Affinity entities with TCR-like antibody binding domains and uses thereof

Abstract

The present invention provides a plurality of affinity binding entities having T cell receptor (TCRL)-like binding domains and methods of using the same. More specifically, the plurality of compositions bind to cells of HLA-A2 / WT1+, HLA-A2 / MAGE-A4, HLA-A2 / MAGE-A9, HLA-A2 / PAP or HLA-A2 / TyrD+, and thus can be used for diagnosis and treatment.

Description

[0001] This application is a divisional application of application number CN201680040163.X (PCT application number PCT / IL2016 / 050600), application date June 8, 2016, and invention name "Affinity entity with TCR-like antibody binding domain with high affinity and excellent specificity and its use".

[0002] Technical field and background technology

[0003] The present invention, in some embodiments thereof, relates to a plurality of affinity binding entities, including a class of TCR antibody binding domains with high affinity and excellent specificity, and uses thereof.

[0004] Multiple cells in tumors and virus infections are expressed by multiple CD8 + Cytotoxic T cells recognize and, in response, + Cytotoxic T cells are activated to destroy these cells. In order to be activated, the clonal T cell receptor (TCR) needs to encounter a specific peptide antigen presented by the major tissue compatibility complex (MHC) molecule on the cell membrane surface. After malignant transformation or viral infection, multiple cells express tumor-associated antigens or viral proteins derived from multiple first-class MHC molecules of the cells. Therefore, multiple disease-specific MHC-peptide complexes are ideal targets for multiple immunotherapy methods. One such method transforms the unique excellent specificity but low inherent affinity of multiple TCRs for MHC peptide complexes into multiple high-affinity soluble antibody molecules with a class of TCR specificity for multiple tumor or viral epitopes. The multiple antibodies, called multiple TCR antibodies, are developed as a new class of immunotherapeutic drugs that can target multiple tumors and virus-infected cells and mediate their specific killing. In addition to their multiple therapeutic capabilities, TCR-like antibodies are developed as multiple diagnostic reagents for cancer and multiple infectious diseases, as well as multiple valuable research tools for studying the expression of the first-class MHC antigens. Summary of the invention

[0005] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0006]

[0007]

[0008] The affinity binding entity is capable of binding to HLA-A2 / TyrD in a human major histocompatibility complex (MHC)-restricted manner 369-377 .

[0009] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0010]

[0011] The affinity binding entity is capable of binding HLA-A2 / TyrD in an MHC-restricted manner 369-377 .

[0012] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0013]

[0014] The affinity binding entity is capable of binding to HLA-A2 / WT1 in an MHC-restricted manner 126-134 .

[0015] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, characterized in that the affinity binding entity comprises an antigen binding region, wherein the antigen binding region comprises a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0016]

[0017] The affinity binding entity is capable of binding HLA-A2 / MAGE-A4 in an MHC-restricted manner 328-343 .

[0018] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0019]

[0020] The affinity binding entity is capable of binding HLA-A2 / MAGE-A9 in an MHC-restricted manner 344-359 .

[0021] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0022]

[0023] The affinity binding entity is capable of binding to HLA-A2 / PAP in an MHC-restricted manner 360-375 .

[0024] According to some embodiments of the present invention, the affinity binding entity is selected from the group consisting of an antibody, a chimeric antigen receptor (CAR) and a T cell receptor (TCR).

[0025] According to some embodiments of the invention, the affinity binding entity is an antibody.

[0026] According to some embodiments of the invention, the affinity binding entity is a TCR.

[0027] According to some embodiments of the invention, the affinity binding entity is a CAR.

[0028] According to some embodiments of the invention, the affinity binding entity is a soluble entity.

[0029] According to some embodiments of the invention, the affinity binding entity is a humanized antibody.

[0030] According to some embodiments of the invention, the affinity binding entity comprises a therapeutic moiety.

[0031] According to some embodiments of the invention, the affinity binding entity comprises a detectable moiety.

[0032] According to some embodiments of the present invention, the antibody is a single-chain antibody, a bispecific antibody or a full-length antibody.

[0033] According to one aspect of some embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the affinity binding entity.

[0034] According to one aspect of some embodiments of the present invention, an expression vector is provided, wherein the polynucleotide is operably linked to a cis-acting regulatory element.

[0035] According to one aspect of some embodiments of the present invention, a cell is provided, comprising the polynucleotide or the expression vector.

[0036] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising the affinity binding entity, the carrier, or the cell.

[0037] According to one aspect of some embodiments of the present invention, a method for detecting a cancer cell is provided, the method comprising: contacting the antibody with the cell under conditions allowing the formation of an immune complex, wherein the presence of the immune complex or a level of the immune complex is used to indicate the cancer cell.

[0038] According to an aspect of some embodiments of the present invention, there is provided a method for detecting and treating cancer in a subject in need of detecting and treating cancer, characterized in that the method comprises:

[0039] (a) detecting the presence of a plurality of cancer cells in the subject according to the method;

[0040] (b) when a plurality of cancer cells are detected, determining that the subject has cancer; and

[0041] (c) administering to said subject an anti-cancer therapy.

[0042] According to one aspect of some embodiments of the present invention, a method for detecting and treating cancer in a subject in need of cancer detection and treatment is provided, the method comprising: contacting a cell of the subject with the antibody under conditions allowing the formation of immune complexes, wherein the presence of the immune complex or a level of the immune complex is used to indicate the cancer.

[0043] According to some embodiments of the invention, the cell is a skin cell.

[0044] According to one aspect of some embodiments of the present invention, there is provided a method for treating cancer, the method comprising: administering a therapeutically effective amount of the affinity binding entity, the vector or the cell to a subject in need thereof, thereby treating cancer.

[0045] According to an aspect of some embodiments of the present invention, there is provided a use for preparing a drug for treating cancer, the use using the affinity binding entity, the carrier or the cell.

[0046] According to some embodiments of the invention, when the affinity binding entity is for TyrD, the cancer is selected from the group consisting of melanoma and glioblastoma.

[0047] According to some embodiments of the present invention, when the affinity binding entity is used for Wilm's tumor protein 1 (WT1), the cancer is selected from chronic myeloid leukemia, multiple myeloma (MM), acute lymphocytic leukemia (ALL), acute myeloid / myeloid leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancer, such as colorectal cancer adenocarcinoma, thyroid cancer, breast cancer, lung cancer (such as non-small cell lung cancer), melanoma, osteosarcoma, endometrial cancer, prostate cancer and glioma.

[0048] According to some embodiments of the present invention, when the affinity binding entity is used for melanoma associated antigen 4 (MAGE-A4), the cancer is selected from melanoma, ovarian cancer, T cell leukemia / lymphoma (such as ATLL), testicular cancer, head and neck cancer, bladder cancer and esophageal cancer.

[0049] According to some embodiments of the present invention, when the affinity binding entity is used for melanoma associated antigen 9 (MAGE-A9), the cancer is selected from the group consisting of renal cell carcinoma, bladder cancer, breast cancer and hepatocellular carcinoma.

[0050] According to some embodiments of the invention, when the affinity binding entity is for prostatic acid phosphatase (PAP), the cancer is selected from the group consisting of prostate cancer.

[0051] Unless otherwise specified, all technical terms and / or scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. Although embodiments of the invention may be implemented or tested by any methods and materials similar or equivalent to those described in the embodiments of the invention, embodiments of the invention, the methods and / or materials listed are described below. In the event of a conflict, the definitions included in this patent specification will control. In addition, materials, methods, and embodiments are exemplary only and are not necessarily intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Some embodiments of the invention described herein are described by way of example only and with reference to the accompanying drawings, and by describing in detail the specific references to the drawings, it should be emphasized that the details shown are only examples for the purpose of illustrating the embodiments of the invention. Based on this, the embodiments of the invention can be clearly implemented by those skilled in the art in conjunction with the drawings and description.

[0053] In the attached figure:

[0054] Figure 1: Surface binding affinity determination of TCR-like antibodies targeting multiple HLA-A2 / tyrosinase complexes. The multiple purified IgGs are indirectly immobilized on the surface plasmon resonance (SPR) sensor chip through anti-mouse or human IgG. The analyte is a multiple purified recombinant single-chain HLA-A2 / tyrosinase complex produced by in vitro refolding of multiple scHLA-A2 complexes expressed by Escherichia coli.

[0055] Figure 2 : Epitope specificity determination for multiple TCR-like antibodies by alanine scanning. Multiple positions 1, 2, 3, 4, 5, 6, 7 and 8 of the tyrosinase peptide sequence were substituted with alanine, and the multiple alanine (Ala) mutant peptides were synthesized and tested at a concentration of 10 -4 Up to 10 -5 Molar concentrations were added to multiple T2 cell antigen presenting cells (APC) at 37°C for 12 hours. Binding at a concentration of 10 μg / mL of TCR-like antibody was obtained by flow cytometry, and the binding intensity measured by mean fluorescence intensity was measured and compared with the binding intensity of the wild-type (WT) native tyrosinase peptide. The relative impact of each Ala substituted position was evaluated as a percentage of the binding of the WT peptide.

[0056] Figure 3 : Binding of multiple TCR-like antibodies D11 and D7 to multiple T2 APCs with tyrosinase peptides and multiple control HLA-A2 restricted peptides. Multiple T2 cells were added with a concentration of 10 -4 Up to 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 hours.

[0057] Binding was monitored by flow cytometry using a secondary anti-mouse IgG labeled with thiolated phycoerythrin (PE). Monoclonal antibody (MAb) BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0058] Figure 4 : Binding of multiple TCR-like antibodies D11 and D7 to multiple T2 APCs with tyrosinase peptides and multiple control HLA-A2 restricted peptides. Multiple T2 cells were added with a concentration of 10 -4 Up to 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 hours. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0059] Figure 5 : Binding of TCR-like antibody D11 to T2 APCs loaded with tyrosinase peptide and control HLA-A2 restricted peptides. T2 cells were loaded with 10 -4 Up to 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 hours. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0060] Figure 6 : Binding of TCR-like antibody D7 to T2APCs with tyrosinase peptide and control HLA-A2 restricted peptides. T2 cells were added with a concentration of 10 -4 Up to 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 hours. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0061] Figure 7 : Binding of the TCR-like antibody MC1 to T2 APCs loaded with tyrosinase peptides and control HLA-A2 restricted peptides. T2 cells were loaded with a concentration of 10 -4 Up to 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 hours. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0062] Figure 8 : Binding of the TCR-like antibody MC1 to melanoma cells expressing HLA-A2 and tyrosinase. Binding of multiple melanoma cells to the TCR-like antibody MC1 was monitored by flow cytometry using a secondary PE-labeled anti-human IgG. MAbBB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0063] Fig. 9: Binding of TCR-like antibody MC1 to multiple cells that are HLA-A2+ and tyrosinase positive or negative. Binding of multiple tumor cells expressing HLA-A2 and tyrosinase positive or negative to TCR-like antibody MC1 was monitored by flow cytometry using secondary PE-labeled anti-human IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0064] Fig.10 : Binding of multiple TCR-like antibodies D11 and D7 to multiple cells that are HLA-A2+ and tyrosinase positive or negative. Binding of multiple tumor cells expressing HLA-A2 and tyrosinase positive or negative to the TCR-like antibody MC1 was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0065] Fig.11 : Binding of multiple TCR-like antibodies D11 and D7 to multiple cells that are HLA-A2+ and tyrosinase negative. Binding of multiple tumor cells expressing HLA-A2 and tyrosinase negative to the TCR-like antibody MC1 was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0066] Fig.12 : Multiple comparative binding of multiple TCR-like antibodies D11, D7 and MC1 to multiple cells that are HLA-A2+ and tyrosinase positive or negative. The binding of multiple tumor cells expressing HLA-A2 and tyrosinase positive or negative to TCR-like antibodies D11, D7 and MC1 was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG.

[0067] Fig.13 : Binding of TCR-like antibody D11 to multiple normal primary cells that are HLA-A2+ / tyrosinase negative. The histological origins of multiple normal primary cells are as indicated, and the binding of multiple tumor cells expressing HLA-A2 and tyrosinase negative to TCR-like antibody D11 was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. MAb BB7.2 was used to monitor HLA-A2 expression.

[0068] Fig.14: Binding of TCR-like antibody D11 to multiple normal primary cells that are negative for HLA-A26 / tyrosinase. The histological origins of multiple normal primary cells are as indicated, and the binding of multiple tumor cells expressing HLA-A2 and negative for tyrosinase to TCR-like antibody D11 was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG.

[0069] Fig.15 : Binding of TCR-like antibody D7 to multiple normal primary cells that are HLA-A2+ / tyrosinase negative. The histological origins of multiple normal primary cells are as indicated, and the binding of multiple tumor cells expressing HLA-A2 and tyrosinase negative to TCR-like antibody D7 was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG.

[0070] Fig.16 : Binding of BB7.2 to multiple normal primary cells. The histological origins of multiple normal primary cells were as indicated, and the expression of HLA-A2 of the histological origins of multiple normal primary cells was monitored by flow cytometry using MAb BB7.2 and secondary PE-labeled anti-mouse IgG.

[0071] Fig.17 : Binding of multiple TCR-like antibodies MC1, D11 and D7 to multiple normal peripheral blood mononuclear cells (PBMCs). The HLA-A2 homology or heterozygosity of PBMCs was identified by PCR. Binding of multiple TCR-like antibodies was monitored using secondary PE-labeled anti-mouse IgG.

[0072] Fig.18 : Summary of the selectivity of TCR-like antibody D11. Binding of multiple cells positive and negative for HLA-A2+ antigen to TCR-like antibody D11 was monitored by using PE-labeled anti-mouse IgG. + / - indicates gene expression of messenger RNA (mRNA) of tyrosinase measured by PCR. MAb BB7.2 was used to monitor the expression of HLA-A2.

