSAVYCYYYGYGYGYGQQQQQQQQGSQSLKQSLKQUQKQQGQGQGQGQQGGGSGGGSGGGSGGGSGGGSGGQQQQQQQQQQQQQGGSkVkVUSkSGUGGGGYTMNKHGKHGLEWMGLVQKKKKKKKQQQSQSQSQSQSQSQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ (SEQ ID NO: (SEQ ID NO: 38), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID No. 38 capable of binding CD3 and PSMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSAGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVLEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 39), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:39 that is capable of binding CD3 and BCMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSAGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVL (SEQ ID NO: 40), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:40 that is capable of binding CD3 and BCMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 41), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:41 that is capable of binding CD3 and mesothelin.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK (SEQ ID NO: 42), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:42 that is capable of binding CD3 and mesothelin.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWYAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLSPEDTALYFCARGWFVEPLSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVLEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 43), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:43 capable of binding CD3 and GPC 3.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWYAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLSPEDTALYFCARGWFVEPLSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL (SEQ ID NO: 44), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:44 that is capable of binding CD3 and GPC 3.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDPFLHYWGQGTLVTGGGGSGGGGSGGGGSEIELTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTFTFGPGTKVEIEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 45), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:45 that is capable of binding CD3 and EpCAM.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDPFLHYWGQGTLVTGGGGSGGGGSGGGGSEIELTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTFTFGPGTKVEI (SEQ ID NO: 46), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:46 that is capable of binding CD3 and EpCAM.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKREPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 47), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:47 capable of binding CD3 and GD 2.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKR (SEQ ID NO: 48), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:48 that is capable of binding CD3 and GD 2.
In some cases, the encoded bispecific antibody also contains a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence: MDFQVQIFSFLLISASVIMSRG (SEQ ID NO: 49).
Thus, in some embodiments, the encoded bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 50), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:50 that is capable of binding CD3 and Her 2.
Thus, in some embodiments, the encoded bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ ID NO: 51), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:51 capable of binding CD3 and Her 2.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQIQLVQSGPELKKPGETVKISCKASGYTFTEYPIHWVKQAPGKGFKWMGMIYTDIGKPTYAEEFGRFAFSLETSASTAYLQINNLKNEDTATYFCVRDRYDSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKEPKSCDKTHTCP (SEQ ID NO: 52), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:52 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQIQLVQSGPELKKPGETVKISCKASGYTFTEYPIHWVKQAPGKGFKWMGMIYTDIGKPTYAEEFGRFAFSLETSASTAYLQINNLKNEDTATYFCVRDRYDSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 53), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:53 that is capable of binding CD3 and EGFR.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGEVQLVESGAEVKKPGDSLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSETTYSPAFQGDVTISVDKSISTAYLQWNSLKASDTGIYYCARRRGNYYMDVWGNGTLVTVSSLKSGGGGSGGGGSGGGGSRSTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRHVFGTGTQLTVLGEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 54), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:54 that is capable of binding CD3 and CSPG 4.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQIQLVQSGPELKKPGETVKISCKASGYTFTEYPIHWVKQAPGKGFKWMGMIYTDIGKPTYAEEFGRFAFSLETSASTAYLQINNLKNEDTATYFCVRDRYDSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKEPKSCDKTHTCP (SEQ ID NO: 55), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:55 that is capable of binding CD3 and CSPG 4.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 56), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:56 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 57), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:57 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 58), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:58 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK (SEQ ID NO: 59), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:59 that is capable of binding CD3 and CD 20.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQQVQQFQFLLISASVSRSRQSRQSRRFSSSSLDLNWYSLQKLSCKKQFKQFQFQFQFQFQFQGQGQGQGQGVGQGQGGQGGQQQQGVQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQGGGGGGGGGGGGGGGGGGGGGGGGGGUGGQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQSQQQQQQQQSQQQQQQQSQQQQQQQQQQQQSQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ (SEQ ID NO: (SEQ ID NO: 60), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID No. 60 capable of binding CD3 and PSMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQQVQFSFLLISASVSRQTTSSLSSRQTQSSLSSSASLGDRQTQDIRNYLNQQKQQQKQQQQQQQQQQQQQQQQGTQQQQQQGGTQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQstage stage (SEQ ID NO: (SEQ ID NO: 61), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID No. 61 capable of binding CD3 and PSMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSAGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVLEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 62), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:62 that is capable of binding CD3 and BCMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSAGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVL (SEQ ID NO: 63), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:63 that is capable of binding CD3 and BCMA.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 64), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:64 that is capable of binding CD3 and mesothelin.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK (SEQ ID NO: 65), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:65 that is capable of binding CD3 and mesothelin.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWYAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLSPEDTALYFCARGWFVEPLSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVLEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 66), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:66 that is capable of binding CD3 and GPC 3.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWYAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLSPEDTALYFCARGWFVEPLSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL (SEQ ID NO: 67), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:67 that is capable of binding CD3 and GPC 3.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDPFLHYWGQGTLVTGGGGSGGGGSGGGGSEIELTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTFTFGPGTKVEIEPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 68), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:68 that is capable of binding CD3 and EpCAM.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDPFLHYWGQGTLVTGGGGSGGGGSGGGGSEIELTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTFTFGPGTKVEI (SEQ ID NO: 69), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:69 that is capable of binding CD3 and EpCAM.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKREPKSCDKTHTCPMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTV (SEQ ID NO: 70), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:70 that is capable of binding CD3 and GD 2.
Thus, in some embodiments, the bispecific antibody has the amino acid sequence: MDFQVQIFSFLLISASVIMSRGMADIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKRAGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVEPKSCDKTHTCPQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKR (SEQ ID NO: 71), or a fragment or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO:71 that is capable of binding CD3 and GD 2.
A variety of known assays can be used to screen for activity of candidate engineered antibodies for inclusion in the fusion polypeptide or the fusion polypeptide itself. For example, screening assays to determine binding specificity are well known in the art and are routinely performed. For a complete discussion of such assays, see Harlow et al (eds.), ANTIBODIES: A LABORATORY MANUAL; cold Spring Harbor Laboratory; cold Spring Harbor, n.y.,1988, chapter 6.
In some embodiments, the bispecific antibody may be subject to alteration such that it is less immunogenic when administered to a human. Such alterations may include one or more of the techniques commonly referred to as chimeric, humanized, CDR grafting, deimmunization, and/or mutating framework region amino acids to correspond to the closest human germline sequence (germline). Thus, an altered bispecific antibody will remain administerable for a longer period of time with reduced or no immune response related side effects compared to a corresponding bispecific antibody that has not undergone any such one or more alterations. One of ordinary skill in the art will understand how to determine whether and to what extent antibodies must be altered to prevent them from eliciting an unwanted host immune response.
Pharmaceutical composition
Also disclosed are pharmaceutical compositions comprising the disclosed molecules in a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These will most typically be standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Suitable carriers and formulations thereof are described, for example, in remington: the Science and Practice of Pharmacy (21 ed.) ed.pp. Gerbinao, lippincott Williams & Wilkins, philiadelphia, pa.2005. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition being administered.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, and the like, which are physiologically compatible with the bispecific antibodies of the present invention. Examples of suitable aqueous and nonaqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose gum solutions, tragacanth, and injectable organic esters such as ethyl oleate and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
Therapeutic method
The disclosed adoptive transfer of ready T cells can be used to treat a variety of diseases and disorders in an allogeneic subject, such as cancer, autoimmune diseases, diabetes, neurological diseases, chronic viral infections, bacterial infections, parasitic infections, alzheimer's disease, heart disease. The disclosed T-cells can also be genetically engineered to express one or more CARs.
The disclosed T-cells may be administered alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2, IL-15 or other cytokines or cell populations. Briefly, a pharmaceutical composition may comprise a target cell population as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise: buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. In some embodiments, the compositions used in the disclosed methods are formulated for intravenous administration. The pharmaceutical composition may be administered in any suitable manner for treating MM. The amount and frequency of administration will be determined by factors such as the condition of the patient and the severity of the patient's disease, although the appropriate dosage may be determined by clinical trials.
When "immunologically effective amount", "antineoplastic effective amount", "tumor inhibiting effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered may be determined by a physician taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). In general, it can be said that a pharmaceutical composition comprising the T cells described herein can be 10 4 To 10 9 Administration of a dose of individual cells/kg body weight, e.g. 10 5 To 10 6 Individual cells/kg body weight, including all integer values within these ranges. T cell compositions may also be administered at these doses multiple times. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., rosenberg et al, new eng. J. Of med.319:1676,1988). Those skilled in the medical arts can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's signs of disease and adjusting the treatment accordingly.
Administration of the disclosed compositions may be performed in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by intravenous injection. The composition may also be injected directly into a tumor, lymph node or infection site.
In certain embodiments, the disclosed off-the-shelf T cells are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic regimens including, but not limited to, thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells can be combined with chemotherapy, radiation therapy, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK 506), antibodies, or other immune scavengers (such as CAM PATH, anti-CD 3 antibodies, or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation). In some embodiments, the CAR-modified immune effector cells are administered to the patient in combination (e.g., before, simultaneously with, or after) bone marrow transplantation, T-cell ablation therapy (using a chemotherapeutic agent such as fludarabine, external irradiation radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH, etc.). In another embodiment, the cell composition of the invention is administered after B cell ablative therapy, such as an agent that reacts with CD20 (e.g., rituxan). For example, in some embodiments, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives infusion of the expanded immune cells of the invention. In further embodiments, the expanded cells are administered before or after surgery.