[0073] Fig.19 : Summary of the selectivity of TCR-like antibody D7. Binding of TCR-like antibody D7 to multiple cells positive and negative for HLA-A2+ antigen was monitored using PE-labeled anti-mouse IgG. + / - indicates gene expression of tyrosinase mRNA measured by PCR. MAb BB7.2 was used to monitor HLA-A2 expression.

[0074] Fig. 20 : Multiple TCR-like antibodies MC1, D11 and D7 bind to multiple T2 APCs with tyrosinase peptides and multiple HLA-A2-restricted peptides similar to tyrosinase. Multiple T2 cells are added with a concentration of 10-4 Equivalent concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG.

[0075] Fig.21 : TCR-like antibody D11 binds to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to tyrosinase peptides. Multiple T2 cells are added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0076] Fig. 22 : TCR-like antibody D11 binds to multiple T2 APCs with multiple HLA-A2-restricted peptides similar to tyrosinase peptides. Multiple T2 cells are added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0077] Fig.23 : TCR-like antibody D11 binds to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to tyrosinase peptides. Multiple T2 cells are added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0078] Fig.24 : TCR-like antibody D11 binds to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to tyrosinase. Multiple T2 cells were added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0079] Fig.25 : TCR-like antibody D7 binds to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to tyrosinase. Multiple T2 cells were added with a concentration of 10 -5Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0080] Fig.26 : TCR-like antibody D7 binds to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to tyrosinase. Multiple T2 cells were added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0081] Fig. 27 : TCR-like antibody D7 binds to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to tyrosinase. Multiple T2 cells were added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0082] Fig.28 : TCR-like antibody D7 binds to multiple T2 APCs with multiple HLA-A2-restricted peptides similar to tyrosinase identified after alanine scanning. Multiple T2 cells are added with a concentration of 10 -5 Tyrosinase peptides and the indicated peptides were screened from multiple Ala mutant peptides based on epitope recognition specificity of D7 at molar concentrations for 12 hours at 37°C. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0083] Fig.29 : Surface binding affinity determination of TCR-like antibody B47B6 targeting multiple HLA-A2 / WT1 complexes. Multiple purified IgGs were indirectly immobilized on the surface plasmon resonance (SPR) sensor chip through anti-mouse or human IgG. The analyte was multiple purified recombinant single-chain HLA-A2 / WT1 complexes produced by in vitro refolding of multiple scHLA-A2 complexes expressed by Escherichia coli.

[0084] Fig.30: Binding of multiple TCR-like antibodies B47 and ESK1 to multiple T2 APCs loaded with WT1 HLA-A2-restricted peptides. Multiple T2 cells were added with a concentration of 10 -4 Up to 10 -5 Molar concentrations of tyrosinase peptide and various indicated peptides were incubated at 37°C for 12 hours. Binding was monitored by flow cytometry using PE-labeled anti-mouse IgG (for B47) or human IgG (for ESK1). MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0085] Fig.31 : Binding of TCR-like antibodies B47 and ESK1 to T2 APCs loaded with WT1 peptide and control HLA-A2 restricted peptides. T2 cells were loaded with 10 -4 Molar concentrations of WT1 peptide and various indicated peptides were incubated at 37°C for 12 hours. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG (for B47) or human IgG (for ESK1). MAb BB7.2 was used to monitor HLA-A2 expression, expressed as mean fluorescence intensity (MFI).

[0086] Fig.32 : Binding of multiple TCR-like antibodies B47 and ESK1 to multiple T2 APCs loaded with multiple HLA-A2-restricted peptides similar to WT1 peptides. Multiple T2 cells were added with a concentration of 10 -4 Up to 10 -5 Molar concentrations of WT1 peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG (for B47) or human IgG (for ESK1). Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0087] Fig.33 : Binding of TCR-like antibody B47 to T2 APCs loaded with WT1 peptide or multiple control HLA-A2 restricted peptides. Multiple T2 cells were loaded with a concentration of 10 -4 Molar concentrations of WT1 peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using a secondary PE-labeled anti-mouse IgG. Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0088] Fig.34 : Binding of TCR-like antibody B47 to multiple T2 APCs loaded with HLA-A2 restricted peptides similar to WT1 peptides. Multiple T2 cells were added with a concentration of 10-4 Up to 10 -5 Molar concentrations of WT1 peptide and various indicated peptides were incubated at 37°C for 12 h. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG (for B47) or human IgG (for ESK1). Binding of MAb BB7.2 ensured measurement of peptide incorporation efficiency.

[0089] Fig.35 : Binding of multiple TCR-like antibodies B47 and ESK1 to multiple cells that are HLA-A2 positive and do not express WT1. Binding was monitored by flow cytometry using secondary PE-labeled anti-mouse IgG (for B47) or human IgG (for ESK1). MAb BB7.2 was used to monitor HLA-A2 expression.

[0090] Fig.36 : Summary of the selectivity of TCR-like antibody B47. Binding of TCR-like antibody B47 to multiple cells positive and negative for HLA-A2+ antigen was monitored by using PE-labeled anti-mouse IgG. + / - indicates gene expression of WT1 mRNA measured by PCR. MAb BB7.2 was used to monitor the expression of HLA-A2.

[0091] Fig.37 : Determination of epitope specificity for multiple TCR-like antibodies by alanine scanning. Multiple positions 1, 3, 4, 5, 7 and 8 of the WT1 peptide sequence were substituted with alanine, and the multiple alanine (Ala) mutant peptides were synthesized and added to multiple antigen presenting cells (APCs). The binding of the TCR-like antibody ESK1 was obtained by flow cytometry, and the binding intensity measured by the mean fluorescence intensity was measured and compared with the binding intensity of the wild-type (WT) natural WT1 peptide. The relative impact of each Ala-substituted position was evaluated as the percentage of the binding of the WT peptide. Data from Dao et al. Science Translational Medicine 5:176ra33 (2013).

[0092] Fig.38 : Binding of multiple TCR-like antibodies D11, D7 and biotinylated MC1 to multiple T2 APCs with tyrosinase peptides and multiple HLA-A2-restricted peptides similar to tyrosinase, S17 to S23 are multiple similarity peptides based on alanine. Multiple T2 cells were added with a concentration of 10 -5Molar concentrations of tyrosinase peptide and the indicated peptides were incubated at 37°C for 12 hours. Cells were stained with T cell receptor-like (TCRL) antibodies at a concentration of 10 μg / mL followed by secondary PE-labeled streptavidin / anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0093] Fig.39 : Binding of multiple TCR-like antibodies D11, D7 and MC1 to multiple T2 APCs with tyrosinase peptides and multiple HLA-A2-restricted peptides similar to tyrosinase, KIAA0355, S7, S17 to S23 are multiple similarity peptides based on alanine. Multiple T2 cells were added with a concentration of 10 -5 Molar concentrations of tyrosinase peptide and the indicated peptides were incubated at 37°C for 12 hours. Cells were stained with 10 μg / mL TCRL antibodies followed by secondary PE-labeled streptavidin / anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0094] Fig.40A To C: Multiple TCR-like antibodies D11 ( Fig.40A ), D7( Fig.40B ) and biotinylated MC1 ( Fig.40C ) binding to HLA-A2+, tyrosinase antigen positive or negative cells. Tyrosinase mRNA expression was tested by qPCR on multiple tumor and normal primary cells expressing HLA-A2. Multiple tumor cells were stained with multiple TCRL antibodies at a concentration of 10 μg / mL followed by secondary PE-labeled streptavidin / anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0095] Fig.41 : Killing of multiple HLA-A2+ / tyrosinase+ (positive) and HLA-A2+ / tyrosinase- (negative) cell lines by a bispecific (BS) TCRL having one anti-CD-3 arm and one D11 arm, the TCRL is called Tyr D11BS TCRL. Tyr D11 BS TCRL was cultured with multiple melanoma HLA-A2+ / tyrosinase+ cells and HLA-A2+ / tyrosinase- control tumor cells, and multiple cells were incubated with the Tyr D11 BS TCRL and multiple unactivated melanoma cells isolated from multiple healthy individuals. PBMCs were cultured at a 10:1 E:T ratio (10:1 effector:target ratio) for 24 hours, and cytotoxicity was determined by lactate dehydrogenase (LDH) release assay.

[0096] Fig.42 : Killing of HLA-A2+ / tyrosinase- normal primary cells by Tyr D11, BS D11 was cultured together with multiple melanoma HLA-A2+ / tyrosinase+ cells and multiple normal primary cells HLA-A2+ / tyrosinase- as a control group, and the multiple cells were cultured with the Tyr D11 BS TCRL and multiple unactivated PBMCs isolated from multiple healthy individuals at a ratio of 10:1E:T (10:1 effector:target ratio) for 24 hours.

[0097] Fig.43 : Killing of multiple HLA-A2+ / Tyrosinase+ and HLA-A2+ / Tyrosinase- cell lines by Tyr D7 BS TCRL. D7 BS was cultured with multiple melanoma HLA-A2+ / Tyrosinase+ cells and multiple control group tumor cells of HLA-A2+ / Tyrosinase-. The multiple cells were cultured with the Tyr D7 BS and multiple unactivated PBMCs isolated from multiple healthy individuals at a ratio of 10:1E:T (10:1 effector:target ratio) for 24 hours.

[0098] Fig.44 : Killing of HLA-A2+ / tyrosinase- normal primary cells by D7 BS, D7 BS was cultured together with melanoma HLA-A2+ / tyrosinase+ cells and HLA-A2+ / tyrosinase- normal primary cells as a control group, and the cells were cultured with the Tyr D11 BS TCRL and unactivated PBMCs isolated from healthy individuals at a 10:1E:T ratio (10:1 effector:target ratio) for 24 hours.

[0099] Fig.45 The in vivo efficacy of D7 BS in preventing tumor formation of SC501A melanoma in NOD / SCID mice was shown.

[0100] Fig.46 : Binding of multiple TCR-like antibodies biotinylated ESK1 and B47B6 to multiple T2 APCs loaded with WT1 peptide and multiple other HLA-A2 restricted peptides. Multiple T2 cells were added with a concentration of 10 -5 WT1 peptide and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL TCRL antibodies followed by secondary PE-labeled streptavidin / anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0101] Fig.47 : Binding of multiple TCR-like antibodies biotinylated ESK1 and B47B6 to multiple T2 APCs loaded with WT1 peptide and multiple HLA-A2 restricted peptides similar to WT1. S2, S6 and S7 are multiple alanine-based similarity peptides. S11 is a heteroclitic peptide. Multiple T2 cells were added with a concentration of 10 -5 WT1 peptide and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL TCRL antibodies ESK1 and B47B6 followed by secondary PE-labeled streptavidin / anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0102] Fig.48 : Affinity-targeting surface binding affinity determination of TCR-like antibodies ESK1 and B47B6 for multiple HLA-A2 / WT1 complexes by SPR. Purified recombinant biotinylated single-chain HLA-A2 / WT1 complexes were generated by in vitro refolding of multiple scHLA-A2 complexes expressed in E. coli, which were indirectly immobilized on the SPR sensor chip with NeutrAvidin. Purified multiple antigen binding domains (Fab) of ESK1 and B47B6 TCRL were used as multiple analytes.

[0103] Fig.49 : Epitope specificity was determined by alanine scanning mutagenesis. Multiple mutant WT1 peptides with alanine substitutions at positions 1, 2, 3, 4, 5, 7, 8, and 9 were synthesized and expressed at a concentration of 10 -5 10 μg / ml of Ala was added to T2 antigen presenting cells (APC) at 37°C for 12 hours. Cells were stained with a TCR-like antibody B47B6 at a concentration of 10 μg / ml and analyzed by flow cytometry. The relative impact of each Ala substitution position was expressed as a percentage of the bound wild-type peptide.

[0104] Fig.50 : Binding of multiple TCR-like antibodies B47 and ESK1 to multiple cells that are HLA-A2+ and WT1 mRNA positive or negative. Multiple tumor cells expressing HLA-A2 were tested for WT1 mRNA expression by qPCR. Multiple tumor cells were stained with 10 μg / ml of multiple TCRL antibodies ESK1 and B47B6 followed by secondary PE-labeled streptavidin, expressed as mean fluorescence intensity (MFI). Data also showing mRNA expression and cell killing by multiple antibodies in bispecific format (with anti-CD3) as described herein.

[0105] Fig.51A :By comparing B47B6 BS and ESK1 BS to kill multiple normal primary cells of HLA-A2+ / WT1+ and HLA-A2+ / WT1-.B47B6 BS and ESK1 BS were cultured with normal primary cells of HLA-A2+ / WT1+ or HLA-A2+ / WT1-, and multiple cells were cultured with the B47B6 BS or ESK1 BS and multiple non-activated PBMCs isolated from multiple healthy individuals at a ratio of 10:1E:T for 24 hours.Cytotoxicity was determined by lactate dehydrogenase (LDH) release test.

[0106] Fig.51B : By comparing the killing of multiple cell lines of HLA-A2+ / WT1+ and HLA-A2+ / WT1- by B47B6 BS and ESK1 BS. B47B6 BS and ESK1 BS were cultured with multiple tumor cells of HLA-A2+ / WT1+ or HLA-A2+ / WT1-, and multiple cells were cultured with the B47B6 BS or ESK1 BS and non-activated PBMCs isolated from multiple healthy individuals at a ratio of 10:1E:T for 24 hours (#F3-format-wherein the single-chain variable region fragment (scFv) of the anti-differentiation cluster 3 (CD3) was fused with the light chain variable region and light chain constant region (VLCL) of the Fab).

[0107] Fig.52 :TCR-like antibody C106B9 and MAGE-A4 230-239 (also known as MAGE-A4 peptide) peptide and multiple other HLA-A2 restricted peptides. Multiple T2 cells were added to a concentration of 10 -5 Molar concentrations of MAGE-A4 and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL of TCRL antibody C106B9 followed by secondary PE-labeled anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0108] Fig.53 : Binding of TCR-like antibody C106B9 to MAGE-A4 peptide and multiple HLA-A2-restricted peptides similar to MAGE-A4. Multiple T2 cells were added to 10 -5 10 μg / mL of MAGE-A4 and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL of TCRL antibody C106B9 followed by secondary PE-labeled anti-mouse IgG and analyzed by flow cytometry.

[0109] Fig.54 : Affinity by SPR - Surface binding affinity determination of TCR-like antibody C106B9 targeting multiple HLA-A2 / MAGE-A4 complexes. Purified recombinant biotinylated HLA-A2 / MAGE-A4 complexes were generated by in vitro refolding of multiple scHLA-A2 complexes expressed in E. coli, and the HLA-A2 / MAGE-A4 complexes were indirectly immobilized on the SPR sensor chip with neutravidin. Purified C106B9 TCRL Fab was used as the analyte.

[0110] Fig.55 : Epitope specificity was determined by alanine scanning mutagenesis. The multiple mutant MAGE-A4 peptides with alanine substitutions at positions 1, 2, 3, 4, 5, 7, 8 and 9 were synthesized, and multiple possible anchoring positions were represented by a gray star. The multiple native and mutant MAGE-A4 peptides were subjected to a concentration of 10 -5 The molar concentrations were added to multiple T2 cell antigen presenting cells (APC) at 37°C for 12 hours. Multiple cells were stained with a concentration of 10 μg / ml of TCR-like antibody C106B9 and analyzed by flow cytometry. The MFI values ​​of multiple cells added with multiple mutants and wild-type peptides were compared, and the relative impact of each Ala substitution position was expressed as a percentage of the bound wild-type peptide.

[0111] Fig.56 : Binding of the TCR-like antibody C106B9 to multiple cells that are HLA-A2+ and MAGE-A4 antigen positive or negative. Expression of MAGE-A4 mRNA in multiple cells was confirmed by qPCR. Multiple tumor cells were stained with 10 μg / mL of C106B9 followed by a secondary PE-labeled anti-mouse antibody, expressed as mean fluorescence intensity (MFI). Also shown are data for mRNA expression and cell killing by multiple antibodies in a bispecific format (with anti-CD3), as described herein.

[0112] Fig.57 : Killing of multiple HLA-A2+ / MAGE-A4+ and HLA-A2+ / MAGE-A4- cell lines by C106B9 BS. C106B9 BS was cultured with multiple HLA-A2+ / MAGE-A4+ cells and multiple HLA-A2+ / MAGE-A4- cells of the control group. The multiple cells were cultured with the C106B9 BS and multiple non-activated PBMCs isolated from multiple healthy individuals at a ratio of 10:1E:T for 24 hours.

[0113] Fig.58: Killing of multiple normal primary cell lines of HLA-A2+ / MAGE-A4- by C106B9 BS. C106B9 BS was cultured together with multiple normal primary cells of HLA-A2+ / MAGE-A4-. Multiple cells were cultured with the C106B9 BS and multiple unactivated PBMCs isolated from multiple healthy individuals at a ratio of 10:1E:T for 24 hours.

[0114] Fig.59 : In vivo efficacy of MAGE-A4 BS C106B9 BS in preventing tumor formation of SC melanoma in NOD / SCID mice.

[0115] Fig.60 :TCR-like antibody F184C7 and MAGE-A9 223-231 peptide (also known as MAGE-A9 peptide) and multiple other HLA-A2 restricted peptides. Multiple T2 cells were added to 10 -5 Molar concentrations of MAGE-A9 and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL of TCRL antibody F184C7 followed by secondary PE-labeled anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0116] Fig.61 : Binding of TCR-like antibody F184C7 to MAGE-A9 peptide and multiple HLA-A2-restricted peptides similar to MAGE-A9. S8 is an alanine-based similarity peptide. Multiple T2 cells were added to 10 -5 Molar concentrations of MAGE-A9 and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL of TCRL antibody F184C7 followed by secondary PE-labeled anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0117] Fig.62 : Epitope specificity was determined by alanine scanning mutagenesis. The multiple mutant MAGE-A9 peptides having alanine substitutions at positions 2, 3, 4, 5, 6, 7, 8 and 9 were synthesized. The multiple Ala mutations and the native peptides were mixed at a concentration of 10 -5 The molar concentrations were added to T2 antigen presenting cells (APC) at 37°C for 12 hours. Cells were stained with a 10 μg / ml TCR-like antibody B47B6 and analyzed by flow cytometry. The relative impact of each Ala substitution position was expressed as a percentage of the bound wild-type peptide.

[0118] Fig.63 : Binding of TCR-like antibody F184C7 to multiple HLA-A2+ normal primary cells. Multiple normal primary cells were stained with 10 μg / mL of multiple TCRL antibody F184C7 followed by secondary PE-labeled anti-mouse antibody, expressed as mean fluorescence intensity (MFI).

[0119] Fig.64 :TCR-like antibody D10A3 and PAP 112-120 Peptide (also known as PAP peptide) and multiple other HLA-A2 restricted peptides. Multiple T2 cells were added to a concentration of 10 -5 Molar concentrations of PAP and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL of TCRL antibody C106B9 followed by secondary PE-labeled anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0120] Fig.65 : Binding of TCR-like antibody D10A3 to peptides containing PAP and multiple HLA-A2-restricted peptides similar to PAP. Multiple T2 cells were added to a concentration of 10 -5 Molar concentrations of PAP and the indicated peptides were incubated at 37°C for 12 h. Cells were stained with 10 μg / mL of TCRL antibody D10A3 followed by secondary PE-labeled anti-mouse IgG and analyzed by flow cytometry, expressed as mean fluorescence intensity (MFI).

[0121] Fig.66 : Epitope specificity was determined by alanine scanning mutagenesis. Multiple mutant PAP peptides with alanine substitutions at positions 1, 3, 4, 6, 7, 8 and 9 were synthesized and expressed at a concentration of 10 -5 The molar concentrations were added to multiple T2 cell antigen presenting cells (APC) at 37°C for 12 hours. Multiple cells were stained with a concentration of 10 μg / ml of TCR-like antibody D10A3, and the MFI values ​​of multiple cells added with multiple mutants and wild-type peptides were compared. The relative impact of each Ala substitution position was expressed as a percentage of the bound wild-type peptide.

[0122] Fig.67 : Binding of TCR-like antibody D10A3 to multiple HLA-A2+ normal primary cells. Multiple normal primary cells were stained with 10 μg / ml of multiple TCRL antibody D10A3 followed by secondary PE-labeled anti-mouse antibody, and the mean fluorescence intensity (MFI) was expressed.

[0123] Fig.68: Multiple amino acids and multiple nucleic acids of D11 antibody (SEQ ID NO: 280-295).

[0124] Fig.69 : Multiple amino acids and multiple nucleic acids of D7 antibody (SEQ ID NO: 296-311).

[0125] Fig.70 : Multiple amino acids and multiple nucleic acids of the B47B6 antibody (SEQ ID NO: 312-327).

[0126] Fig.71 : Multiple amino acids and multiple nucleic acids of the C106B9 antibody (SEQ ID NOs: 328-343).

[0127] Fig.72 : Multiple amino acids and multiple nucleic acids of the F184C7 antibody (SEQ ID NOs: 344-359).

[0128] Fig.73 : Multiple amino acids and multiple nucleic acids of the D10A3 antibody (SEQ ID NOs: 360-375). Specific embodiments

[0129] The present invention, in some embodiments thereof, relates to a plurality of affinity binding entities, including a class of TCR antibody binding domains with high affinity and excellent specificity, and uses thereof.

[0130] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not necessarily limited to the details of the present invention described below or the multiple embodiments exemplified. The present invention can be implemented or applied in other embodiments or in various ways.

[0131] T cell receptor (TCR)-like (TCRL) is endowed with a class of TCR specificity for multiple tumor epitopes. Unlike multiple TCRs that show low affinity for MHC peptide antigen complexes, multiple TCRLs have the property that even the soluble form of the multiple TCRLs has affinity. TCRLs are developed as a novel therapeutic class for targeting multiple tumor cells and mediating their specific killing. In addition, the multiple antibodies are multiple valuable research reagents, allowing the study of human class I peptide-MHC ligand expression and TCR-peptide-MHC multiple interactions.

[0132] The inventors have now identified novel TCRLs through laborious screening and experimentation, which exhibit unprecedentedly good specificity for TyrD-HLA-A2 (D7 and D11), WT1-HLA-A2 (B47), MAGE-A4-HLA-A2 (C106B9), MAGE-A9-HLA-A2 (F184C7) and PAP (D10A3). The CDRs of the antibodies can be implanted in any affinity binding entity, such as having an effector activity (e.g., CAR and TCR).

[0133] Therefore, according to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0134]

[0135] The affinity binding entity is capable of binding to HLA-A2 / TyrD in a human major histocompatibility complex (MHC)-restricted manner 369-377 .

[0136] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0137]

[0138] The affinity binding entity is capable of binding HLA-A2 / TyrD in an MHC-restricted manner 369-377 .

[0139] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0140]

[0141] The affinity binding entity is capable of binding to HLA-A2 / WT1 in an MHC-restricted manner 126-134 .

[0142] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, characterized in that the affinity binding entity comprises an antigen binding region, wherein the antigen binding region comprises a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0143]

[0144] The affinity binding entity is capable of binding HLA-A2 / MAGE-A4 in an MHC-restricted manner 328-343 .

[0145] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0146]

[0147] The affinity binding entity is capable of binding HLA-A2 / MAGE-A9 in an MHC-restricted manner 344-359 .

[0148] According to one aspect of some embodiments of the present invention, there is provided an affinity binding entity, the affinity binding entity comprising an antigen binding region, the antigen binding region comprising a plurality of complementarity determining region (CDR) sequences arranged from N-terminus to C-terminus:

[0149]

[0150] The affinity binding entity is capable of binding to HLA-A2 / PAP in an MHC-restricted manner 360-375 .

[0151] As used herein, a "T cell receptor-like antibody" or "TCRL" refers to an antibody that binds to an MHC complexed with an HLA-restricted peptide antigen. The binding of TCRL to the TCRL target has an MHC-restricted specificity. In the absence of the complexed peptide, the TCRL antibody does not bind to the MHC, and in the absence of the MHC, the antibody does not bind to the peptide.

[0152] As used herein, "binding" or "binds" refers to binding in an antibody-antigen format, wherein typically, in the case of clinically relevant TCRL, K is measured by surface plasmon resonance analysis (SPR). D The concentration is below 20 nanomolar.

[0153] The affinity of the antigen binding domain to the antigen of the antigen binding domain is determined using the soluble form of the antibody from which the CDRs of the antigen binding domain of the antibody are derived. For affinity assessment, the antigen is a soluble form of the antigen, e.g., a single chain MHC peptide complex as described further below.

[0154] As used herein, the term "K D ” refers to the equilibrium dissociation constant between the antigen binding domain and the corresponding antigen of the antigen binding domain.

[0155] It will be appreciated that the affinity of the affinity binding entity is determined by the plurality of CDRs. However, the affinity can be improved using a variety of methods known in the art, such as affinity maturation.

[0156] As used herein, an "affinity binding entity" refers to a binding moiety that binds to a specific antigen with a higher affinity than to a non-specific antigen and confers an affinity of at least 10 -6 Molar concentration is as determined by a variety of tests known in the art, including surface plasmon resonance (SPR).

[0157] According to a specific embodiment, the affinity is 500 nanomolar-0.5 nanomolar, 100 nanomolar-1 nanomolar, 50 nanomolar-1 nanomolar, 20 nanomolar-1 nanomolar, 10 nanomolar-1 nanomolar.

[0158] The affinity portion can be selected from the group consisting of TCR, CAR-T and antibody.

[0159] According to a specific embodiment, the affinity binding entity is an antibody. Although the references to the plurality of antibodies are detailed compared to other affinity binding entities, the description of this embodiment should not be construed as limiting, and the invention is equally relevant to the plurality of binding entities described herein, particularly in the sense of the cell therapy described further below.

[0160] The term "antibody" used in the present invention includes multiple complete molecules and multiple functional fragments of the complete molecules, such as Fab, F(ab')2, Fv, scFv, dsFv or multiple single domain molecules, such as VH and VL, which can bind to an epitope of an antigen in an MHC-restricted form. As a more basic statement, the term "antibody" is intended to include any affinity binding entity that binds to a molecule expressed on the surface of a cell with an MHC-restricted specificity. Therefore, multiple CDRs of the antibodies of some embodiments of the present invention can be implanted in multiple artificial molecules, such as T cell receptors or multiple CARs, as further described below.

[0161] Suitable antibody fragments for implementing some embodiments of the present invention include a complementarity determining region (CDR) of an immunoglobulin light chain (referred to herein as "light chain"), a complementarity determining region of an immunoglobulin heavy chain (referred to herein as "heavy chain"), a light chain variable region, a heavy chain variable region, a light chain, a heavy chain, a Fd fragment, and the antibody fragments substantially include light chains and heavy chains, such as an Fv region, a single-chain Fv (scFv), a disulfide bond-stabilized Fv (dsFv), a Fab, a Fab' and a F(ab')2.

[0162] As used herein, the terms "complementarity determining region" or "CDR" are used interchangeably to refer to the multiple antigen binding regions found within the variable regions of the multiple heavy and light chain polypeptides. Generally, multiple antibodies include three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3); and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3). Multiple examples of the multiple CDR sequences are provided for D7 and D11-TCRL prepared according to Example 1 below. Additional examples include WT1 B47B6, MAGE-A4 C106B9, MAGE-A9 F184C7, PAP D10A3 (such as Figures 68-73 shown).