In some embodiments, the method is for treating cancer in an allogeneic subject. In some aspects, the cancer may be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor. Representative but non-limiting lists of cancers that the disclosed compositions can be used to treat include lymphomas, B-cell lymphomas, T-cell lymphomas, mycosis fungoides, hodgkin's disease, myelogenous leukemia, bladder cancer, brain cancer, cancers of the nervous system, head and neck cancer, head and neck squamous cell carcinoma, lung cancer such as renal cancer, small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, oral squamous cell carcinoma, laryngeal cancer, lung cancer, endometrial cancer, cervical cancer, breast cancer, epithelial cancer, kidney cancer, genitourinary system cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic system cancer; testicular cancer; colon and rectal cancer, prostate cancer and pancreatic cancer.
The disclosed T-cells may be used in combination with any compound, moiety or group that has cytotoxic or cytostatic effects. The pharmaceutical moiety includes chemotherapeutic agents, which may act as tubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those for the treatment of cancer.
The disclosed T-cells may be used in combination with checkpoint inhibitors. Two known inhibitory checkpoint pathways involve signaling through cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 co-signaling family of molecules and play an important role in various stages of T cell function. The PD-1 receptor (also known as CD 279) is expressed on the surface of activated T cells. Its ligands PD-L1 (B7-H1; CD 274) and PD-L2 (B7-DC; CD 273) are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the primary ligand, while the expression pattern of PD-L2 is more restricted. When the ligand binds to PD-1, an inhibitory signal is transmitted to T cells, thereby reducing cytokine production and inhibiting T cell proliferation. Checkpoint inhibitors include, but are not limited to, antibodies blocking PD-1 (Nivolumab) (BMS-936558 or MDX 1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB 0010718C), PD-L2 (rHIgM 12B 7), CTLA-4 (Ipilimumab) (MDX-010), tremelimumab (Tremelimumab) (CP-675,206)), IDO, B7-H3 (MGA 271), B7-H4, TIM3, LAG-3 (BMS-986016).
Human monoclonal antibodies directed against programmed death 1 (PD-1) and methods of treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents are described in U.S. patent No. 8,008,449, which is incorporated by reference for these antibodies. anti-PD-L1 antibodies and their use are described in U.S. patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agents comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. patent No. 8,617,546, which is incorporated by reference for these antibodies.
In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds to PD1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb) or MEDI4736 (AstraZeneca). Human monoclonal antibodies directed against PD-1 and methods of treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents are described in U.S. patent No. 8,008,449, which is incorporated by reference for these antibodies. anti-PD-L1 antibodies and their use are described in U.S. patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agents comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. patent No. 8,617,546, which is incorporated by reference for these antibodies.
The disclosed T-cells may be used in combination with other cancer immunotherapies. There are two different types of immunotherapy: passive immunotherapy utilizes components of the immune system to direct targeted cytotoxic activity against cancer cells, not necessarily eliciting an immune response in a patient; active immunotherapy actively triggers an endogenous immune response. Passive strategies include monoclonal antibodies (mabs) produced by B cells in response to specific antigens. The development of hybridoma technology and the identification of tumor-specific antigens in the 70 s of the 20 th century enabled the development of drugs that could specifically target tumor cells for mabs destroyed by the immune system. Monoclonal antibodies have so far been the most successful case of immunotherapy. The first three most popular anticancer drugs in 2012 are monoclonal antibodies. Among these are rituximab (Rituxan, genentech), which binds to CD20 protein that is highly expressed on the surface of B cell malignancies such as non-hodgkin's lymphoma (NHL). Rituximab is approved by the FDA for the treatment of NHL and Chronic Lymphocytic Leukemia (CLL) in combination chemotherapy. Another important monoclonal antibody is trastuzumab (Herceptin; genentech), which completely alters the treatment of HER2 (human epidermal growth factor receptor 2) positive breast cancer by targeting HER2 expression.
T-cell receptor activation and co-stimulation are also required to generate optimal "killer" CD 8T cell responses, which can be provided by linking members of the tumor necrosis factor receptor family, including OX40 (CD 134) and 4-1BB (CD 137). OX40 is of particular interest because treatment with an activating (agonist) anti-OX 40 mAb enhances T cell differentiation and cytolytic function, thereby enhancing anti-tumor immunity against a variety of tumors.
In some embodiments, such additional therapeutic agents may be selected from antimetabolites, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine (dacarbazine), hydroxyurea, asparaginase, gemcitabine, or cladribine.
In some embodiments, such additional therapeutic agents may be selected from alkylating agents, such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives, such as carboplatin.
In some embodiments, such additional therapeutic agents may be selected from antimitotic agents, for example, taxanes (such as docetaxel and paclitaxel), and vinca alkaloids (such as vindesine, vincristine, vinblastine, and vinorelbine).
In some embodiments, such additional therapeutic agents may be selected from topoisomerase inhibitors, such as topotecan or irinotecan, or cytostatic drugs, such as etoposide and teniposide.
In some embodiments, such additional therapeutic agents may be selected from growth factor inhibitors, for example, an ErbB1 (EGFR) inhibitor (such as an EGFR antibody, for example, zafiuximab, cetuximab, panitumumab or nituzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER 2/neu), such as a HER2 antibody, for example, trastuzumab-DM l or pertuzumab, or an inhibitor of both EGFR and HER2, such as lapatinib.
In some embodiments, such additional therapeutic agents may be selected from tyrosine kinase inhibitors, such as imatinib (glicc, gleevec STI 571) or lapatinib.
Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanolimumab, adalimumab (danatumumab), ranibizumab (ranibizumab), nituzumab (nimotuzumab), panitumumab (panitumumab), hu806, daclizumab (daclizumab) (Zenapax), basiliximab (basiliximab) (Simulect), infliximab (Remicade), adalimumab (adalimumab) (Humira), natalizumab (natalizumab) (tyrabi), omalizumab (omalizumab) (xolazumab), efalizumab (efalizumab) and/or rituximab.
In some embodiments, the therapeutic agent used in combination with off-the-shelf T cells to treat the disorder described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2 b), IFN, GM-CSF, CD40L, flt3 ligand, stem cell factor, anxisetin (Ancestim), and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR) -negative chemokines, such as IP-10, MCP-3, MIG, and SDF-1a from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments and cytokine fusion proteins.
In some embodiments, the therapeutic agent used in combination with an off-the-shelf T cell to treat a disorder as described above may be a cell cycle control/apoptosis modulator (or "modulator"). Cell cycle control/apoptosis modulators may include molecules that target and modulate cell cycle control/apoptosis modulators, such as (i) cdc-25 (such as NSC 663284); (ii) Cyclin-dependent kinases that overstimulate the cell cycle, such as Huang Bi alcohol (L868275, HMR 1275), 7-hydroxysteroid (UCN-01, KW-2401) and roscovitine (R-roscovitine, CYC 202); and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions such as described in US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with the apoptotic pathway include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFN, and antisense Bcl-2.
In some embodiments, the therapeutic agent used in combination with off-the-shelf T cells to treat the disorder as described above may be a hormonal regulator, such as an agent useful in anti-androgens and anti-estrogenic therapies. Examples of such hormone modulators are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estradiol, antiandrogens such as flutamide (flutamide)/eulexin, progestins such as medroxyprogesterone, medroxyprogesterone/megestrol acetate (megace), adrenocorticosteroids such as hydrocortisone, prednisone, luteinizing hormone releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), aromatase inhibitors such as anastrozole/rilidine (arimide), aminoglutethimide/cytraden, exemestane, or hormone inhibitors such as octreotide/edestatin.
In some embodiments, the therapeutic agent used in combination with off-the-shelf T cells to treat the disorder described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.
As noted above, the combined administration may be simultaneous, separate or sequential. For simultaneous administration, the agents may be administered as one composition or as separate compositions, as the case may be.
In some embodiments, the disclosed off-the-shelf T cells are administered in combination with radiation therapy. Radiation therapy may include providing radiation or related radiopharmaceutical administration to a patient. The radiation source may be external or internal to the patient undergoing treatment (radiation therapy may be in the form of, for example, external Beam Radiation Therapy (EBRT) or Brachytherapy (BT)). Radioactive elements that may be used to practice such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium 111.
In some embodiments, the disclosed off-the-shelf T cells are administered in conjunction with surgery.
Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Examples
Example 1
FIGS. 1A-1C show in vitro CD3/TCRαβ expression after treatment with: CD19 BiTE or Her2 BiTE (fig. 1A); her2 bite+αher2BB, her2 car+αcd3εil15RA, or Her2 car+αcd3εscfv (fig. 1B); and CD19 bite+41BBLCD80, CD19 tripe 1 (aCD 28) or CD19 tripe 2 (41 BBL) (fig. 1C).
Fig. 2A and 2B show GvDH model tests. FIG. 2A shows NSG mice treated with whole body irradiation and 1e7 Her2 BiTE-T or CD19 BiTE-T cells. Figure 2B shows NSG mice treated with chemotherapy and 1e7ctx+car-T or ctx+bite-T cells.
FIG. 3 shows in vivo CD 3/TCR. Alpha. Beta. Expression in the absence of antigen. NSG mice received irradiation and then received T cells. Donor T cells in peripheral blood were evaluated at certain time points. D12, day 12 after T cell injection.
FIG. 4 shows in vivo CD 3/TCR. Alpha. Beta. Expression in the presence of antigen. NSG mice were injected with tumor cells and then received T cell therapy. Donor T cells in peripheral blood or tumors were evaluated at certain time points.
Example 2
Next, an in vitro continuous killing assay was established to evaluate tcrαβ and CD3 on BiTE-T cells in the presence of target antigen. T cells are repeatedly challenged with target cells until they fail to kill. TCR αβ and CD3 epsilon expression was assessed every 48 hours during this period. For both CD19 and Her2 targets, biTE-T cells maintained low tcrαβ/CD3 in the challenge assay in contrast to CAR-T and UT cells. In vivo, her2 BiTE-T cells were evaluated using a solid tumor model expressing Her 2. NSG mice were first vaccinated with Her2 expressing human melanoma cells a375 (a 375. Her2) and then treated with BiTE-T or UT. Blood T cells and tumor infiltrating T cells were evaluated by flow cytometry. In peripheral blood, her2 BiTE-T cells showed limited presence and low CD3 epsilon. Tumor infiltrating Her2 BiTE-T cells also showed lower TCRαβ/CD3 than CAR-T cells. These results indicate that BiTE-T cells maintain low CD3 epsilon and tcrαβ in the presence of target antigen.