[0163] The identity of the amino acid residues that make up a variable region or a CDR in a particular antibody can be determined using methods known in the art and include, for example, the method of sequence variability defined by Kabat et al. (see, e.g., Kabat et al., 1992, Protein Sequences of Immunity Related, 5th Ed., Public Health Service, National Institutes of Health, Washington, D.C.), the positions of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989), using Newton's Molecular AbM antibody modeling software (now See Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268; and the global website www.bioinf-org.uk / abs) A compromise between Kabat and Chothia, as defined by the contact definition available in complex crystal structures (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996), the "configurational definition" (see, e.g., Makabe et al., J. Biol. Chem. 283:1156-1164, 2008), and IMGT [LeFRanc MP et al. (2003) IMGT unique numbering for multiple variable regions of immunoglobulins and T cell receptors and individual class V regions of the Ig superfamily, Dev. Comp. Immunol. 27:55-77].

[0164] As used herein, the "variable regions" and "CDRs" may refer to variable regions and CDRs defined by any method known in the art including a combination of methods.

[0165] According to a specific embodiment, the plurality of CDRs are determined according to Kabat et al. (supra).

[0166] Functional antibody fragments include all or substantially all of the variable regions of the light chain and the heavy chain and are defined as follows:

[0167] (1) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH), expressed as a double chain;

[0168] (2) Single-chain variable region fragment Fv ("scFv"), a genetically engineered single-chain molecule comprising the variable region of the light chain and the variable region of the heavy chain, wherein the genetically engineered single-chain molecule is a genetically fused single-chain molecule via a suitable polypeptide linker.

[0169] (3) A disulfide-stabilized Fv ("dsFv"), a genetically engineered antibody comprising the variable region of the light chain and the variable region of the heavy chain, wherein the genetically engineered antibody is connected by a genetically engineered disulfide bond.

[0170] (4) Fab, a fragment of an antibody molecule comprising a monovalent antigen-binding portion of an antibody molecule; the fragment of the antibody molecule can be obtained by treating the whole antibody with the papain to produce the complete light chain and the Fd fragment of the heavy chain, wherein the Fd fragment of the heavy chain consists of the variable region and the CH1 region of the Fd fragment of the heavy chain;

[0171] (5) Fab', a fragment of an antibody molecule comprising the monovalent antigen-binding portion of an antibody molecule, which can be obtained by treating a whole antibody with pepsin followed by reduction (two Fab' fragments are obtained per antibody molecule);

[0172] (6) F(ab')2, a fragment of an antibody molecule comprising a monovalent antigen-binding portion of an antibody molecule, which can be obtained by treating a whole antibody with the enzyme pepsin (i.e., a dimer of multiple Fab' fragments held together by two disulfide bonds); and

[0173] (7) Multiple single-domain antibodies or nanobodies are composed of a single VH or VL region with sufficient affinity for the antigen.

[0174] Methods for producing polyclonal and monoclonal antibodies, and fragments thereof, are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0175] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0176] According to some embodiments of the present invention, multiple antibody fragments can be prepared by proteolysis of the antibody or by expression of the fragment encoded by DNA in Escherichia coli or multiple mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems). Multiple antibody fragments can be obtained by cutting multiple whole antibodies by pepsin or papain by multiple conventional methods. For example, multiple antibody fragments can be produced by enzymatic cleavage of multiple antibodies by pepsin, with a 5S fragment provided being represented as F (ab'). The fragment can be further cleaved by a thiol reducing agent, and optionally a blocking group for multiple sulfhydryl groups produced by the cleavage of multiple disulfide bonds to produce multiple 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and Fc fragments. The multiple methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and multiple references contained in the multiple patents, the entirety of which is incorporated herein by reference. See also Porter, RR [Journal of Biochemistry 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques may also be used, as long as the fragments bind to the antigen recognized by the intact antibody.

[0177] Multiple Fv fragments include a linking of multiple VH and VL chains. The linking can be non-covalent, as described by Inbar et al. [Proceedings of the National Academy of Sciences of the United States of America 69: 2659-62 (19720)]. Alternatively, the multiple variable chains can be linked by a disulfide bond between molecules or cross-linked by multiple chemicals such as glutaraldehyde. Preferably, the multiple Fv fragments include multiple VH and VL chains connected by a peptide linker. The multiple single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene containing multiple DNA sequences encoding the multiple VH and VL regions connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as Escherichia coli. The multiple recombinant host cells synthesize a single polypeptide chain having a connector bridging the two V domains. A variety of methods for producing sFvs are described, for example, in [Whitlow and Filpula, Methods 2:97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio / Science 11:1271-77 (1993); and U.S. Pat. No. 4,746,778, the entirety of which is incorporated herein by reference.

[0178] Another form of an antibody fragment is a peptide that encodes a single complementarity determining region (CDR). Multiple CDR peptides ("multiple minimum recognition units") can be obtained by constructing multiple genes encoding the CDR of an antibody of interest. The multiple genes are prepared, for example, by synthesizing the variable regions from RNA of multiple antibody-producing cells using the polymerase chain reaction. See, for example, Larrick and FRy [Methods, 2: 106-10 (1991)].

[0179] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains, or fragments of immunoglobulin chains (e.g., Fv, Fab, Fab', F(ab') branch 2, or other antigen-binding subsequences), wherein the humanized forms contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies comprise human immunoglobulins (recipient antibodies), wherein residues forming a complementary determining region (CDR) of the receptor within the human immunoglobulins are replaced with residues from a CDR of non-human species (donor antibodies), such as mouse, rat, or rabbit, wherein the residues of the non-human species have the desired specificity, affinity, and capacity. In some examples, the Fv framework residues of the human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies may also include residues or framework sequences that are not found in either the receptor antibody or the introduced CDR, and residues in the receptor antibody that are not found in the introduced CDR or framework sequence. In general, the humanized antibody may substantially comprise at least one of all, and typically two, multiple variable regions, wherein all or substantially all of the CDR regions correspond to the multiple CDR regions of a non-human immunoglobulin, and all or substantially all of the FR regions correspond to the multiple FR regions of a non-human immunoglobulin consensus sequence. The humanized antibody ideally also includes at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Current Opinion in Structural Biology, 2: 593-596 (1992)].

[0180] Methods for humanizing non-human antibodies are known in the art. Typically, a humanized antibody has one or more amino acid residues introduced from a non-human source. The non-human amino acid residues are often referred to as introduced residues, which are typically taken from an introduced variable region. Humanization can be performed essentially according to the method of Winter et al. [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); Verhoeyen et al., Science, 239:1534-1652 (1988)] by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable region has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0181] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, Journal of Mol. Biol., 227:381 (1991); Marks et al., Journal of Mol. Biol., 222:581 (1991)], and the techniques of Cole et al. and Boerner et al. can also be used for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., Journal of Immunology, 147(1):86-95 (1991)]. Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, the production of human antibodies was observed that closely resembled those seen in humans in all aspects, including gene rearrangement, combination, and antibody repertoires. The method is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Journal of Bio / Technology: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and: Lonberg and Huszar, International Reviews of Immunology 13, 65-93 (1995).

[0182] In one embodiment, the antibody is a full-length antibody, and the multiple heavy chains and the multiple light chains of an antibody of the present invention can be full-length (e.g., an antibody can include at least one, and preferably two complete heavy chains and at least one or two complete light chains) or can include an antigen binding portion (a Fab, F(ab') branch 2, Fv or a single chain variable region fragment ("scFv"). In other embodiments, the heavy chain constant region of the antibody is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. In some embodiments, the isotype of the immunoglobulin is selected from IgG1, IgG2, IgG3 and IgG4, more specifically, IgG1 (e.g., human IgG1) or IgG4 (e.g., human IgG4). The choice of the antibody type will depend on the function of the immune effect that the antibody is designed to elicit.

[0183] Also contemplated herein are multiple bispecific configurations of multiple antibodies. A bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and thus binds to two different types of antigens. According to a specific embodiment, the BsMAb is designed to simultaneously bind to a cytotoxic cell (e.g., using a receptor similar to CD3) and a target such as a tumor cell to be destroyed (described further below).

[0184] As used herein, the phrase "chimeric antigen receptor (CAR)" refers to a recombinant or synthetic molecule that combines antibody-based specificity for a desired antigen with an activated intracellular region of a T cell receptor to produce a chimeric protein that exhibits cellular immune activity against the specific antigen.

[0185] As used herein, the phrase "T cell receptor" or "TCR" refers to soluble as well as insoluble forms of recombinant T cell receptors.

[0186] As used herein, the phrase "MHC (or HLA) restricted peptide" refers to a peptide potentially expressed on an MHC molecule. The peptide can be detected by "wet" laboratory procedures such as mass spectrometry or by computer simulation analysis. An MHC (or HLA) expressed peptide refers to a peptide that is confirmed to be expressed by an MHC molecule in vitro or in vivo.

[0187] According to a specific embodiment, the MHC-restricted peptide is from WT1 and the affinity binding entity comprises the multiple CDRs of B47B6.

[0188] According to a specific embodiment, the MHC-restricted peptide is from TyrD and the affinity binding entity comprises the CDRs of D7 or D11.

[0189] According to a specific embodiment, the MHC-restricted peptide is from WT1 and the affinity binding entity comprises the multiple CDRs of C106B9.

[0190] According to a specific embodiment, the MHC-restricted peptide is from MAGE-A9 and the affinity binding entity comprises the CDRs of F184C7.

[0191] According to a specific embodiment, the MHC-restricted peptide is from PAP and the affinity binding entity comprises the CDRs of D10A3.

[0192] The multiple CDRs of the above-mentioned multiple affinity binding entities are described in Figures 68-73 middle.

[0193] Multiple homology sequences are also contemplated, for example, at least 90% homology, 95% homology or even at least 99% homology, as long as the binding affinity and optionally specificity to the respective target is maintained or even improved.

[0194] Therefore, according to one aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the affinity binding entity described herein.

[0195] The present invention also provides an expression vector comprising a polynucleotide operably linked to a cis-acting regulatory element.

[0196] The nucleic acid constructs of some embodiments of the present invention (also referred to herein as an "expression vector") comprise additional sequences that render the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, typical cloning vectors may also comprise a transcriptional and translational initiation sequence, a transcriptional and translational terminator, and a polyadenylation signal. As an example, the constructs will typically comprise a 5'LTR, a tRNA binding site, a packaging signal, a source for second-strand DNA synthesis, and a 3'LTR or a portion of the 3'LTR.

[0197] The nucleic acid construct of some embodiments of the invention typically comprises a signal sequence for secreting or expressing the affinity binding entity from a host cell in which the antibody is located. Preferably, the signal sequence for this purpose is a mammalian signal sequence.

[0198] The eukaryotic promoter typically comprises two types of recognition sequences, the TATA box and multiple upstream promoters. The TATA box, located 25-30 base pairs upstream of the transcription start site, is believed to be involved in directing RNA polymerase to start RNA synthesis. The other upstream promoter components measure the rate after transcription initiation.

[0199] Preferably, the promoter used in the nucleic acid construct of some embodiments of the present invention is activated in the specific cell population transformed. Examples of multiple cell type-specific and / or tissue-specific promoters include promoter components, such as liver-specific albumin [Pinkert et al., (1987) Genes and Development 1: 268-277], multiple lymphoid-specific promoters [Calame et al., (1988) Advances in Immunology 43: 235-275]; in multiple specific promoters of multiple T cell receptors [Winoto et al., (1989) EMBO 8: 729-733] and multiple immunoglobulins; [Ba nerji et al. (1983) Cell 33:729-740], multiple neuron-specific promoters, such as the neurofilament promoter [Byrne et al., (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], multiple pancreas-specific promoters [Edlunch et al., (1985) Science 230:912-916] or multiple mammary gland-specific promoters, such as the milk whey protein promoter (U.S. Pat. No. 4,973,316 and European Application Publication No. 264,166).

[0200] Multiple enhancer components can stimulate transcription to 1,000 times from multiple linked homologous or heterologous promoters. Multiple enhancers are activated when they are located downstream or upstream of the transcription start site. Many enhancer components derived from multiple viruses have a wide host range and are active in various tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter compositions suitable for some embodiments of the present invention include long terminal repeats derived from polyoma virus, human or murine cytomegalovirus (CMV), derived from various retroviruses, such as murine leukemia virus, murine or golden sarcoma virus and HIV. See, "Multiple Enhancers and Eukaryotic Expression", Cold Spring Harbor Press, Cold Spring Harbor, New York 1983, which is incorporated herein by reference.

[0201] In the construction of the expression vector, the promoter is preferably located at about the same distance from the heterologous transcription start site as the transcription start site in the natural setting of the promoter. However, as known in the art, some variation of the distance can be tolerated without loss of promoter function.

[0202] A polyadenylation sequence may also be added to the expression vector to increase the efficiency of TCRL mRNA transcription. Two distinct sequence components are required for accurate and efficient polyadenylation: multiple GU or U-rich sequences located downstream of the polyadenylation site and a highly conserved sequence of 6 nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals suitable for use in some embodiments of the present invention include the multiple termination and polyadenylation signals derived from SV40.

[0203] In addition to the components already described, the expression vectors of some embodiments of the present invention may generally contain other specialized components to increase the expression level of multiple cloned nucleic acids or to facilitate the recognition of multiple cells carrying the recombinant DNA. For example, many animal viruses contain multiple DNA sequences that facilitate the extrachromosomal replication of the viral genome in multiple permissive cell types. As long as multiple appropriate factors are provided by the genes carried on the plasmid or the genome of the host cell, the plasmid carrying the multiple viral replicons can be replicated episomally.

[0204] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, the vector is amplified in eukaryotic cells using the appropriate selectable marker. If the vector does not include a eukaryotic replicon, episomal amplification is not possible. Instead, the recombinant DNA is integrated into the genome of the designed cell, where the promoter directs expression of the desired nucleic acid.

[0205] Also provided are a plurality of cells comprising the plurality of polynucleotides / plurality of expression vectors described herein.

[0206] The cells are usually selected for high expression of recombinant proteins (e.g., bacteria, plants or eukaryotic cells, e.g., CHO, HEK-293 cells), but when, for example, the CDRs of the TCRL are implanted into a T cell receptor or CAR in the cells, they may also be host cells (e.g., T cells or NK cells) with a specific immune effector activity. As further described below.

[0207] The high specificity of the affinity binding entity renders it particularly suitable for a variety of diagnostic and therapeutic applications.

[0208] Thus, according to one aspect of the present invention, a method of detecting a cell expressing an HLA-restricted peptide antigen of interest is provided. The method comprises contacting the cell with the affinity binding entity (e.g., antibody) of the present invention having specificity for the HLA-restricted peptide antigen of interest. The contacting is performed under conditions that allow the formation of immune complexes, wherein the expression of the immune complexes or the level of the immune complexes indicates that the cell expresses the HLA-restricted peptide antigen of interest.

[0209] As used herein, the term "detecting" refers to "detecting," "perceiving," "uncovering," "exposing," "visualizing," or "identifying" the action of a cell. The precise detection method relies on the detectable moiety (also referred to herein as the recognizable moiety) to which the antibody is bound as further described herein.

[0210] According to the teachings of the present invention, multiple single cells can be used as multiple cells. For example, the multiple cells can come from any biological sample, such as a cell line, a primary (such as a tumor culture) and multiple cell samples, such as various surgical specimens, including incision or resection specimens, fine needle aspirates, etc. Multiple methods of biopsy are known in the art.

[0211] The above detection method can be used for the diagnosis of multiple diseases, and the diagnosis of the disease is determined by the normal expression or distribution of the HLA peptide complex in different tissues.

[0212] The term "diagnosing" as used herein refers to classifying a disease, determining a severity (grade or stage) of a disease, monitoring progression, predicting an outcome of the disease and / or prospects for recovery.

[0213] The subject may be a healthy subject (e.g., human) undergoing a routine health check-up. Alternatively, the subject may be at risk of the disease. Additionally, alternatively, the method may be used to monitor the efficacy of a treatment.

[0214] The TCRL may comprise, for example, conjugated to a recognizable moiety. Alternatively or additionally, the TCRL (or a complex comprising the same TCRL) may be recognized indirectly, for example by using a secondary antibody.

[0215] The contacting can be performed in vitro (ie, in a cell line, a plurality of primary cells), in vivo or ex vivo.

[0216] As described above, the methods of the present invention are sufficient to form an immune complex (e.g., a complex between the antibodies of the present invention and the peptide complexed with the MHC, generally when the cells are not lysed) under a plurality of conditions; the plurality of conditions (e.g., a plurality of suitable concentrations, a plurality of buffers, a plurality of temperatures, a plurality of reaction times) and methods for optimizing the plurality of conditions are known to those skilled in the art, and a plurality of examples are disclosed herein.

[0217] The plurality of affinity binding entities (eg, antibodies) of the present invention are particularly suitable for the treatment of such cancers.

[0218] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0219] The cancer can be a hematological malignancy, a solid tumor, a primary or metastatic tumor. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, chronic lymphocytic leukemia (CLL), leukemia, lung cancer, etc. Additional non-limiting examples of cancers that can be treated by the methods of some embodiments of the present invention are provided in Table 1 above.

[0220] A plurality of cancers that can be treated include tumors that are not vascularized, or are not substantially vascularized, and a plurality of vascularized tumors. The cancer may include a plurality of non-solid tumors (e.g., a plurality of blood tumors, e.g., leukemias and lymphomas) or may include a plurality of solid tumors. The types of cancers treated by the plurality of antibodies of the present invention include, but are not limited to, cancers, embryonic tumors, and sarcomas, as well as some leukemias or lymphomas, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas. A plurality of adult tumors / cancers and a plurality of pediatric tumors / cancers are also included.

[0221] A variety of blood cancers are a variety of cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemias, (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphoma (indolent, high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplastic syndrome.

[0222] Solid tumors are abnormal tissues that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcomas, cancers, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovial sarcoma, mesothelioma, and sarcomas, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, adrenal sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, Chloroepithelial carcinoma, Wilms' tumor, cervical cancer, testicular tumors, testicular seminoma, bladder cancer, melanoma, and central nervous system tumors (e.g., gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, central nervous system (CNS) lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, neuroblastomas, meningiomas, retinoblastomas, and brain metastases).

[0223] According to some embodiments of the invention, the pathology is a solid tumor.

[0224] According to some embodiments of the present invention, the drugs prepared by the methods of some embodiments of the present invention have an anti-tumor effect.

[0225] The term "anti-tumor effect" as used herein refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life span, or an improvement in various physiological symptoms associated with cancerous conditions. An "anti-tumor effect" can also be manifested by the ability of the drug of the present invention to prevent tumor occurrence in the first line.

[0226] According to specific embodiments, when said affinity binding entity is for tyrosinase (TyrD), said cancer is selected from said group consisting of melanoma and glioblastoma.

[0227] According to a specific embodiment, when the affinity binding entity is for WT1, the cancer is selected from:

[0228] Table 1

[0229] leukemia Multiple Myeloma (MM) Acute lymphoblastic leukemia (ALL) Acute Myeloid Leukemia (AML) Myelodysplastic syndrome (MDS) Mesothelioma Ovarian cancer Gastrointestinal cancers, such as colorectal cancer, adenocarcinoma, and thyroid cancer Breast cancer Lung cancer (such as non-small cell lung cancer) Melanoma Osteosarcoma Endometrial cancer

[0230] According to a specific embodiment, when the affinity binding entity is for MAGE, the cancer is selected from:

[0231] Table 2

[0232]

[0233]

[0234] Table 3

[0235] MAGE-A9 Renal cell carcinoma Bladder cancer Breast cancer Primary hepatocellular carcinoma.

[0236] According to a specific embodiment, when said affinity binding entity is for PAP, said cancer is prostate cancer.

[0237] The above categories are all related to diagnosis and treatment.

[0238] Determining the presence or level of the immune complex of the present invention depends on the detectable moiety to which the antibody is attached.

[0239] Examples of detectable moieties useful in the present invention include, but are not limited to, radioisotopes, phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes, fluorescent polypeptides, and epitope tags. The detectable moiety can be a component of a binding pair that can be recognized by the interaction of the component with an additional component of the binding pair and a label that can be directly observed. In one example, the component of the binding pair is an antigen recognized by a corresponding labeled antibody. In one example, the label is a fluorescent protein or an enzyme that produces a colorimetric reaction.

[0240] Other examples of detectable moieties include those detectable by positron emission tomography (PET) and magnetic resonance imaging (MRI), all of which are known to those skilled in the art.

[0241] When the detectable moiety is a polypeptide, the immunolabel (i.e., the antibody coupled to the detectable moiety) can be produced by a variety of recombinant means, or chemically synthesized, for example, by the stepwise addition of one or more amino acid residues in a defined order using a variety of solid phase peptide synthesis techniques. Examples of polypeptide detectable moieties that can be bound to the antibodies of the invention using recombinant DNA technology (wherein the polynucleotide encoding the TCRL is transcriptionally fused to the detectable moiety) include a variety of fluorescent polypeptides, a variety of phosphorescent polypeptides, a variety of enzymes and a variety of epitope tags.

[0242] Alternatively, a chemical linkage of a detectable moiety of the antibodies of the invention may be performed using any suitable chemical bond, directly or indirectly, such as through a peptide bond (when the detectable moiety is a polypeptide), or through a covalent bond of an intermediate linking component, such as a linker peptide or other chemical moiety, such as an organic polymer. The chimeric peptides may be linked by bonding at the carboxyl (C) or amino (N) termini of the peptides, or by bonding to internal chemical groups such as linear, branched or cyclic side chains, internal carbon or nitrogen atoms, etc. One of ordinary skill in the art can readily identify and prepare the modified peptides using known peptide synthesis methods and / or covalent bonding of peptides. A description of fluorescent labeling of antibodies is provided in detail in U.S. Patent Nos. 3,940,475, 4,289,747 and 4,376,110.

[0243] An exemplary method for coupling two peptide moieties is described below:

[0244] SPDP coupling:

[0245] Any SPDP coupling method known to those skilled in the art can be used. For example, in one exemplary embodiment, a modification of the method of Cumber et al. (1985, Methods in Enzymology 112: 207-224) is used as described below.

[0246] A peptide, such as an identifiable or therapeutic moiety, (1.7 mg / mL) is mixed with a 10-fold excess of SPDP (50 mM in ethanol), the antibody is mixed with a 25-fold excess of SPDP in 20 mM sodium phosphate, 0.10 M sodium hydroxide (NaCl) pH 7.2, and each reaction is left at room temperature for, e.g., 3 hours. The reaction is then dialyzed against PBS.

[0247] The peptide is reduced, for example, with 50 mM DTT for 1 hour at room temperature. The peptide is then purified by G-25 column (up to 5% sample / column volume) in potassium dihydrogen phosphate (KH 2 PO 4 ) at pH 6.5, the reduced peptide was combined with the SPDP-antibody at a molar ratio of 1:10 antibody:peptide, and allowed to stand at 4° C. overnight to form a peptide-antibody conjugate.

[0248] Glutaraldehyde coupling:

[0249] The conjugation of a peptide (e.g., a recognizable or therapeutic moiety) to an antibody can be achieved by a number of methods known to those skilled in the art using glutaraldehyde. For example, in one exemplary embodiment, the conjugation method described below by GT Hermanson (1996 "Antibody Modification and Conjugation, in Bioconjugation Technology, Academic Press, San Diego) is used.

[0250] The antibody and the peptide (1.1 mg / ml) were mixed with a 10-fold excess of 0.1M phosphate and 0.15M sodium chloride (pH 6.8) solution with 0.05% glutaraldehyde and allowed to react at room temperature for 2 hours. 0.01M lysine may be added to block multiple excess sites. After the reaction, the excess glutaraldehyde was removed using a G-25 column equilibrated with PBS (10% v sample / column volume).

[0251] Carbodiimide coupling:

[0252] The coupling of a peptide to an antibody can be achieved by a number of methods known to those skilled in the art using a dehydrating agent such as a carbodiimide. Most preferably, the carbodiimide is used in the presence of 4-dimethylaminopyridine. As known to those skilled in the art, carbodiimide coupling can be used to form a covalent bond between a carboxyl group of a peptide and a hydroxyl group of an antibody (resulting in the formation of an ester bond), or an amino group of an antibody (resulting in the formation of an amide bond) or a thiol group of an antibody (resulting in the formation of a thioester bond).

[0253] Likewise, carbodiimide coupling can be used to form similar covalent bonds between a carbon group of an antibody and a hydroxyl, amino or sulfhydryl group of the peptide. See, generally, J. March, Advanced Organic Chemistry: Mechanism and Structure of Reactions, pp. 349-50 & 372-74 (3d ed.), 1985. By way of illustration, and not limitation, the peptide is coupled to the antibody via a covalent bond using a carbodiimide, such as dicyclohexylcarbodiimide. See generally, the multiple coupling methods of B. Neises et al. (1978, Angewandte Chemie, Int. Engl. Ed., 17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); EP Boden et al. (1986, J. Org. Chem. 50:2394) and LJ Mathias (1979, Synthesis 561). The level of the immune complex can be compared with a control sample from a non-diseased subject, wherein upregulation of immune complex formation is indicative of melanoma. Preferably, the subject is, for example, the same species as a human, preferably matched to the same age, weight, sex, etc. It should be understood that the control sample can also be from a healthy tissue of the same subject, before disease progression or after disease remission.

[0254] According to one embodiment, the detection is performed by FACS.

[0255] As described above, the affinity binding entities, eg, antibodies, of the invention may also be used in a variety of therapeutic applications, wherein the antibody comprises a therapeutic moiety.

[0256] The therapeutic moiety can be a component of the antibody, for example, in the case of a whole antibody, the Fc region or antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a cell-mediated immune defense mechanism in which a target cell whose membrane-surface antigens have been bound by specific antibodies is actively lysed by an effector cell of the immune system. This is one of the mechanisms by which antibodies act as part of the humoral immune response and can serve to limit and contain infection. Classical ADCC is mediated by natural killer (NK) cells, and macrophages, neutrophils, and eosinophils can also mediate ADCC. For example, eosinophils can kill some parasites known as helminths through IgE-mediated ADCC. Because ADCC relies on a prior antibody response, ADCC is part of the adaptive immune response.

[0257] Alternatively or additionally, the antibody may be a bispecific antibody wherein the therapeutic moiety is a T cell engager, such as an anti-CD3 antibody or an anti-CD16a, alternatively, the therapeutic moiety may be an anti-immune checkpoint molecule (anti-PD-1).

[0258] Alternatively or additionally, the antibody can be linked to a heterologous therapeutic moiety (eg, multiple conjugation methods as described above). The therapeutic moiety can be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety, a drug.

[0259] The antibody may be in a soluble or insoluble form.

[0260] Insoluble forms may be those in which a molecule comprising the CDRs of the antibody is anchored to or displayed on a cell or a particle (the latter being useful for therapeutic as well as diagnostic applications).

[0261] Examples of the plurality of cells include a plurality of immune cells, a plurality of T cells, a plurality of B cells, a plurality of dendritic cells, cytokine-induced killer cells (CIK), natural killer T cells (NKT), natural killer cells (NK) (autologous, allogeneic, xenogeneic).

[0262] According to a specific embodiment, the antibody (or indeed the CDRs of the antibody) forms a chimeric antigen receptor (CAR) (as described above) or an artificial T cell receptor. Thus, a polynucleotide encoding such a molecule is introduced into a cell of interest.