Allogeneic OTS T cells are expected to overcome host rejection in order to last longer and maximize their efficacy. An important mechanism of host rejection is that host T cells recognize non-self antigens via TCRs. CD19 BiTE-T cells were co-cultured with HLA-mismatched T cells for 72 hours and BiTE-T was found to reduce CD3 and TCR. Alpha. Beta. On recipient T cells. Similar results were also observed when alloMLR assays were established by co-culturing CD19 or Her2 BiTE-T cells with HLA-mismatched PBMC (FIG. 8). These data indicate that donor BiTE-T cells have the potential to overcome host rejection by releasing bites that bind tcrαβ/CD3 on host T cells.
Other groups have demonstrated anti-cancer activity of BiTE-T cells (Iwahori, K.et al mol Ther 2015:171-178;Choi,B.D.et al.Nat Biotechnol 2019 37:1049-1058;Liu,X.et al.Protein Cell 2017 8:514-526). The anticancer activity of the BiTE-T and second generation CAR-T cells were compared side by side. CD19 or Her2 BiTE-T and CAR-T cells sharing the same scFv were evaluated. For both targets, biTE-T cells showed comparable (if not better) killing power in vitro as CAR-T cells (fig. 5A, 5C). BiTE-T cells produced only a small fraction of the cytokines produced by CAR-T cells (FIGS. 5B, 5D), including IFNγ, IL2, IL6, and TNF α. This suggests that BiTE-T cells may be safer than CAR-T cells. However, CAR-T cells outperformed BiTE-T cells in continuous killing assays (fig. 9A-9D) and also provided better survival, persistence, and efficacy in NSG models transplanted with NALM6 or a375.Her2 cells (fig. 9E-9H). This is not surprising, as the second generation CAR-T cells have a co-stimulatory domain built in.
One potential advantage of BiTE-T cells is the ability to mobilize bystander T cells or host T cells. Thus, a mouse model was used to model the environment with pre-existing host T cells (Mo, F.et al Nat Biotechnol 2021 39:56-63). NSG mice were irradiated and then implanted with activated HLA-A2+ human T cells. The mice were then injected with tumor cells and subsequently treated with HLA-A2-BiTE-T cells or CAR-T cells (fig. 5E). In the NSG NALM6 model with pre-existing T cells, both CD 19-targeting BiTE-T cells and CAR-T cells showed survival benefits comparable to UT cells (fig. 5F). Although host T cells showed expansion in CAR-T treated groups (fig. 5G, 5H), donor CD19 CAR-T cell expansion was also inhibited in this model. In the her2+ tumor model with pre-existing host T cells (fig. 5I), CAR-T cells were unable to inhibit tumor growth, while BiTE-T cells were still viable (fig. 5J). Both Her 2-targeted BiTE-T and CAR-T cells showed very limited persistence in peripheral blood (fig. 5K). However, her2 BiTE-T cells induced greater expansion of host T cells than CAR-T cells (fig. 5L).
To enhance efficacy, co-stimulation was added to BiTE-T cells. T cells were co-transduced with a combination of BiTE, 4-1BBL and CD80 (Velasquez, M.P.et al. Cancer Immunol Res 20175:860-870) to prepare next generation BiTE-T cells. These enhanced Her2 BiTE-T cells showed better proliferation than Her2 CAR-T cells in the continuous killing assay while maintaining low levels of CD3/tcrαβ (fig. 6A-6C). In vivo (fig. 6D), her2 BiTE-T cells showed comparable tumor inhibition, blood persistence, tumor infiltration, and weight to CAR-T cells (fig. 6E-6H). Similarly, in the NALM6 model (fig. 6I), the enhanced CD19 BiTE-T cells had efficacy comparable to CAR-T cells and possibly better blood persistence than CAR-T cells (fig. 6J-6L, fig. 10). Enhanced CD19 BiTE-T cells maintained low CD3/TCRαβ in vivo (FIG. 6M). BiTE-T cells are also enhanced by engineering with a combination of IL7 and IL15 genes. In an in vitro continuous killing assay, biTE T cells engineered with cytokines outperformed CAR-T cells (fig. 6N, fig. 6O).
This study proposes a new strategy for allogeneic OTS T cell therapy. CAR-T cell therapy is one of the most powerful cancer therapies currently approved by the FDA. Recent clinical trials have shown that allogeneic CAR-T cells are likely to replace autologous allogeneic cells, although some efficacy is sacrificed. BiTE-T cells may have some advantages over allogeneic OTS CAR-T cells. First, the production of BiTE-T cells is less complex and is a natural "pure" product. BiTE-T cell production is essentially identical to autologous CAR-T production, whereas allogeneic CAR-T cells require gene editing and tcrαβ negative purification. Unlike current CAR-T products with "dormant" non-transduced T cells, the BiTE-T cell product will be fully equipped with BiTE, whether transduced or not, and thus the final product will have 100% activity. Second, biTE-T cells have the potential to overcome host rejection without lymphocyte depletion or further engineering. Current allogeneic CAR-T cells require stringent lymphocyte depletion protocols to achieve a longer therapeutic window or more engineering, such as alloimmune defenses receptors (Mo, f.et al, nat Biotechnol 2021 39:56-63) to kill host T cells. In contrast, biTE-T cells have the potential to expand and re-target host T cells, and thus may not require lymphocyte depletion or require only slight lymphocyte depletion while providing additional killing. Third, biTE-T cells are safer because they release less cytokine when killing the target. One concern with BiTE-T cell products that have not been TCR alpha beta negative purified is that contaminated untransduced T cells may lead to GvHD. However, high doses of human BiTE-T cells with reasonable non-transduced T cell contamination did not induce GvHD in the immunodeficient NSG mouse model. The risk of BiTE-T causing GvHD is lower if used without or at lower levels of lymphocyte clearance. Another potential disadvantage of BiTE-T cells is that they are not as potent as CAR-T cells. This can be solved to a large extent by adding co-stimulators or cytokines.
In summary, biTE-T cells provide a novel and simple method for "off-the-shelf allogeneic T cell therapy, worth further evaluation in clinical trials.
Method
Study design
The aim of this study was to develop a new allogeneic "off-the-shelf" T cell therapy. CD19 and Her2 targeted BiTE engineered T cells were evaluated for CD3/TCRαβ expression. The risk of GvHD of BiTE-T cells was assessed using chemotherapy or systemically irradiated NSG mice. Side-by-side comparisons of cytotoxicity, cytokines, continuous killing, and in vivo efficacy with or without host T cells were also made for BiTE-T cells and CAR-T cells. Two xenograft mouse models were used to compare the efficacy of BiTE-T cells and CAR-T cells, including hematology and solid tumors. A variety of healthy donors are used to generate engineered T cells. All experiments were independently repeated at least twice.
Cells and culture media
NALM6GL cell lines expressing GFP and firefly luciferases were purchased from ATCC and cultured in RPMI1640 medium supplemented with 10% FBS, L-glutamine and Pen/Strep (RPMI 10). The human melanoma A375 cell line was presented by Cecilia Ramello doctor and cultured in DMEM medium supplemented with 10% FBS, L-glutamine and Pen/Strep. Her2 expressing a375 (a 375. Her2) was prepared by transducing a375 cells with a retrovirus expressing truncated Her 2. Human T cells were cultured in RPMI10 supplemented with 10ng/ml IL7 and 5ng/ml IL 15.
Retroviral constructs, gamma-retrovirus production and T cell transduction
The DNA of human Her2 CAR, human CD19 BiTE, her2 BiTE, 4-1BBL-CD80 and other constructs were synthesized by Genewiz (South Plainfield, NJ) and subcloned into SFG retroviral vectors. Human CD19 CAR has been previously described (Li, g.et al jci Insight 2018 3). Her2 CAR and Her2 BiTE used the same scFv derived from trastuzumab. CD19 CAR and CD19 BiTE used the same scFv derived from FMC63 clones. Recombinant gamma-retrovirus production has been previously described (Li, G., et al methods Mol Biol 2017 1514:111-118). Briefly, retroviral constructs containing CAR or BiTE were transiently transfected into H29 cells. The supernatant of transfected H29 was used to transduce RD114 cells to prepare stable producer cell lines. Retrovirus supernatant from RD114 cells was harvested, filtered at 0.45 μm, and cryopreserved for future use. For T cell transduction, healthy donors leukopaks were purchased from Stemcell Technologies (vancomus canadensis) and ALLCELLS (ala Mi Da, california). PBMCs were isolated using standard Ficoll methods and cryopreserved for future use. On day 0, T cells were isolated from cryopreserved PBMCs using a human T cell isolation kit (Stemcell Technologies). Human CD3/CD28 dynabeads (ThermoFisher Scientific, waltham, mass.) were then used to activate T cells. On days 1 and 2, T cells were transduced with fresh BiTE or CAR retrovirus at 2000g at 32 ℃ for 1 hour in RetroNectin (Takara Bio, mountain View, CA) -coated plates. Fresh medium containing cytokines was added on day 3. Dynabeads were removed on day 6 and T cells were further expanded in complete medium containing 10ng/ml human IL7 and 5ng/ml human IL 15. Media and cytokines were supplemented every 2-3 days. T cells on days 8-14 were used for in vivo studies.
Flow cytometry
The following antibody clones were used for flow cytometry. From BD: CD3 (clone SK 7), CD8 (clone RPA-T8), CD45 (clone HI 30), TCRαβ (clone T10B9.1A-31), CD45RA (HI 100), PD1 (clone EH12.2H7), CD69 (clone FN 50), CD25 (clone 2A 3). From Biolegend: CD4 (clone OKT 4), TCRαβ (clone IP 26), CCR7 (clone G043H 7), CD62L (clone DREG-56). Fc receptor binding inhibitors (ThermoFisher) are routinely used for staining. Whole blood samples were stained and lysed using BD FACS lysate. CountBIght absolute count beads (ThermoFisher) were added for cell number measurement. The flow data was collected on a BD LSRII or BD FACSymphony flow cytometer. Data was analyzed using FlowJo version 10.