[0263] According to some embodiments of the present invention, the cell is a T cell, a natural killer cell, a cell that exerts an effector killing function on a target cell, a cell that exerts an inhibitory function on an effector T cell, a designed cell with an effector killing function, or a designed cell with an inhibitory function.

[0264] According to some embodiments of the present invention, the cell is a T cell, or an αβT cell or a γδT cell.

[0265] According to some embodiments of the invention, the cell is a natural killer (NK) cell.

[0266] According to some embodiments of the present invention, the natural killer cells are used to target cancer

[0267] According to some embodiments of the invention, the T cell is a cytotoxic T cell (effector T cell).

[0268] According to some embodiments of the invention, cytotoxic T cells (effector T cells) are used to treat multiple cancer antigens.

[0269] According to some embodiments of the invention, the cytotoxic T cells are used to treat a pathology caused by or associated with a cancer.

[0270] According to some embodiments of the present invention, the T cells include Treg cells).

[0271] According to some embodiments of the present invention, the T cells include CD3 T cells

[0272] According to some embodiments of the invention, the T cells include CD4 T cells.

[0273] According to some embodiments of the invention, the T cells include CD8 T cells.

[0274] According to some embodiments of the invention, the antigen binding domain comprises a single chain variable region fragment (scFv) molecule.

[0275] The cytoplasmic region (also referred to as "intracellular signaling region") of the CAR molecule of the present invention is responsible for at least one of the multiple normal effector functions in the immune cell, wherein the CAR has been placed in the immune cell.

[0276] The term "effector function" refers to a specific function of a cell. The effector function of a T cell, for example, can be a cytolytic activity or a helper activity comprising the secretion of the plurality of cytokines. Thus, the term "intracellular signaling region" refers to the portion of a protein, wherein the portion converts the actuator function signal and directs the cell to perform a dedicated function. Although the entire intracellular signaling region can generally be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling region is used, the truncated portion can be used to replace the complete chain as long as the truncated portion transduces the effector function signal. Thus, the term intracellular signaling region refers to any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0277] Multiple examples of multiple intracellular signaling regions used in the CAR molecules of the present invention include the multiple cytoplasmic sequences of the T cell receptor (TCR) and multiple co-receptors that synergistically trigger signal transduction after antigen-receptor engagement, as well as any derivatives or variants of the multiple sequences, and any synthetic sequences with the same functional capabilities.

[0278] It is known that the signals generated by the TCR alone are not sufficient to fully activate the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: those that provide an antigen-dependent primary activation method triggered by the TCR (primary cytoplasmic signaling sequences) and those that provide a secondary or co-stimulatory signal in an antigen-independent manner (secondary cytoplasmic signaling sequences).

[0279] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0280] Examples of ITAMs with multiple major cytoplasmic signal sequences used in the present invention include those derived from TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. It is particularly preferred that the cytoplasmic signal molecule in the CAR of the present invention includes a cytoplasmic signal sequence derived from CD3 zeta.

[0281] In a preferred embodiment, the cytoplasmic region of the CAR can be designed to include the CD3-zeta signal region itself or in combination with any other desired (multiple) cytoplasmic regions in the context of the CAR applicable to the present invention. For example, the cytoplasmic region of the CAR may include a CD3 zeta chain portion and a costimulatory signal region. The costimulatory signal region refers to a portion of the CAR that includes the intracellular region of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than a ligand of an antigen receptor required for an effective response of an antigen receptor or multiple lymphocytes to an antigen. Multiple examples of the multiple molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand specifically bound to CD83, etc. Therefore, although the present invention mainly uses 4-1BB as the costimulatory signal component, other costimulatory components are within the scope of the present invention.

[0282] According to some embodiments of the present invention, the intracellular region includes a co-stimulatory signal region and a zeta chain portion. The co-stimulatory signal region refers to the intracellular region in which a portion of the CAR molecule includes a co-stimulatory molecule. Multiple co-stimulatory molecules are multiple cell surface molecules other than ligands of multiple antigen receptors required for an effective response of multiple antigen receptors or multiple lymphocytes to antigens.

[0283] As used herein, a "costimulatory ligand" includes a molecule on an antigen presenting cell [e.g., an aAPC (artificial antigen presenting cell), a dendritic cell, a B cell, etc.] that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of a TCR / CD3 complex to an MHC molecule to which a peptide is added. A co-stimulatory ligand may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM-1), CD 30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a receptor of Toll ligand, and a ligand that specifically binds to B7-H3. A co-stimulatory ligand also includes, in particular, an antibody that specifically binds to a co-stimulatory molecule expressed on a T cell, such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, light, NKG2C, B7-H3 and a ligand that specifically binds to CD83.

[0284] A "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Multiple costimulatory molecules include, but are not limited to, a class I MHC molecule, BTLA, and a Toll ligand receptor.

[0285] As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in T cell proliferation and / or up-regulation or down-regulation of multiple key molecules.

[0286] The term "stimulation" refers to a primary response, triggered by a stimulatory molecule (e.g., a TCR / CD3 complex) binding to its cognate ligand, which is mediated by a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate the altered expression of some molecules, such as downregulation of TGF-β, and / or the reorganization of multiple cytoskeletal structures, etc.

[0287] A "stimulatory molecule," as the term is used herein, refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand displayed on an antigen presenting cell.

[0288] A "stimulatory molecule", as the term is used herein, refers to a ligand that, when presented on an antigen presenting cell (e.g., aAPC, a dendritic cell, a B cell, etc.), can specifically bind to a cognate binding partner on a T cell (referred to herein as a "stimulatory molecule"), through which the T cell mediates a primary response, including but not limited to, activation, initiation of an immune response, proliferation, etc. A variety of stimulatory ligands are known in the art, and include, among others, a class I MHC molecule coupled to a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0289] Regarding the cytoplasmic region, the CAR molecule of some embodiments of the present invention can be designed to include the signal region of the CD28 and / or 4-1BB itself or to be combined with any other desired (multiple) cytoplasmic regions in the context of the CAR applicable to the present invention. It is useful in the environment of the CAR molecule of some embodiments of the present invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further include the signal region of CD3-zeta. For example, the cytoplasmic region of the CAR can include but is not limited to multiple signal modules and multiple combinations of CD3-zeta, 4-1BB and CD28.

[0290] According to some embodiments of the present invention, the intracellular region includes at least one, for example, at least two, at least three, at least four, at least five, for example, at least six of the multiple polypeptides selected from the group consisting of CD3ζ (CD247, CD3z), CD28, 41BB, ICOS, OX40 and CD137.

[0291] According to some embodiments of the invention, the intracellular region includes the CD3ζ-chain [CD247 molecule, also known as "CD3-zeta" and "CD3z"; Genbank Accession Nos. NP_000725.1 and NP_93170.1], which is the main transmitter of signals from multiple endogenous TCRs.

[0292] According to some embodiments of the present invention, the intracellular region includes various co-stimulatory protein receptors in the cytoplasmic tail of the CAR to provide additional multiple signals to the T cells (second-generation CAR) examples include but are not limited to CD28 [e.g., Genbank accession numbers NP_001230006.1, NP_001230007.1, NP_006130.1], 4-1BB [tumor necrosis factor receptor superfamily, member 9 (TNFRSF 9), also known as "CD137", e.g., Genbank accession number NP_001552.2], and ICOS [inducible T cell co-stimulator, e.g., Genbank accession number NP_036224.1]. Multiple preclinical studies have shown that the second-generation CAR design improves the anti-tumor activity of multiple T cells.

[0293] According to some embodiments of the present invention, the intracellular region includes multiple signal regions, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance efficacy. The term "OX40" refers to tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), such as Genbank accession number NP_003318.1 (multiple "third generation" CARs).

[0294] According to some embodiments of the present invention, the intracellular region includes CD28-CD3z, CD3z, CD28-CD137-CD3z. The term "CD137" refers to tumor necrosis factor receptor superfamily, member 9 (TNFRSF9), such as Genbank accession number NP_001552.2.

[0295] According to some embodiments of the present invention, when the CAR molecule is designed for a natural killer cell, the signal region may be CD28 and / or CD3ζ.

[0296] The transmembrane region may be obtained from a natural or synthetic source. In the case where the source is natural, the region may be derived from any membrane-bound or transmembrane protein. The multiple transmembrane regions of specific use in the present invention may be derived from the alpha, beta or zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 of the T cell receptor (i.e., at least including the (multiple) transmembrane regions). Alternatively, the transmembrane region may be synthetic, in which case the transmembrane region mainly includes hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane region. Alternatively, a short oligo- or polypeptide connector, preferably between 2 and 10 amino acids in length, can form the connection between the transmembrane region and the cytoplasmic signal region of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0297] According to some embodiments of the present invention, the transmembrane region included in the CAR molecule of some embodiments of the present invention is a transmembrane region naturally associated with one of the multiple regions of the CAR. According to some embodiments of the present invention, the transmembrane region can be screened or modified by amino acid substitution to avoid the multiple regions from binding to the multiple transmembrane regions of the same or different surface membrane proteins to minimize multiple interactions with other members of the receptor complex.

[0298] According to some embodiments, between the extracellular region and the transmembrane region of the CAR molecule, or between the cytoplasmic region and the transmembrane region of the CAR molecule, a spacer region may be included. As used herein, the term "spacer region" generally refers to any oligo- or polypeptide, which links the transmembrane region to the extracellular region or the cytoplasmic region in the polypeptide chain. A spacer region may include up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0299] According to an aspect of some embodiments of the present invention, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to said subject said affinity binding entity, thereby treating said cancer in said subject.

[0300] Also provided is a use of said affinity binding entity, as defined herein, in the preparation of a medicament for treating a pathology, such as cancer.

[0301] The selection of the TCRL will naturally depend on the performance of the TCRL in pathologies. A number of exemplary TCRLs and the association of the TCRL with a number of pathologies are provided in the table above.

[0302] The term "treat" refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and / or causing the reduction, alleviation or return of a pathology. Those skilled in the art will appreciate that various methods and tests can be used to obtain the development of a pathology, and similarly, various methods and assays can be used to obtain the reduction, alleviation or return of a pathology.

[0303] As used herein, the term "subject" includes a variety of mammals, preferably humans at any age suffering from the pathology.

[0304] The antibodies of some embodiments of the present invention may be administered to an organism per se, or in a pharmaceutical composition mixed with suitable carriers or excipients.

[0305] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0306] The term "active ingredient" herein refers to the antibody responsible for the biological effect.

[0307] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to indicate a carrier or a diluent that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0308] The term "excipient" herein refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starch types, various cellulose derivatives, gelatin, vegetable oils, and various polyethylene glycols.

[0309] Techniques for the preparation and administration of various drugs may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., latest edition, which is incorporated herein by reference.

[0310] A variety of suitable routes of administration may, for example, include oral, rectal, mucosal, especially nasal, enteral or parenteral administration, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal injections, directly into the cardiac cavity, intracardiac, for example, into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal or intraocular injections.

[0311] A variety of conventional approaches are used to deliver drugs to the central nervous system (CNS) including: neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular manipulation of the agent (e.g., preparation of a chimeric fusion protein comprising a transport peptide having an affinity for an endothelial cell surface molecule to bind an agent that cannot itself cross the blood-brain barrier (BBB)) in an attempt to exploit one of the BBB's endogenous transport pathways; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., coupling water-soluble agents to lipid or cholesterol carriers); and temporary disruption of the integrity of the BBB by hyperosmotic disruption (by injection of a mannitol solution into the carotid artery or the use of a bioactive agent such as angiotensin). However, each of the strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by limitations inherent in the endogenous delivery system, potential undesirable biological side effects associated with systemic administration of a chimeric molecule comprising a carrier motif that may be activated outside the CNS, and the potential risk of brain damage in areas where the BBB of the brain is disrupted, making this approach a less preferred method of delivery.

[0312] Alternatively, the pharmaceutical composition may be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue area of ​​a patient.

[0313] The term "tissue" refers to a part of an organism designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, liver tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, heart tissue, brain tissue, vascular tissue, kidney tissue, lung tissue, gonadal tissue, hematopoietic tissue.

[0314] The pharmaceutical compositions of some embodiments of the present invention can be prepared by methods known in the art, for example, by conventional mixing, dissolving, granulating, sugar-coating, spraying, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0315] Therefore, the multiple pharmaceutical compositions used according to some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, wherein the carriers contain multiple excipients and multiple auxiliaries, and the multiple excipients and multiple auxiliaries facilitate the process of preparing the active ingredients for pharmaceutical use, and the appropriate formulation depends on the selected route of administration.

[0316] For injection, the active ingredients of the pharmaceutical composition can be formulated into aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution or physiological saline buffer. For mucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0317] For oral administration, the pharmaceutical composition can be easily formulated by combining the multiple active compounds with multiple pharmaceutically acceptable carriers known in the art. The multiple carriers allow the pharmaceutical composition to be formulated into tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a patient. Multiple pharmaceutical preparations for oral administration can be prepared using a solid excipient, optionally grinding the resulting mixture, and if necessary, processing into multiple tablets or multiple sugar-coated cores after adding multiple suitable excipients. Multiple suitable excipients, in particular, fillers such as multiple sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium methylcellulose, and / or multiple physiologically acceptable polymers such as polyvinyl pyrrolidone (PVP). If necessary, multiple disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or salts of the alginic acid such as sodium alginate can be added.

[0318] A plurality of sugar coating cores have a plurality of suitable coatings.For this purpose, a plurality of concentrated sugar solutions can be used, and the plurality of concentrated sugar solutions can optionally contain gum arabic, talcum (talc), polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution and suitable organic solvent or a plurality of solvent mixtures.A plurality of dyes or a plurality of pigments can be added into the plurality of tablets or the sugar coating, for identifying or characterizing the different combinations of a plurality of active compound dosages.

[0319] Pharmaceutical compositions for oral administration include push-in capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-in capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate and, optionally, a stabilizer. In the soft capsules, the active ingredients may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin or a variety of liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should have a dosage suitable for the selected route of administration.

[0320] For oral administration, the composition may take the form of tablets or lozenges formulated in conventional manner.

[0321] For inhalation through the nose, the multiple active ingredients used according to some embodiments of the present invention are conveniently delivered in the form of an aerosol spray from a pressurized package, or using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be measured by providing a valve for delivering a metered amount. For example, gelatin can be formulated into a powder mixture containing the compound and a suitable powder base such as lactose or starch for capsules and boxes for a dispenser.

[0322] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by intravenous injection or continuous infusion. Multiple formulations for injection can be presented in unit dosage form, for example in ampoules or multiple multi-dose containers, with, optionally, an added preservative. The multiple compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain multiple formulating agents, such as suspending agents, stabilizers and / or dispersants.

[0323] A plurality of pharmaceutical compositions for parenteral administration include a plurality of aqueous solutions of the active agent in a water-soluble form. Additionally, the suspension of the plurality of active ingredients can be prepared as a suitable oily or water-based injection suspension. A variety of suitable lipophilic solvents or a variety of carriers include a variety of mineral oils such as sesame oil, or a plurality of synthetic fatty acid esters such as ethyl oleate, a variety of triglycerides or a variety of liposomes. Aqueous injection suspensions can contain a variety of substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain a plurality of suitable stabilizers or agents that increase the solubility of the plurality of active ingredients to allow the preparation of the plurality of highly concentrated solutions.

[0324] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, a sterile, pyrogen-free, water-based solution, before use.

[0325] The pharmaceutical compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, for example, with conventional suppository bases such as cocoa butter or other glycerides.

[0326] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention comprise a plurality of ingredients, wherein the plurality of active ingredients are included in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount refers to an amount of the plurality of active ingredients (TCRL antibodies) that is effective to prevent, alleviate or ameliorate the symptoms of a disease (e.g., cancer) or prolong the survival of the subject being treated.

[0327] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0328] For any formulation used in the methods of the invention, the therapeutically effective amount or dose can be initially estimated from a variety of in vitro and cell culture assays. For example, a dose can be formulated in a variety of animal models to obtain a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0329] The toxicity and therapeutic efficacy of the multiple active ingredients described herein can be determined by multiple standard pharmaceutical processes in vitro, in multiple cell cultures or multiple experimental animals. The data obtained from the multiple in vitro and cell culture tests and animal studies can be used to formulate a range of dosages for humans. The dosage can vary depending on the dosage form used and the route of administration used. The exact formulation, route of administration and dosage can be selected by the individual physician based on the patient's condition. (See, e.g., Fingl et al., 1975, in "Pharmacological Basis of Drugs", Chapter 1, page 1).

[0330] The number and interval of doses can be adjusted individually to provide levels of TCRL (the TCRL tissue) in the active ingredient sufficient to induce or inhibit the biological effect (minimum effective concentration, MEC). The MEC used in each formulation will be different, but can be estimated from in vitro data. The dose required to achieve the MEC will depend on individual characteristics and route of administration. Multiple detection assays can be used to determine multiple plasma concentrations.

[0331] Depending on the severity and responsiveness of the condition to be treated, administration may be a single or multiple administrations, continuing for a duration ranging from several days to several weeks, or until a cure or therapeutic disease state is achieved.

[0332] Of course, the amount of a composition administered will depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0333] If desired, multiple compositions of some embodiments of the present invention may also be provided in a package or a dispenser device, such as an FDA-approved kit (diagnostic or therapeutic), which may contain one or more unit dosage forms containing the active ingredients. The package may, for example, include metal or plastic foil, such as a blister pack. The package or dispenser device may be accompanied by multiple instructions for administration. The package or dispenser may also be accompanied by a notice associated with the container, which is in the form of a drug regulated by a government agency for manufacture, use or sale, and the notice reflects the approval of the multiple ingredients or the form by human or veterinary regulatory agencies. For example, the notice may be an approved product insert for multiple prescription drugs or multiple prescription drugs approved by the U.S. Food and Drug Administration. Multiple compositions including a formulation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as described in further detail above.

[0334] It is expected that during the patent life of the present application maturing, many related TCRLs will be developed; the scope of the term TCRLs is intended to include all similar prior art.

[0335] As used herein, the term "about" refers to ± 10%.

[0336] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0337] The term "consisting of" means "including and not limited to".

[0338] The term “essentially consisting of” means that the composition, method or structure may include additional ingredients, steps and / or components, but only if the additional ingredients, steps and / or components do not substantially alter the basic and novel characteristics of the claimed composition, method or structure.

[0339] As used herein, the singular forms "a", "an", and "at least one" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0340] Throughout this application, various embodiments of the present invention may be presented in the form of a range. It should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a hard limitation on the scope of the present invention. Therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range.

[0341] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integer) within the indicated range. The terms, "range between" a first indicated numeral and a second indicated numeral, and "range from" a first indicated numeral to" a second indicated numeral" are interchangeable herein and are meant to include the first and second indicated numerals, and all fractions and integers therebetween.

[0342] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a particular task, including but not limited to those manners, means, techniques and procedures that are known or readily developed by practitioners in the chemical, pharmacological, biological, biochemical and medical fields from known manners, means, techniques or procedures.

[0343] As used herein, the term "treating" includes halting, substantially inhibiting, slowing or reversing the progression of a disorder, substantially ameliorating the clinical or psychological symptoms of a disorder or substantially preventing the appearance of the clinical or psychological symptoms of a disorder.

[0344] When reference is made to multiple specific sequence listings, the reference will be understood to also include multiple sequences that substantially correspond to the complementary sequence of the reference, including multiple minor sequence changes due to, for example, multiple sequencing errors, multiple cloning errors or other changes caused by base substitutions, base deletions or base additions, and the frequency of the multiple changes is less than one in fifty nucleotides, less than one in one hundred nucleotides, or, alternatively, less than one in two hundred nucleotides, less than one in five hundred nucleotides, or less than one in a thousand nucleotides, or, less than one in five thousand nucleotides, or, less than one in ten thousand nucleotides.

[0345] It will be appreciated that certain features of the invention, which for the sake of clarity are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for the sake of clarity are described in the context of a single embodiment, may also be provided individually or in any suitable subcombination or in any other described implementation suitable for the invention. Some features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment does not function without those components.

[0346] Various embodiments and aspects of the present invention are described in the following examples and find experimental support as claimed in the claims section below.

[0347] Multiple Examples

[0348] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0349] Generally, the terms used herein and the laboratory methods used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. The multiple techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I to III, Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", American Science Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Volumes I to IV, Cold Spring Harbor Laboratory Press, New York (1998); in U.S. Patent Nos. 4,666,828; 4,683 ,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Manual", Volumes I to III, Cellis, JE, ed. (1994); "Animal Cell Culture - A Manual of Basic Techniques" by Freshney, Wiley-Liss, New York (1994), Third Edition; "Current Protocols in Immunology", Volumes I to III, Coligan J.E., ed. (1994); Stites et al. (eds), "Basic Immunology and Clinical Immunology" (Eighth Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Multiple Selection Methods in Cellular Immunology", WH Freeman and Co., New York (1980); Existing immunoassays are widely described in patents and scientific literature.See, e.g., U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1986). SJ, eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "Molecular Cloning - A Practical Guide" Perbal, B., (1984) and "Methods in Enzymology" Volume 1-317, Academic Press; "PCR Protocol: A Guide to Methods and Applications", Academic Press, San Diego, California (1990); Marshak et al., "Protein Purification and Identification Strategies - A Laboratory Course Manual" CSHL Press (1996); All of the above are incorporated herein by reference. Other general references are provided in this document. The various steps described in the various documents are believed to be known in the art and are provided for the convenience of the reader. All information contained herein is incorporated herein by reference.

[0350] Multiple commonly used materials and methods

[0351] Preparation of biotinylated single-chain MHC-peptide complexes

[0352] Multiple single-chain MHC (scMHC) 3 The scMHC-peptide complex was generated by in vitro refolding from multiple inclusion bodies in Escherichia coli under the induction of isopropyl β-D-thiogalactoside (IPTG). 2The extracellular regions of the microglobulin and the HLA-A2 gene are linked to each other via a flexible linker, and the scMHC is designed to contain a BirA recognition sequence for site-specific biotinylation at the C-terminus (scMHC-BirA). In vitro refolding is performed in the presence of a peptide as described. Correctly folded MHC-peptide complexes are separated and purified by anion exchange Q-Sepharose chromatography (GE Healthcare Life Sciences) and then site-specifically biotinylated using the BirA enzyme (affinity) for the production of single-chain MHC-peptide complexes. A more detailed description is provided in Denkberg et al. (2002) PNAS, 99: 9421-9426.

[0353] Flow cytometry

[0354] Multiple TB hybridized T2 cells were washed with serum-free RPMI 1640 medium and then washed with 10 -4 Up to 10 -5 Molar concentration of tyrosinase 369-377 YMDGTMSQV (SEQ ID NO: 1) / WT1 126-134 (RMFPNAPYL, SEQ ID NO: 141) peptide / MAGE-A4 230-239 SEQ ID NO: 176 / MAGE-A9 223-231 203 / PAP 112-120 The culture medium containing the peptide of SEQ ID NO: 230 or related control peptides (listed in Table 15 below) was incubated overnight. The ratio between the MFI of HLA-A2-binding antibody BB7.2 on T2 cells loaded with peptides and the MFI of unloaded T2 cells (>1) was used to verify the peptide loading efficiency, data not shown.

[0355] Multiple T2 or primary cells or cell lines (10 6 ) were incubated with 10 μg / mL of specific antibody (Ab) (with or without biotinylation) at 4°C for 1 hour, followed by incubation with PE-labeled anti-mouse / human / streptavidin Ab at 4°C for 45 minutes. It should be understood that anti-mouse secondary antibodies or streptavidin work to obtain similar results as D11 and B47B6. Finally, the cells were washed and analyzed:

[0356] FACS 1:

[0357] Machine:BD FACS calibur

[0358] Analysis software:CELLQuest

[0359] FACS 2:

[0360] Machine:Beckman Coulter NAVIOS

[0361] Analysis software: Kaluza version 1.3

[0362] Production of HLA-A2 / Tyrosinase 369-377 / WT1 using the hybridoma technology 126-134 / MAGE-A4 230-239 / MAGE-A9 223-231 / PAP 112-120 Multiple TCR-like antibodies

[0363] HHD mice were immunized by 5-6 injections of 50 μg / mouse of HLA-A2-peptide complexes. The first 2-3 injections were administered subcutaneously with QuilA adjuvant. Hybridoma clones were generated by fusion of spleen cells isolated from mice immunized with the complexes with NSO myeloma cells (as described above Weidanz et al. 2011, Int. Rev. Immunol. 30:328-340) and screened and isolated by differential ELISA assays as described below. For example, for screening of tyrosinase TCRL, the relevant TyrD369-377 peptide HLA-A2 complexes were used and compared with the irrelevant p68-DDX5 control peptide (SEQ ID NO: 2YLLPAIVHI) HLA-A2 complexes. ELISA with purified HLA-A2-Tyr complexes and control HLA-A2 complexes showing other HLA-A2-restricted peptides (Table 15) was used to screen for specific clones, and isolated hybridoma clones were subcloned and sequenced. Two clones, 906-11-D11 (referred to as D11, Fig.68 ) and 905-2-D7 (referred to as D7, Fig.69 ) is described.

[0364] Hybridomas were grown in HAT DMEM or serum-free DCCM2 medium until confluence>80% and supernatants were collected. Purified IgG was isolated from the culture supernatant by affinity chromatography using a protein A column. SDS-PAGE analysis of the purified protein showed homogeneous, pure IgG with an expected molecular weight of about 150 kilodaltons.

[0365] Construction of the entire IgG Ab

[0366] The H and L Fab genes (only for MC1) were cloned for expression as human IgG1κAb into the eukaryotic expression vectors pOptiVEC and pcDNA3.3-TOPO, respectively. Each shuttle expression vector carried a different gene selection (DHFR / HT- for pOptiVEC and Geneticin for pcDNA3.3). Expression was promoted by co-transfection of the two constructs into the dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) derived from DG44 cells in suspension culture using the FreeStyle MAX reagent (Invitrogen). After co-transfection, cells were cultured in selective medium. Clones that reacted specifically to JY T2 cells with the tyrosinase 369-377 peptide were adjusted to grow in 0.5% serum and further purified using protein A affinity chromatography. SDS-PAGE analysis of the purified protein showed homogeneous, pure IgG with an expected molecular weight of ~150 kilodaltons.

[0367] ELISA with supernatant or purified multiple Abs

[0368] The binding specificity of each supernatant or purified TCRL antibodies was determined by ELISA using biotinylated scMHC-peptide complexes. Maxi sorp 96-well ELISA plates (Nunc #442404) were coated with BSA-biotin (1 μg / well) overnight. After being washed, the plates were primed with streptavidin (1 μg / well) (1 hour, room temperature), washed extensively, and primed with 0.25 μg of MHC / peptide complex (1 hour, room temperature). The plates were blocked with PBS / 2% skim milk for 30 minutes at room temperature, followed by 1 mg / well of supernatant or purified TCRL antibodies for 1 hour at room temperature. After washing, the plates were primed with HRP-conjugated / anti-human or mouse Ab. The multiple HLA-A2-restricted peptides were used for multiple specificity tests of the purified supernatant or purified multiple TCRL antibodies using TMB tetramethylbenzidine reagent (DAKO, S1599) for detection.