ELISA
The Ella machine (ProteinSimple, san Jose) was used for ELISA. The 24 to 72 hour supernatants from cytotoxicity or TCR stimulation assays were added to the ELLA cassettes for cytokine measurement.
TCR/CD3 stimulation assay
For the human T cell stimulation assay, non-tissue culture treated 24-well plates were coated overnight with 1 μg/ml human tcrαβ antibody (clone IP 26). T cells were added and cultured in plates. For activation tagging, T cells were subjected to flow analysis 5 hours after stimulation. Supernatants were collected 24 hours after stimulation and cytokines were measured by ELISA. For BiTE stimulation, human CD19 BiTE (Blinatumomab) was purchased from BPS Bioscience (San Diego, CA). T cells were co-cultured with 3T3.Hcd19 or 3T3.Mcd19 cells and Blinatumomab concentration was increased. For mouse T cell stimulation, T cells were isolated from the spleen and added to a 24-well plate coated with mouse CD3 epsilon antibody (clone 145-2C 11). After 5 hours, the cells were analyzed by flow cytometry.
Cytotoxicity and continuous killing assay
xcelligent RTCA instrument (Agilent Technologies, santa Clara, CA) was used for cytotoxicity assays. T cells were co-cultured with target cells in different E: T ratios in RTCA E plates and real-time cell killing was recorded on the instrument. For continuous killing assays, T cells were co-cultured with target cells in a 5:1e:t ratio in non-tissue culture treated 24 well plates. Every two days or longer, a small fraction of T cells were analyzed for phenotype and cell count by flow cytometry while the remaining cells were transferred to a new plate containing fresh medium and target cell challenge.
Mouse study
All mouse protocols were approved by the institutional animal care and use committee of the university of south florida. NSG mice were purchased from jackson laboratories and raised in USF animal facilities. In vivo studies were performed using eight to twelve week old male NSG mice. For GvHD evaluation, NSG mice received 2.5Gy of whole body irradiation or 250mg/kg Cyclophosphamide (CTX) followed by 1000 ten thousand doses of T cells the following day. Mice were monitored for survival and any signs of GvHD (humpback posture, slow-moving, weight loss, etc.). Mice with weight loss of more than 20% and other GvHD symptoms were sacrificed. For efficacy studies in hematological tumor models, NSG mice were intravenously injected with 50 ten thousand NALM6GL cells. Three days later, mice were given 1000 ten thousand CAR-T, biTE-T or UT cells intravenously. Survival was monitored twice a week. For the solid tumor model, NSG mice were subcutaneously injected on the right with 50 ten thousand a375.her2 cells. Seven to ten days later, mice were intravenously injected with 1000 ten thousand CAR-T, biTE-T or UT cells. Blood was collected weekly and tumor tissue was collected at the endpoint. For pre-existing T cell mice, NSG mice were subjected to 1.2Gy whole body irradiation, followed by implantation of activated T cells. Mice were injected with tumor cells and treated with HLA-mismatched T cells. For detailed information, please refer to the schedule in the figures. Tumor tissue was treated on gentlMACS Octo Dissociator (Miltenyi Biotec, bergisch Gladbach, germany) and digested with Liberase and DNase I (Roche, mannheim, germany). Flow assays were performed on isolated tumor cells.
Statistical methods.
GraphPad Prism 8 was used for statistical analysis as shown in the legend. Data are expressed as mean ± standard deviation. Two sets of comparisons were made using the unpaired parameter t-test. Multiple sets of comparisons were made using one-way anova. Survival was compared using a log rank test.
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 the disclosed invention belongs. Publications cited herein and the materials to which they refer are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence listing
<110> H. Lim. Murphy cancer center and institute Co., ltd
<120> compositions and methods for reducing therapeutic T cytotoxicity
<130> 320803-2800
<150> US 63/159,222
<151> 2021-03-10
<150> US 63/209,094
<151> 2021-06-10
<150> US 63/225,715
<151> 2021-07-26
<160> 71
<170> PatentIn version 3.5
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Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
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Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
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Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met
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Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe
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Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
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Gly Ala Gly Thr Thr Val Thr Val
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
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Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
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Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
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Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
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Pro Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met
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Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala Glu Glu Phe
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Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu
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Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Val
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Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
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Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
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Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
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Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Asp
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
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Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
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Gly Ile Ile Tyr Pro Gly Asp Ser Glu Thr Thr Tyr Ser Pro Ala Phe
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Gln Gly Asp Val Thr Ile Ser Val Asp Lys Ser Ile Ser Thr Ala Tyr
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Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Gly Ile Tyr Tyr Cys
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Leu Val Thr Val Ser Ser Leu Lys Ser
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<212> PRT
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Arg Ser Thr Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
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Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val
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Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
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Pro Lys Leu Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser
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Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
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Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
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Thr Ser Ser Ser Thr Arg His Val Phe Gly Thr Gly Thr Gln Leu Thr
100 105 110
Val Leu Gly
115
<210> 9
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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
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Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
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Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
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Ala Gly Thr Thr Val Thr Val Ser Ala
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<210> 10
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<212> PRT
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Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
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Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
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Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
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Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys
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Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp
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Gly Ala Gly Thr Thr Val Thr Val Ser Ser
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Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
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Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met
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Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
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Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr
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Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Met Ala Arg Phe Ser Ser Ser Ser Leu Asp Leu Asn Trp Tyr Ser Leu
1 5 10 15
Gly Leu Gln Xaa Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
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Tyr Phe Asp Ile Asn Trp Leu Arg Gln Arg Pro Glu Gln Gly Leu Glu
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Trp Ile Gly Val Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Asn Glu
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Asn Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Thr Thr
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Ala Tyr Ile Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr
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Phe Cys Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp
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Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Lys
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Leu
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Asp Ile Val Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ile Leu Gly
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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser
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Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser
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Pro Lys Leu Leu Ile Tyr Asn Val Ser Asn Leu Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser
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Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
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Ala Asp Ala Ala Ala Ala Gly Ser
115 120
<210> 15
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<212> PRT
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Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
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Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
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Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ile Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Asp Gly Ala Val Ala Gly Leu Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120
<210> 16
<211> 107
<212> PRT
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<220>
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Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr
1 5 10 15
Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His
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Trp Tyr Gln Gln Pro Pro Gly Gln Ala Pro Val Val Val Val Tyr Asp
35 40 45
Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn
50 55 60
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp
65 70 75 80
Glu Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val
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Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 17
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<212> PRT
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<220>
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<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala
1 5 10 15
Ser Val Gln Val Ser Cys Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr
20 25 30
Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln
50 55 60
Gly Arg Val Thr Leu Thr Asn Asp Thr Ser Thr Asn Thr Val Tyr Met
65 70 75 80
Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Trp Ala Leu Trp Gly Asp Phe Gly Met Asp Val Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 18
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 19
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<212> PRT
<213> artificial sequence
<220>
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<400> 19
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Tyr
35 40 45
Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Leu Tyr Phe Cys
85 90 95
Ala Arg Gly Trp Phe Val Glu Pro Leu Ser Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 20
<211> 110
<212> PRT
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<400> 20
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30
Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95
Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 21
<211> 112
<212> PRT
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<220>
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<400> 21
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Phe Leu His Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
<210> 22
<211> 110
<212> PRT
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<400> 22
Glu Ile Glu Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile
100 105 110
<210> 23
<211> 119
<212> PRT
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<223> synthetic construct
<400> 23
Gln Val Gln Leu Val Glu Ser Gly Pro Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Ile Ser Cys Ala Val Ser Gly Phe Ser Val Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Ile Thr Asn Tyr Asn Ser Ala Phe Met
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 24
<211> 105
<212> PRT
<213> artificial sequence
<220>
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<400> 24
Glu Ile Val Met Thr Gln Thr Pro Ala Thr Leu Ser Val Ser Ala Gly
1 5 10 15
Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Asn Arg Tyr Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Tyr Gly Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly
85 90 95
Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 25
<211> 5
<212> PRT
<213> artificial sequence
<220>
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<400> 25
Gly Gly Gly Gly Ser
1 5
<210> 26
<211> 12
<212> PRT
<213> artificial sequence
<220>
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<400> 26
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
1 5 10
<210> 27
<211> 515
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Gly Ser Thr
100 105 110
Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Glu Val Gln Leu
115 120 125
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
130 135 140
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp
145 150 155 160
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr
165 170 175
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe
180 185 190
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn
195 200 205
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly
210 215 220
Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val
225 230 235 240
Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
245 250 255
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Asp Ile
260 265 270
Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg
275 280 285
Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn
290 295 300
Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr
305 310 315 320
Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly
325 330 335
Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp
340 345 350
Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe
355 360 365
Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly
370 375 380
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln
385 390 395 400
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser
405 410 415
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val
420 425 430
Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro
435 440 445
Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr
450 455 460
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser
465 470 475 480
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr
485 490 495
Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr
500 505 510
Val Thr Val
515
<210> 28
<211> 503
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 28
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
260 265 270
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val
275 280 285
Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
290 295 300
Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
305 310 315 320
Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
325 330 335
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr
340 345 350
Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
355 360 365
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Glu
370 375 380
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
385 390 395 400
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr
405 410 415
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
420 425 430
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys
435 440 445
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu
450 455 460
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
465 470 475 480
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly
485 490 495
Thr Leu Val Thr Val Ser Ser
500
<210> 29
<211> 515
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 29
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Pro Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met
35 40 45
Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu
65 70 75 80
Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Val
85 90 95
Arg Asp Arg Tyr Asp Ser Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
130 135 140
Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
145 150 155 160
Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
165 170 175
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
180 185 190
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe
210 215 220
Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
245 250 255
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Asp Ile
260 265 270
Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg
275 280 285
Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn
290 295 300
Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr
305 310 315 320
Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly
325 330 335
Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp
340 345 350
Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe
355 360 365
Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly
370 375 380
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln
385 390 395 400
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser
405 410 415
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val
420 425 430
Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro
435 440 445
Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr
450 455 460
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser
465 470 475 480
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr
485 490 495
Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr
500 505 510
Val Thr Val
515
<210> 30
<211> 515
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 30
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys
260 265 270
Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
275 280 285
Thr Glu Tyr Pro Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Phe
290 295 300
Lys Trp Met Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala
305 310 315 320
Glu Glu Phe Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr
325 330 335
Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr
340 345 350
Phe Cys Val Arg Asp Arg Tyr Asp Ser Leu Phe Asp Tyr Trp Gly Gln
355 360 365
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu
385 390 395 400
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
405 410 415
Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr
420 425 430
Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser
435 440 445
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
450 455 460
Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
465 470 475 480
Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly
485 490 495
Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His
500 505 510
Thr Cys Pro
515
<210> 31
<211> 522
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 31
Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Asp
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Ser Glu Thr Thr Tyr Ser Pro Ala Phe
50 55 60
Gln Gly Asp Val Thr Ile Ser Val Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Gly Ile Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Gly Asn Tyr Tyr Met Asp Val Trp Gly Asn Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Leu Lys Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Thr Gln Ser Ala Leu Thr
130 135 140
Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser
145 150 155 160
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp
165 170 175
Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val
180 185 190
Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser
195 200 205
Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu
210 215 220
Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Arg His Val
225 230 235 240
Phe Gly Thr Gly Thr Gln Leu Thr Val Leu Gly Glu Pro Lys Ser Cys
245 250 255
Asp Lys Thr His Thr Cys Pro Glu Pro Lys Ser Cys Asp Lys Thr His
260 265 270
Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
275 280 285
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
290 295 300
Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
305 310 315 320
Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
325 330 335
Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
340 345 350
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
355 360 365
Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys
370 375 380
Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
385 390 395 400
Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
405 410 415
Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
420 425 430
Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu
435 440 445
Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln
450 455 460
Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
465 470 475 480
Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
485 490 495
Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp
500 505 510
Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520
<210> 32
<211> 522
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 32
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys
260 265 270
Pro Gly Asp Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe
275 280 285
Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu
290 295 300
Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Glu Thr Thr Tyr Ser
305 310 315 320
Pro Ala Phe Gln Gly Asp Val Thr Ile Ser Val Asp Lys Ser Ile Ser
325 330 335
Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Gly Ile
340 345 350
Tyr Tyr Cys Ala Arg Arg Arg Gly Asn Tyr Tyr Met Asp Val Trp Gly
355 360 365
Asn Gly Thr Leu Val Thr Val Ser Ser Leu Lys Ser Gly Gly Gly Gly
370 375 380
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Thr Gln Ser
385 390 395 400
Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile
405 410 415
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr
420 425 430
Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile
435 440 445
Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly
450 455 460
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala
465 470 475 480
Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr
485 490 495
Arg His Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu Gly Glu Pro
500 505 510
Lys Ser Cys Asp Lys Thr His Thr Cys Pro
515 520
<210> 33
<211> 501
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 33
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln Ser Pro
130 135 140
Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg
145 150 155 160
Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly
165 170 175
Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly
180 185 190
Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
195 200 205
Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala
245 250 255
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
260 265 270
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr
275 280 285
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
290 295 300
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly
305 310 315 320
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
325 330 335
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
340 345 350
Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly
355 360 365
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln
370 375 380
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys
385 390 395 400
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn
405 410 415
Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile
420 425 430
Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys
435 440 445
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu
450 455 460
Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser
465 470 475 480
Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly
485 490 495
Thr Thr Val Thr Val
500
<210> 34
<211> 501
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 34
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys
260 265 270
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
275 280 285
Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu
290 295 300
Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn
305 310 315 320
Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
325 330 335
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
340 345 350
Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn
355 360 365
Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly
370 375 380
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser
385 390 395 400
Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met
405 410 415
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln
420 425 430
Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu
435 440 445
Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser
450 455 460
Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr
465 470 475 480
Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr
485 490 495
Lys Leu Glu Ile Lys
500
<210> 35
<211> 502
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 35
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys
85 90 95
Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp
100 105 110
Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser
130 135 140
Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
145 150 155 160
Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln Lys Lys Pro
165 170 175
Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser
180 185 190
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
195 200 205
Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
210 215 220
Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu
225 230 235 240
Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met
245 250 255
Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu
260 265 270
Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn
275 280 285
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu
290 295 300
Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser
305 310 315 320
Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
325 330 335
Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
340 345 350
Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly
355 360 365
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val
370 375 380
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met
385 390 395 400
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
405 410 415
Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu
420 425 430
Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp
435 440 445
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
450 455 460
Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
465 470 475 480
Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala
485 490 495
Gly Thr Thr Val Thr Val
500
<210> 36
<211> 502
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 36
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
260 265 270
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
275 280 285
Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu
290 295 300
Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn
305 310 315 320
Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
325 330 335
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp
340 345 350
Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe
355 360 365
Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu
385 390 395 400
Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr
405 410 415
Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln
420 425 430
Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn
435 440 445
Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
450 455 460
Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr
465 470 475 480
Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly
485 490 495
Thr Lys Leu Glu Ile Lys
500
<210> 37
<211> 518
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa can be any naturally occurring amino acid
<400> 37
Met Ala Arg Phe Ser Ser Ser Ser Leu Asp Leu Asn Trp Tyr Ser Leu
1 5 10 15
Gly Leu Gln Xaa Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Tyr Phe Asp Ile Asn Trp Leu Arg Gln Arg Pro Glu Gln Gly Leu Glu
35 40 45
Trp Ile Gly Val Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Asn Glu
50 55 60
Asn Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Thr Thr
65 70 75 80
Ala Tyr Ile Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr
85 90 95
Phe Cys Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Lys
115 120 125
Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Asp Ile Val Met Thr
130 135 140
Gln Ile Pro Leu Ser Leu Pro Val Ile Leu Gly Asp Gln Ala Ser Ile
145 150 155 160
Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser Asn Gly Asn Thr Tyr
165 170 175
Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
180 185 190
Tyr Asn Val Ser Asn Leu Phe Ser Gly Val Pro Asp Arg Phe Ser Gly
195 200 205
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
210 215 220
Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr His Val Pro Thr
225 230 235 240
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Ala
245 250 255
Ala Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met
260 265 270
Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu
275 280 285
Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn
290 295 300
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu
305 310 315 320
Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser
325 330 335
Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
340 345 350
Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
355 360 365
Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly
370 375 380
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val
385 390 395 400
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met
405 410 415
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
420 425 430
Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu
435 440 445
Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp
450 455 460
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
465 470 475 480
Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
485 490 495
Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala
500 505 510
Gly Thr Thr Val Thr Val
515
<210> 38
<211> 518
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> misc_feature
<222> (279)..(279)
<223> Xaa can be any naturally occurring amino acid
<400> 38
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Met Ala Arg Phe Ser Ser Ser Ser Leu Asp Leu Asn Trp
260 265 270
Tyr Ser Leu Gly Leu Gln Xaa Lys Leu Ser Cys Lys Ala Ser Gly Tyr
275 280 285
Thr Phe Thr Tyr Phe Asp Ile Asn Trp Leu Arg Gln Arg Pro Glu Gln
290 295 300
Gly Leu Glu Trp Ile Gly Val Ile Ser Pro Gly Asp Gly Asn Thr Asn
305 310 315 320
Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser
325 330 335
Ser Thr Thr Ala Tyr Ile Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser
340 345 350
Ala Val Tyr Phe Cys Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met
355 360 365
Asp Ser Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr
370 375 380
Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Asp Ile
385 390 395 400
Val Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ile Leu Gly Asp Gln
405 410 415
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser Asn Gly
420 425 430
Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys
435 440 445
Leu Leu Ile Tyr Asn Val Ser Asn Leu Phe Ser Gly Val Pro Asp Arg
450 455 460
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg
465 470 475 480
Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr His
485 490 495
Val Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp
500 505 510
Ala Ala Ala Ala Gly Ser
515
<210> 39
<211> 504
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 39
Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
20 25 30
Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ile Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg His Asp Gly Ala Val Ala Gly Leu Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro
130 135 140
Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly
145 150 155 160
Gly Asn Asn Ile Gly Ser Lys Ser Val His Trp Tyr Gln Gln Pro Pro
165 170 175
Gly Gln Ala Pro Val Val Val Val Tyr Asp Asp Ser Asp Arg Pro Ser
180 185 190
Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr
195 200 205
Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Val Tyr Tyr Cys
210 215 220
Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly Gly Gly Thr
225 230 235 240
Lys Leu Thr Val Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
245 250 255
Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
260 265 270
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile
275 280 285
Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys
290 295 300
Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys
305 310 315 320
Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn
325 330 335
Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr
340 345 350
Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
355 360 365
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
370 375 380
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
385 390 395 400
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
405 410 415
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met
420 425 430
Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe
435 440 445
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
450 455 460
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
465 470 475 480
Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
485 490 495
Gly Ala Gly Thr Thr Val Thr Val
500
<210> 40
<211> 504
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 40
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
260 265 270
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
275 280 285
Ser Ser Gly Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys
290 295 300
Gly Leu Glu Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ile Thr Tyr Tyr
305 310 315 320
Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
325 330 335
Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
340 345 350
Val Tyr Tyr Cys Ala Arg His Asp Gly Ala Val Ala Gly Leu Phe Asp
355 360 365
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu
385 390 395 400
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile
405 410 415
Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His Trp Tyr Gln
420 425 430
Gln Pro Pro Gly Gln Ala Pro Val Val Val Val Tyr Asp Asp Ser Asp
435 440 445
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn
450 455 460
Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Val
465 470 475 480
Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val Val Phe Gly
485 490 495
Gly Gly Thr Lys Leu Thr Val Leu
500
<210> 41
<211> 500
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 41
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala
1 5 10 15
Ser Val Gln Val Ser Cys Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr
20 25 30
Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln
50 55 60
Gly Arg Val Thr Leu Thr Asn Asp Thr Ser Thr Asn Thr Val Tyr Met
65 70 75 80
Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Trp Ala Leu Trp Gly Asp Phe Gly Met Asp Val Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
130 135 140
Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
145 150 155 160
Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
165 170 175
Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
210 215 220
Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
225 230 235 240
Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Asp
245 250 255
Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp
260 265 270
Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu
275 280 285
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr
290 295 300
Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser
305 310 315 320
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu
325 330 335
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr
340 345 350
Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser
355 360 365
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu
370 375 380
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile
385 390 395 400
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp
405 410 415
Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn
420 425 430
Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala
435 440 445
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu
450 455 460
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly
465 470 475 480
Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
485 490 495
Thr Val Thr Val
500
<210> 42
<211> 500
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 42
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg
260 265 270
Pro Gly Ala Ser Val Gln Val Ser Cys Arg Ala Ser Gly Tyr Ser Ile
275 280 285
Asn Thr Tyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu
290 295 300
Glu Trp Met Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln
305 310 315 320
Lys Phe Gln Gly Arg Val Thr Leu Thr Asn Asp Thr Ser Thr Asn Thr
325 330 335
Val Tyr Met Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr
340 345 350
Tyr Cys Ala Arg Trp Ala Leu Trp Gly Asp Phe Gly Met Asp Val Trp
355 360 365
Gly Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
385 390 395 400
Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys
405 410 415
Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys
420 425 430
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala
435 440 445
Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
450 455 460
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr
465 470 475 480
Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys
485 490 495
Leu Glu Ile Lys
500
<210> 43
<211> 501
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 43
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Tyr
35 40 45
Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Leu Tyr Phe Cys
85 90 95
Ala Arg Gly Trp Phe Val Glu Pro Leu Ser Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly
130 135 140
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
145 150 155 160
Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
165 170 175
Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro
180 185 190
Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
195 200 205
Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
210 215 220
Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr
225 230 235 240
Val Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala
245 250 255
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
260 265 270
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr
275 280 285
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
290 295 300
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly
305 310 315 320
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
325 330 335
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
340 345 350
Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly
355 360 365
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln
370 375 380
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys
385 390 395 400
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn
405 410 415
Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile
420 425 430
Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys
435 440 445
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu
450 455 460
Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser
465 470 475 480
Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly
485 490 495
Thr Thr Val Thr Val
500
<210> 44
<211> 501
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 44
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln
260 265 270
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
275 280 285
Ser Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
290 295 300
Glu Trp Tyr Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala
305 310 315 320
Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn
325 330 335
Thr Leu Tyr Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Leu
340 345 350
Tyr Phe Cys Ala Arg Gly Trp Phe Val Glu Pro Leu Ser Trp Gly Gln
355 360 365
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser
385 390 395 400
Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser
405 410 415
Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro
420 425 430
Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser
435 440 445
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
450 455 460
Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
465 470 475 480
Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly Thr Gly Thr
485 490 495
Lys Leu Thr Val Leu
500
<210> 45
<211> 496
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Phe Leu His Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
115 120 125
Ile Glu Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu
130 135 140
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn
145 150 155 160
Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro
165 170 175
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp
180 185 190
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
195 200 205
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu
210 215 220
Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Asp Ile Gln Met Thr
245 250 255
Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile
260 265 270
Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln
275 280 285
Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg
290 295 300
Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr
305 310 315 320
Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
325 330 335
Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly
340 345 350
Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser
355 360 365
Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly
370 375 380
Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala
385 390 395 400
Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser
405 410 415
His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly
420 425 430
Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val
435 440 445
Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser
450 455 460
Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp
465 470 475 480
Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
485 490 495
<210> 46
<211> 496
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 46
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
260 265 270
Pro Gly Ser Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
275 280 285
Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
290 295 300
Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala
305 310 315 320
Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser
325 330 335
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
340 345 350
Tyr Tyr Cys Ala Arg Asp Pro Phe Leu His Tyr Trp Gly Gln Gly Thr
355 360 365
Leu Val Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
370 375 380
Gly Ser Glu Ile Glu Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
385 390 395 400
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu
405 410 415
His Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
420 425 430
Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly
435 440 445
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
450 455 460
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
465 470 475 480
Gln Ala Leu Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile
485 490 495
<210> 47
<211> 498
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 47
Gln Val Gln Leu Val Glu Ser Gly Pro Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Ile Ser Cys Ala Val Ser Gly Phe Ser Val Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Ile Thr Asn Tyr Asn Ser Ala Phe Met
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Thr Pro Ala Thr
130 135 140
Leu Ser Val Ser Ala Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser
145 150 155 160
Gln Ser Val Ser Asn Asp Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln
165 170 175
Ala Pro Arg Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Ser Gly Val
180 185 190
Pro Ala Arg Phe Ser Gly Ser Gly Tyr Gly Thr Glu Phe Thr Phe Thr
195 200 205
Ile Ser Ser Val Gln Ser Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
210 215 220
Asp Tyr Ser Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Glu
225 230 235 240
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Asp Ile Gln
245 250 255
Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val
260 265 270
Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp
275 280 285
Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr
290 295 300
Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser
305 310 315 320
Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile
325 330 335
Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala
340 345 350
Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly
355 360 365
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln
370 375 380
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys
385 390 395 400
Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys
405 410 415
Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr
420 425 430
Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu
435 440 445
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu
450 455 460
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr
465 470 475 480
Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val
485 490 495
Thr Val
<210> 48
<211> 498
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 48
Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser
1 5 10 15
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
35 40 45
Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu
85 90 95
Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly
100 105 110
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr
145 150 155 160
Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly
165 170 175
Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys
180 185 190
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly
225 230 235 240
Ala Gly Thr Thr Val Thr Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Gln Val Gln Leu Val Glu Ser Gly Pro Gly Val Val Gln
260 265 270
Pro Gly Arg Ser Leu Arg Ile Ser Cys Ala Val Ser Gly Phe Ser Val
275 280 285
Thr Asn Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
290 295 300
Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ile Thr Asn Tyr Asn Ser
305 310 315 320
Ala Phe Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr
325 330 335
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr
340 345 350
Tyr Cys Ala Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu Asp Tyr Trp
355 360 365
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Thr
385 390 395 400
Pro Ala Thr Leu Ser Val Ser Ala Gly Glu Arg Val Thr Ile Thr Cys
405 410 415
Lys Ala Ser Gln Ser Val Ser Asn Asp Val Thr Trp Tyr Gln Gln Lys
420 425 430
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr
435 440 445
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Tyr Gly Thr Glu Phe
450 455 460
Thr Phe Thr Ile Ser Ser Val Gln Ser Glu Asp Phe Ala Val Tyr Phe
465 470 475 480
Cys Gln Gln Asp Tyr Ser Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile
485 490 495
Lys Arg
<210> 49
<211> 22
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 49
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly
20
<210> 50
<211> 525
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 50
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly 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 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 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
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Arg Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu
130 135 140
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
145 150 155 160
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys
165 170 175
Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
180 185 190
Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
195 200 205
Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
210 215 220
Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
225 230 235 240
Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met 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 Met Ala Asp Ile Gln Met Thr Gln Thr Thr
275 280 285
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
290 295 300
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
305 310 315 320
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
325 330 335
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
340 345 350
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
355 360 365
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
370 375 380
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
385 390 395 400
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
405 410 415
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
420 425 430
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
435 440 445
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
450 455 460
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
465 470 475 480
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
485 490 495
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
500 505 510
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520 525
<210> 51
<211> 525
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 51
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Asp Ile Gln Met Thr Gln Ser
275 280 285
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
290 295 300
Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys
305 310 315 320
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr
325 330 335
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe
340 345 350
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
355 360 365
Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys
370 375 380
Val Glu Ile Lys Arg Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly
385 390 395 400
Ser Gly Glu Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
405 410 415
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
420 425 430
Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
435 440 445
Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg
450 455 460
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
465 470 475 480
Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
485 490 495
Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
500 505 510
Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
515 520 525
<210> 52
<211> 537
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 52
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Ile Gln Leu Val Gln Ser
275 280 285
Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys
290 295 300
Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Pro Ile His Trp Val Lys Gln
305 310 315 320
Ala Pro Gly Lys Gly Phe Lys Trp Met Gly Met Ile Tyr Thr Asp Ile
325 330 335
Gly Lys Pro Thr Tyr Ala Glu Glu Phe Gly Arg Phe Ala Phe Ser Leu
340 345 350
Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn
355 360 365
Glu Asp Thr Ala Thr Tyr Phe Cys Val Arg Asp Arg Tyr Asp Ser Leu
370 375 380
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly
385 390 395 400
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
405 410 415
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
420 425 430
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn
435 440 445
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu
450 455 460
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
465 470 475 480
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val
485 490 495
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val
500 505 510
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys
515 520 525
Ser Cys Asp Lys Thr His Thr Cys Pro
530 535
<210> 53
<211> 525
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 53
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu
20 25 30
Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
35 40 45
Tyr Thr Phe Thr Glu Tyr Pro Ile His Trp Val Lys Gln Ala Pro Gly
50 55 60
Lys Gly Phe Lys Trp Met Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro
65 70 75 80
Thr Tyr Ala Glu Glu Phe Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser
85 90 95
Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr
100 105 110
Ala Thr Tyr Phe Cys Val Arg Asp Arg Tyr Asp Ser Leu Phe Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln
145 150 155 160
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser
165 170 175
Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu
180 185 190
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
195 200 205
Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
225 230 235 240
Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr
245 250 255
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp
260 265 270
Lys Thr His Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr
275 280 285
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
290 295 300
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
305 310 315 320
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
325 330 335
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
340 345 350
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
355 360 365
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
370 375 380
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
385 390 395 400
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
405 410 415
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
420 425 430
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
435 440 445
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
450 455 460
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
465 470 475 480
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
485 490 495
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
500 505 510
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520 525
<210> 54
<211> 532
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 54
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Val Gln Leu Val Glu Ser Gly Ala Glu
20 25 30
Val Lys Lys Pro Gly Asp Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly
35 40 45
Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly
50 55 60
Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Glu Thr
65 70 75 80
Thr Tyr Ser Pro Ala Phe Gln Gly Asp Val Thr Ile Ser Val Asp Lys
85 90 95
Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp
100 105 110
Thr Gly Ile Tyr Tyr Cys Ala Arg Arg Arg Gly Asn Tyr Tyr Met Asp
115 120 125
Val Trp Gly Asn Gly Thr Leu Val Thr Val Ser Ser Leu Lys Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser
145 150 155 160
Thr Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly
165 170 175
Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
180 185 190
Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys
195 200 205
Leu Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg
210 215 220
Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
225 230 235 240
Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser
245 250 255
Ser Ser Thr Arg His Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu
260 265 270
Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Asp
275 280 285
Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp
290 295 300
Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu
305 310 315 320
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr
325 330 335
Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser
340 345 350
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu
355 360 365
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr
370 375 380
Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser
385 390 395 400
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu
405 410 415
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile
420 425 430
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp
435 440 445
Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn
450 455 460
Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala
465 470 475 480
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu
485 490 495
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly
500 505 510
Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
515 520 525
Thr Val Thr Val
530
<210> 55
<211> 537
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 55
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Ile Gln Leu Val Gln Ser
275 280 285
Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys
290 295 300
Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Pro Ile His Trp Val Lys Gln
305 310 315 320
Ala Pro Gly Lys Gly Phe Lys Trp Met Gly Met Ile Tyr Thr Asp Ile
325 330 335
Gly Lys Pro Thr Tyr Ala Glu Glu Phe Gly Arg Phe Ala Phe Ser Leu
340 345 350
Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn
355 360 365
Glu Asp Thr Ala Thr Tyr Phe Cys Val Arg Asp Arg Tyr Asp Ser Leu
370 375 380
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly
385 390 395 400
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
405 410 415
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
420 425 430
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn
435 440 445
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu
450 455 460
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
465 470 475 480
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val
485 490 495
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val
500 505 510
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys
515 520 525
Ser Cys Asp Lys Thr His Thr Cys Pro
530 535
<210> 56
<211> 523
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 56
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
20 25 30
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45
Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly
50 55 60
Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr
65 70 75 80
Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys
85 90 95
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
100 105 110
Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp
115 120 125
Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile
145 150 155 160
Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys
165 170 175
Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp
180 185 190
Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr
195 200 205
Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser
210 215 220
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala
225 230 235 240
Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly
245 250 255
Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr
260 265 270
His Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
275 280 285
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
290 295 300
Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
305 310 315 320
Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val
325 330 335
Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
340 345 350
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
355 360 365
Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile
370 375 380
Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
385 390 395 400
Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
405 410 415
Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
420 425 430
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu
435 440 445
Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn
450 455 460
Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
465 470 475 480
Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
485 490 495
Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe
500 505 510
Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520
<210> 57
<211> 523
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 57
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Val Gln Leu Gln Gln Pro
275 280 285
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
290 295 300
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln
305 310 315 320
Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn
325 330 335
Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
340 345 350
Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
355 360 365
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly
370 375 380
Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val
385 390 395 400
Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
405 410 415
Ser Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro
420 425 430
Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
435 440 445
Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile
450 455 460
Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly
465 470 475 480
Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala
485 490 495
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro
500 505 510
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
515 520
<210> 58
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 58
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
20 25 30
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45
Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly
50 55 60
Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr
65 70 75 80
Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys
85 90 95
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
100 105 110
Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr
115 120 125
Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
145 150 155 160
Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu
165 170 175
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp
180 185 190
Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala
195 200 205
Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
210 215 220
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp
225 230 235 240
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe
245 250 255
Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys
260 265 270
Thr His Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser
275 280 285
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
290 295 300
Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
305 310 315 320
Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly
325 330 335
Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
340 345 350
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
355 360 365
Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu
370 375 380
Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
385 390 395 400
Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
405 410 415
Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
420 425 430
Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn
435 440 445
Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr
450 455 460
Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
465 470 475 480
Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala
485 490 495
Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr
500 505 510
Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520
<210> 59
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 59
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Glu Val Gln Leu Gln Gln Ser
275 280 285
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
290 295 300
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln
305 310 315 320
Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn
325 330 335
Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
340 345 350
Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
355 360 365
Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly
370 375 380
Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr
385 390 395 400
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
405 410 415
Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser
420 425 430
Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn
435 440 445
Tyr Met Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp
450 455 460
Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
465 470 475 480
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu
485 490 495
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro
500 505 510
Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
515 520
<210> 60
<211> 540
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> misc_feature
<222> (42)..(42)
<223> Xaa can be any naturally occurring amino acid
<400> 60
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Arg Phe Ser Ser Ser Ser Leu Asp
20 25 30
Leu Asn Trp Tyr Ser Leu Gly Leu Gln Xaa Lys Leu Ser Cys Lys Ala
35 40 45
Ser Gly Tyr Thr Phe Thr Tyr Phe Asp Ile Asn Trp Leu Arg Gln Arg
50 55 60
Pro Glu Gln Gly Leu Glu Trp Ile Gly Val Ile Ser Pro Gly Asp Gly
65 70 75 80
Asn Thr Asn Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ile
85 90 95
Asp Lys Ser Ser Thr Thr Ala Tyr Ile Gln Leu Ser Arg Leu Thr Ser
100 105 110
Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Asp Gly Asn Phe Pro Tyr
115 120 125
Tyr Ala Met Asp Ser Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140
Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg
145 150 155 160
Val Asp Ile Val Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ile Leu
165 170 175
Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr
180 185 190
Ser Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln
195 200 205
Ser Pro Lys Leu Leu Ile Tyr Asn Val Ser Asn Leu Phe Ser Gly Val
210 215 220
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
225 230 235 240
Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln
245 250 255
Ser Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270
Arg Ala Asp Ala Ala Ala Ala Gly Ser Glu Pro Lys Ser Cys Asp Lys
275 280 285
Thr His Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser
290 295 300
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
305 310 315 320
Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
325 330 335
Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly
340 345 350
Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
355 360 365
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
370 375 380
Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu
385 390 395 400
Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
405 410 415
Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
420 425 430
Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
435 440 445
Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn
450 455 460
Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr
465 470 475 480
Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
485 490 495
Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala
500 505 510
Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr
515 520 525
Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
530 535 540
<210> 61
<211> 540
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> misc_feature
<222> (301)..(301)
<223> Xaa can be any naturally occurring amino acid
<400> 61
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Met Ala Arg Phe Ser Ser Ser
275 280 285
Ser Leu Asp Leu Asn Trp Tyr Ser Leu Gly Leu Gln Xaa Lys Leu Ser
290 295 300
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe Asp Ile Asn Trp Leu
305 310 315 320
Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Val Ile Ser Pro
325 330 335
Gly Asp Gly Asn Thr Asn Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr
340 345 350
Leu Thr Ile Asp Lys Ser Ser Thr Thr Ala Tyr Ile Gln Leu Ser Arg
355 360 365
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Asp Gly Asn
370 375 380
Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln Gly Thr Ser Val Thr
385 390 395 400
Val Ser Ser Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser
405 410 415
Glu Ala Arg Val Asp Ile Val Met Thr Gln Ile Pro Leu Ser Leu Pro
420 425 430
Val Ile Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
435 440 445
Leu Val Tyr Ser Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys
450 455 460
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Asn Val Ser Asn Leu Phe
465 470 475 480
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
485 490 495
Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe
500 505 510
Cys Ser Gln Ser Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Leu
515 520 525
Glu Ile Lys Arg Ala Asp Ala Ala Ala Ala Gly Ser
530 535 540
<210> 62
<211> 526
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 62
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly
20 25 30
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
35 40 45
Gly Ser Ile Ser Ser Gly Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ile
65 70 75 80
Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp
85 90 95
Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Asp Gly Ala Val Ala Gly
115 120 125
Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
145 150 155 160
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr
165 170 175
Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His
180 185 190
Trp Tyr Gln Gln Pro Pro Gly Gln Ala Pro Val Val Val Val Tyr Asp
195 200 205
Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn
210 215 220
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp
225 230 235 240
Glu Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Val
245 250 255
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Glu Pro Lys Ser Cys
260 265 270
Asp Lys Thr His Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr
275 280 285
Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys
290 295 300
Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys
305 310 315 320
Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
325 330 335
Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr
340 345 350
Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe
355 360 365
Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys
370 375 380
Leu Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
385 390 395 400
Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
405 410 415
Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly
420 425 430
Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly
435 440 445
Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser
450 455 460
Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys
465 470 475 480
Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp
485 490 495
Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp
500 505 510
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520 525
<210> 63
<211> 526
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 63
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Leu Gln Leu Gln Glu Ser
275 280 285
Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr
290 295 300
Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Phe Trp Gly Trp Ile
305 310 315 320
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr Tyr
325 330 335
Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile
340 345 350
Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val
355 360 365
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Asp Gly Ala
370 375 380
Val Ala Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
385 390 395 400
Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
405 410 415
Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro
420 425 430
Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys
435 440 445
Ser Val His Trp Tyr Gln Gln Pro Pro Gly Gln Ala Pro Val Val Val
450 455 460
Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
465 470 475 480
Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu
485 490 495
Ala Gly Asp Glu Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser
500 505 510
Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
515 520 525
<210> 64
<211> 522
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 64
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu
20 25 30
Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser Cys Arg Ala Ser Gly
35 40 45
Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly
50 55 60
Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro Ser Gly Val Thr Ser
65 70 75 80
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr Asn Asp Thr Ser
85 90 95
Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp Gly Asp Phe Gly Met
115 120 125
Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
145 150 155 160
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr
165 170 175
Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr
180 185 190
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser
195 200 205
Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
210 215 220
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala
225 230 235 240
Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly
245 250 255
Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Cys Asp Lys Thr His
260 265 270
Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
275 280 285
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
290 295 300
Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
305 310 315 320
Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
325 330 335
Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
340 345 350
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
355 360 365
Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys
370 375 380
Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
385 390 395 400
Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
405 410 415
Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
420 425 430
Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu
435 440 445
Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln
450 455 460
Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
465 470 475 480
Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
485 490 495
Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp
500 505 510
Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520
<210> 65
<211> 522
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 65
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Val Gln Leu Val Gln Ser
275 280 285
Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser Cys Arg
290 295 300
Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg Gln
305 310 315 320
Ala Pro Gly Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro Ser Gly
325 330 335
Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr Asn
340 345 350
Asp Thr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr Ser
355 360 365
Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp Gly Asp
370 375 380
Phe Gly Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser
385 390 395 400
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
405 410 415
Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp
420 425 430
Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp Leu
435 440 445
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
450 455 460
Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser
465 470 475 480
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
485 490 495
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr
500 505 510
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
515 520
<210> 66
<211> 523
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 66
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly
20 25 30
Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45
Phe Thr Phe Ser Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Tyr Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys
65 70 75 80
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn
85 90 95
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Ser Pro Glu Asp
100 105 110
Thr Ala Leu Tyr Phe Cys Ala Arg Gly Trp Phe Val Glu Pro Leu Ser
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln
145 150 155 160
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys
165 170 175
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln
180 185 190
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln
195 200 205
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr
210 215 220
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp
225 230 235 240
Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly
245 250 255
Thr Gly Thr Lys Leu Thr Val Leu Glu Pro Lys Ser Cys Asp Lys Thr
260 265 270
His Thr Cys Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
275 280 285
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
290 295 300
Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
305 310 315 320
Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val
325 330 335
Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
340 345 350
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
355 360 365
Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile
370 375 380
Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
385 390 395 400
Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
405 410 415
Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
420 425 430
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu
435 440 445
Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn
450 455 460
Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
465 470 475 480
Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
485 490 495
Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe
500 505 510
Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
515 520
<210> 67
<211> 523
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 67
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Val Gln Leu Val Gln Ser
275 280 285
Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala
290 295 300
Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Leu His Trp Val Arg Gln
305 310 315 320
Ala Pro Gly Lys Gly Leu Glu Trp Tyr Ala Ala Ile Ser Tyr Asp Gly
325 330 335
Ser Lys Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Leu Thr Ile Ser
340 345 350
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Ser
355 360 365
Pro Glu Asp Thr Ala Leu Tyr Phe Cys Ala Arg Gly Trp Phe Val Glu
370 375 380
Pro Leu Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
385 390 395 400
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val
405 410 415
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr
420 425 430
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn
435 440 445
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser
450 455 460
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys
465 470 475 480
Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp
485 490 495
Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr
500 505 510
Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu
515 520
<210> 68
<211> 518
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 68
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu
20 25 30
Val Lys Lys Pro Gly Ser Ser Val Arg Val Ser Cys Lys Ala Ser Gly
35 40 45
Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala
65 70 75 80
Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
85 90 95
Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Phe Leu His Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Ile Glu Leu Thr Gln Ser Pro Leu Ser Leu
145 150 155 160
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
165 170 175
Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln
180 185 190
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg
195 200 205
Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
225 230 235 240
Tyr Cys Met Gln Ala Leu Gln Thr Phe Thr Phe Gly Pro Gly Thr Lys
245 250 255
Val Glu Ile Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Met
260 265 270
Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu
275 280 285
Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn
290 295 300
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu
305 310 315 320
Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser
325 330 335
Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
340 345 350
Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
355 360 365
Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Gly Gly
370 375 380
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val
385 390 395 400
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met
405 410 415
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
420 425 430
Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu
435 440 445
Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp
450 455 460
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
465 470 475 480
Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
485 490 495
Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala
500 505 510
Gly Thr Thr Val Thr Val
515
<210> 69
<211> 518
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 69
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Val Gln Leu Val Gln Ser
275 280 285
Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Arg Val Ser Cys Lys
290 295 300
Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln
305 310 315 320
Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe
325 330 335
Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr
340 345 350
Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg
355 360 365
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Phe Leu His
370 375 380
Tyr Trp Gly Gln Gly Thr Leu Val Thr Gly Gly Gly Gly Ser Gly Gly
385 390 395 400
Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Glu Leu Thr Gln Ser Pro
405 410 415
Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg
420 425 430
Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp Trp
435 440 445
Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly
450 455 460
Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
465 470 475 480
Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val
485 490 495
Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Thr Phe Thr Phe Gly Pro
500 505 510
Gly Thr Lys Val Glu Ile
515
<210> 70
<211> 520
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 70
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Val Gln Leu Val Glu Ser Gly Pro Gly
20 25 30
Val Val Gln Pro Gly Arg Ser Leu Arg Ile Ser Cys Ala Val Ser Gly
35 40 45
Phe Ser Val Thr Asn Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly
50 55 60
Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ile Thr Asn
65 70 75 80
Tyr Asn Ser Ala Phe Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu
115 120 125
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met
145 150 155 160
Thr Gln Thr Pro Ala Thr Leu Ser Val Ser Ala Gly Glu Arg Val Thr
165 170 175
Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Thr Trp Tyr
180 185 190
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ser Ala Ser
195 200 205
Asn Arg Tyr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Tyr Gly
210 215 220
Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ser Glu Asp Phe Ala
225 230 235 240
Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly Gln Gly Thr Lys
245 250 255
Leu Glu Ile Lys Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
260 265 270
Pro Met Ala Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
275 280 285
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile
290 295 300
Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys
305 310 315 320
Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys
325 330 335
Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn
340 345 350
Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr
355 360 365
Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
370 375 380
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
385 390 395 400
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
405 410 415
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
420 425 430
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met
435 440 445
Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe
450 455 460
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
465 470 475 480
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
485 490 495
Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
500 505 510
Gly Ala Gly Thr Thr Val Thr Val
515 520
<210> 71
<211> 520
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 71
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Met Ala Asp Ile Gln Met Thr Gln Thr Thr
20 25 30
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
35 40 45
Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
50 55 60
Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser
65 70 75 80
Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
85 90 95
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
100 105 110
Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
180 185 190
Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr
195 200 205
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
245 250 255
Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Glu Pro Lys
260 265 270
Ser Cys Asp Lys Thr His Thr Cys Pro Gln Val Gln Leu Val Glu Ser
275 280 285
Gly Pro Gly Val Val Gln Pro Gly Arg Ser Leu Arg Ile Ser Cys Ala
290 295 300
Val Ser Gly Phe Ser Val Thr Asn Tyr Gly Val His Trp Val Arg Gln
305 310 315 320
Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly Gly
325 330 335
Ile Thr Asn Tyr Asn Ser Ala Phe Met Ser Arg Leu Thr Ile Ser Lys
340 345 350
Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala
355 360 365
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ser Arg Gly Gly His Tyr Gly
370 375 380
Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
385 390 395 400
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
405 410 415
Ile Val Met Thr Gln Thr Pro Ala Thr Leu Ser Val Ser Ala Gly Glu
420 425 430
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val
435 440 445
Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
450 455 460
Ser Ala Ser Asn Arg Tyr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
465 470 475 480
Gly Tyr Gly Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ser Glu
485 490 495
Asp Phe Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly Gln
500 505 510
Gly Thr Lys Leu Glu Ile Lys Arg
515 520

Claims (10)

1. A method for treating cancer in a subject, comprising
Obtaining immune effector cells from an allogeneic donor;
engineering the immune effector cell to express an anti-CD 3 multispecific antibody, wherein the antibody is configured to bind to the CD3 complex on the immune effector cell and a second antigen on another cell in a manner sufficient to activate the CD3 complex; and
Administering engineered immune effector cells to the subject in an amount effective to treat the cancer.
2. The method of claim 1, wherein the antibody is a bispecific antibody.
3. The method of claim 1, wherein the second antigen is a tumor antigen.
4. The method of claim 1, wherein the second antigen is selected from EpCAM, CCR5, CD19, HER-2neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5Ac, MUC5B, MUC7, dhCG, lewis-Y, CD, CD33, CD30, ganglioside GD3, 9-O-acetyl-GD 3, GM2, globo H, fucosyl GM1, poly SA, GD2, carbonic anhydrase IX (MN/CA IX), CD44v6, sonic Hedgehog (Shh), wue-1, plasma cell antigen, (membrane bound) IgE, melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF- α precursor, STEAP, mesothelin, a33 antigen, prostate Stem Cell Antigen (PSCA), ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substances (MIS) type II receptor, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS and CD63.
5. The method of any one of claims 1-4, wherein the immune effector cell is further engineered to express a Chimeric Antigen Receptor (CAR).
6. The method of any one of claims 1 to 5, wherein the immune effector cell is selected from the group consisting of: alpha beta T cells, gamma delta T cells, natural Killer (NK) cells, natural Killer T (NKT) cells, B cells, congenital lymphoid cells (ILC), cytokine Induced Killer (CIK) cells, cytotoxic T Lymphocytes (CTL), lymphokine Activated Killer (LAK) cells, regulatory T cells, or any combination thereof.
7. A method of enhancing CAR-T cells for allogeneic cell transfer, comprising engineering the CAR-T cells to secrete monospecific anti-CD 3 antibodies.
8. The method of claim 7, wherein the monospecific anti-CD 3 antibody is a single chain variable fragment (scFv).
car-T cells engineered to express a monospecific anti-CD 3 antibody.
10. A method of enhancing immune effector cells for allogeneic cell transfer comprising
Engineering the immune effector cell to express a membrane-bound anti-CD 3 antibody,
wherein the anti-CD 3 antibody is configured to bind to a CD3 complex on the immune effector cell in a manner sufficient to automatically activate the CD3 complex.
CN202280034086.2A 2021-03-10 2022-03-10 Compositions and methods for reducing therapeutic T cytotoxicity Pending CN117295506A (en)

Applications Claiming Priority (5)

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US63/159,222 2021-03-10
US63/209,094 2021-06-10
US202163225715P 2021-07-26 2021-07-26
US63/225,715 2021-07-26
PCT/US2022/071070 WO2022192895A1 (en) 2021-03-10 2022-03-10 Compositions and methods to reduce therapeutic t cell toxicity

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