[0369] Proteon XPR36 Surface Plasmon Resonance (SPR) Binding Analysis

[0370] Immobilization of IgG class TCR antibodies was performed on a Generic Layer Medium (GLM) chip (Bio-Rad Laboratories, Hercules, CA, USA) in a vertical orientation at 25°C with a continuous running buffer of PBST (10 mM sodium phosphate, 150 mM sodium chloride, 0.005% Tween 20, pH 7.4). Five channels were activated with 50 μl of a mixture of 0.04 M N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.01 M N-hydroxysulfosuccinimide (Sulfo-NHS) at a flow rate of 30 μl / min, the anti-mouse or human IgG / NeutrAvidin was diluted in 10 mM sodium acetate buffer pH 4.5 to a final concentration of 25 μg / ml, and 150 μl was injected followed by an injection of 150 μl of 1 M ethanolamine hydrochloride pH 8.5. The IgG TCRL antibody / purified single-chain recombinant HLA-A2 / tyrosinase / WT1 / MAGE-A4 / MAGE-A9 / PAP complex ligand was diluted to 5-10 μg / ml in PBST, and 90 μl was injected vertically at a flow rate of 30 μl / min. The sixth channel remained empty as a reference. The analyte purified single-chain recombinant HLA-A2 / tyrosinase / WT1 / MAGE-A4 / MAGE-A9 / PAP complex / Fab TCRL antibody was injected (75 μl at 50 μl / min) in the horizontal direction of the ProteOn using five different concentrations (1000, 500, 250, 125 and 625 nanomolar concentrations), and running buffer was injected simultaneously in the sixth channel for double reference to correct for the loss of the multiple captured antibodies from the chip sensor surface during the experiment. All binding sensorgrams were collected, processed and analyzed using the integrated ProteOn Manager (Bio-Rad Laboratories, Hercules, USA) software. Multiple binding curves were fit using the Langmuir model describing 1:1 binding stoichiometry, or using the Langmuir and mass-shift limited models.

[0371] Multiple functional analyses

[0372] LDH-release test

[0373] In a non-radioactive cytotoxicity assay using (Promega) assay for bispecific TCRL retargeting of target cell killing, the test quantitatively measures lactate dehydrogenase (LDH), an enzyme released upon cell lysis. Released LDH is measured in culture supernatants in a 10-minute coupled enzymatic assay, resulting in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of color produced is proportional to the number of lysed cells.

[0374] Specifically, multiple target cells and multiple effector cells were washed, counted and resuspended in phenol red-free cRPMI medium (1% FBS). Multiple target cells were adjusted to a cell density of 2.5×10 per ml. 5 cells, and the plurality of reaction cells are reacted to a cell density of 2.5X10 per ml. 6 cells. 40 μl (1×10 4 Multiple target cells (cells) were cultured in a 96-well V-shaped plate. A five-fold concentrate of the bispecific TCRL test reagent was prepared at the highest tested concentration, and the concentrate was serially diluted one-tenth in medium without phenol red to obtain other tested concentrations. The bispecific TCRL was then added to the multiple target cells in the test plate at 20 μl per well to give multiple final indicated titers. The test plate containing the multiple target cells was then mixed with the bispecific TCRL and incubated at 37°C / 5% CO 2 After 20 minutes of incubation, 40 μl of multiple effector cells (1×10 5 Cells) were added to each well to produce an effector at a 10:1 (E:T) ratio, control wells were individually set up with effector cells to calculate target spontaneous release, and target cells with a final 80 μg / mL of digitalis to calculate maximal release. Each condition was assayed in triplicate in a final volume of 100 μL. The plates were incubated at 37°C / 5% CO 2 After the incubation period, the plates were centrifuged at 700 x g for 5 minutes and 50 μl was transferred from each well to the corresponding well in a 96-well flat-bottom Maxisorb plate (Nunc) using a 4% ethanol flask according to the manufacturer's instructions. The assay buffer was reconstituted as described The matrix mixture was added and 50 microliters was added to each well of the plate. The plate was covered with aluminum foil and left at room temperature for 10 minutes. The absorbance at 490 nanometers was then recorded in a plate reader. The percentage of cytotoxicity was then calculated using the following formula: specific lysis = [(experimental - spontaneous effector - target effector) / (target maximum - target spontaneous value)] x 100. The multiple PBMCs used for the killing assay were isolated from multiple healthy volunteers and had multiple approvals and multiple written consents from all regulatory IRBs. Multiple effector PBMCs were isolated using the Lymphoprep technology.

[0375] Multiple tumor cell lines and multiple normal primary cells

[0376] Cell lines A375 (melanoma), U2OS (osteosarcoma), TCCSUP (bladder cancer) and Fib (fibroblasts) were cultured in complete DMEM supplemented with 10% FBS (all provided by GIBCO). 501A, SKMel5, Mewo and 1938 (melanoma), Saos2 (osteosarcoma), Pancl (pancreatic cancer), J82 and UMUC3 (bladder), H1703 (non-small cell lung adenocarcinoma), JVM2 (hemoglobin lymphoma), IM9 (multiple myeloma), U266 (myeloma) and SW620 (colorectal cancer) were cultured in complete RPMI supplemented with 10% FBS (all provided by GIBCO). Malme3m (melanoma), JEKO1 (mantle cell lymphoma), SET2 (essential platelet inhibitor) and BV173 (B cell precursor leukemia) were cultured in complete RPMI supplemented with 20% FBS (all provided by GIBCO), thp-1 (AML) was cultured in complete RPMI supplemented with 10% FBS (all provided by GIBCO) and 0.05 mM beta-mercaptoethanol (provided by Thermo-fisher). OVCAR-3 (ovarian adenocarcinoma) was cultured in complete RPMI supplemented with 20% FBS and 0.01 mg / ml bovine insulin (provided by Sigma). All cell lines were maintained at 37°C in a humidified atmosphere of 7.5% CO2 and were purchased from the American Type Culture Collection.

[0377] Normal primary hepatocytes, cardiac myocytes, osteoblasts, astrocytes, bronchial epithelial cells, colon smooth muscle cells, mesothelial cells, and renal epithelial cells were obtained from Sciencell and cultured according to the manufacturer's instructions. All cell lines were maintained at 37°C in a humidified atmosphere of 7.5% CO2.

[0378] Expression and purification of multiple soluble recombinant Fab Abs in the Expi293 system

[0379] The VH-CH1 and VL-CL genes of TyrD11 and D7, MAGE-A4 C106B9, WT1 B47B6 and ESK1 IgGs were cloned in the eukaryotic expression vector pcDNA3.4 for expression as Fab, and a His-tag was linked to the C-terminus of the CH1 region.

[0380] The two constructs (heavy chain and light chain) were co-transfected into the multiple Expi293F human cells (both of which are multiple components of the Expi293 expression system) in the Expi293 expression culture medium by the Fectamine transfection reagent (Life technologies) to promote expression. After co-transfection, multiple cells were cultured for 6 days. After 6 days, multiple cells were centrifuged at 700x g for 5 minutes. After centrifugation, the supernatant containing D11, D7, C106B9, B47B6 or ESK1 Fab was removed from multiple cells and passed through a 0.22μ filter. The supernatant was then dialyzed overnight with PBS.

[0381] The D11, D7, C106B9, B47B6 or ESK1 Fab recombinant protein was purified by metal affinity column (Talon) and dialyzed against PBS overnight. The purified D11, D7, C106B9, B47B6 or ESK1 Fab was analyzed on reducing and non-reducing SDS-PAGE.

[0382] Construction, expression and purification of multiple bispecific TCRLs in the Expi 293 system

[0383] The VH-CH1 and VL-CL genes of TyrD11 and D7, WT1 B47B6 and ESK1 and MAGE-A4 C106B9, IgGs were cloned into the eukaryotic expression vector pcDNA3.4 for use as bispecific (BS) in (multiple sequences such as Figures 68-70, multiple sequences of ESK1 can be obtained from WO 2015 / 0761). For the light chain vectors of Tyr D11, WT1 B47B6 and ESK1 and MAGE-A4 C106B9, anti-CD3 (clone UCHT1) scFv was linked to the N-terminus of the VL region (BS format 3, #F3). For the heavy chain vector, a His-tag was linked to the C-terminus of the CH1 region. For TyrD7, anti-CD3 (clone UCHT1) scFv was linked to the N-terminus of the VH region of the heavy chain (BS format 1, #1) and a His-tag was linked to the C-terminus of the CH1 region.

[0384] The two constructs were co-transfected in the Expi293F human cells (both of which are multiple components of the Expi293 expression system) in the Expi293 expression culture medium by the Fectamine transfection reagent (Life technologies) to promote expression. After co-transfection, multiple cells were cultured for 6 days. After 6 days, multiple cells were centrifuged at 700x g for 5 minutes. After centrifugation, the supernatant containing the multiple TCRL bispecific antibodies was removed from multiple cells and passed through a 0.22 micron filter. The supernatant was then dialyzed overnight with PBS.

[0385] The recombinant proteins of the BS-TCRL were purified in two steps by metal affinity (Talon) and size exclusion chromatography (Superdex 200 10 / 300 GL GE). The purified BS-TCRL were analyzed on SDS-PAGE.

[0386] Multiple in vivo tests

[0387] For 501A melanoma cell line (ATCC, Manassas, VA, USA)

[0388] Cells were cultured in RPMI 1640 growth medium (GIBCO, Waltham MA, USA) supplemented with 10% fetal bovine serum (GIBCO, Waltham MA, USA).

[0389] Human peripheral blood mononuclear cells (PBMCs) were prepared from multiple healthy donors using multiple SepMateTM-50 tubes (Stemcell).

[0390] On day 0, female NOD / SCID mice (Envigo, Israel; n=6-8) aged 8-10 weeks were injected subcutaneously (sc) on one side with 5×10 6501A melanoma cells, + / -25x10 6 PBMCs (effector:tumor cell ratio 5:1) in a final volume of 0.25 ml phosphate buffered solution (PBS); D7 bispecific TCRL (0.1 mg / kg) or vehicle (PBS) was administered intravenously (iv) in a final volume of 0.2 ml 1 hour after sc injection, including four additional doses, administered every 24 hours.

[0391] For A375 melanoma cell line (ATCC, Manassas, VA, USA)

[0392] Cells were cultured in RPMI 1640 growth medium (GIBCO, Waltham MA, USA) supplemented with 10% fetal bovine serum (GIBCO, Waltham MA, USA).

[0393] Multiple activated CD8 T cells were prepared from human peripheral blood mononuclear cells (PBMC) using a rapid expansion protocol (REP) and multiple non-activated CD8 T cells were prepared from the peripheral blood of healthy donors using the SepMateTM-50 tube (Stemcell). PBMCs were then used UntouchedTM Human CD8 T Cell Kit (Invitrogen) was used to enrich CD8 T cells. The purified CD8 T cells were activated in Erlenmeyer flasks pre-coated with monoclonal antibodies against CD3 (OKT3) and CD28 for 72 hours in medium supplemented with 10% FBS and 100 IU / mL human IL-2. The activated cells were then cultured in medium supplemented with 10% FBS, 3000 IU / mL IL-2, 30 mg / mL OKT3, and 2×10 8 of irradiated PBMCs.

[0394] On day 0, female NOD / SCID mice (Envigo, Israel; n = 6-8) aged 8-10 weeks were injected subcutaneously (sc) on one side with 5×10 6 A375 melanoma cells, + / -10x10 6 REP CD8 T cells (effector:tumor cell ratio 2:1) were administered in a final volume of 0.25 ml of phosphate buffered solution (PBS); MAGE-A4 C106B9 bispecific TCRL (0.1 mg / kg), WT1 B47B6 bispecific TCRL (0.1 mg / kg) or vehicle (PBS) was administered intravenously (iv) in a final volume of 0.2 ml 1 hour after sc injection, including four additional doses, administered every 24 hours.

[0395] In both cases (501A and A375) tumors were measured twice weekly with vernier calipers in two perpendicular dimensions and tumor volume was calculated using the following formula:

[0396] Other TCRL antibodies used in this study

[0397] The production of MC1 is described in WO 2008 / 120202.

[0398] The production of ESK1 (Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T, Scott A, Whitten J, Maslak P, Casey E, Tan T, Liu H, Zakhaleva V, Curcio M, Doubrovina E, O'Reilly RJ, Liu C, Scheinberg DA. Targeting intracellular WT1 oncogene products with therapeutic human antibodies. Science Translational Medicine. 2013 Mar 13;5(176):176ra33). Therefore, ESK1 was produced by synthetic gene synthesis according to the disclosed sequence WO 2015 / 07761, ESK1 full-length VH-SEQ ID NO: 128 and ESK1 full-length VL-SEQ ID NO: 130 in the sequence listing of WO2015 / 070061, and the antibody was produced as IgG in multiple HEK293 cells using the Expi293 system described above, and purified from multiple culture supernatants by affinity chromatography using a protein A column.

[0399] Extraction of multiple nucleic acids

[0400] RNeasy Plus Mini (Qiagen) was used from 1*10 according to the manufacturer's instructions. 6 Up to 5*10 6 Total RNA was extracted from cultured cells.

[0401] cDNA synthesis

[0402] According to the manufacturer's instructions cDNA was synthesized from 1-5 μg of RNA using the III First-Strand Synthesis System (Invitrogen) using a combination of oligo dT and random hexamers (1:1). 2 Dilute the cDNA to 1:5.

[0403] Conventional PCR (PCR)

[0404] The PCR cycling conditions were 2 min at 95° C., followed by 40 cycles of 20 sec at 95° C., 1 min at 60° C., and 1 min at 72° C. The PCR ended with a final extension of 10 min at 72° C. Reactions were performed using KAPA HiFi PCR kits (Kapa Biosystems) according to the manufacturer's instructions.

[0405] The following multiple primers are used:

[0406] TYR_S: TTAGCAAAGCATACCATCA (SEQ ID NO: 3) and TYR_AS: CCAGACAAAGAGGTCATAA (SEQ ID NO: 4)

[0407] for tyrosinase expression (expected product size: 117 base pairs) and WT1_s: AGGCTGCAATAAGAGATA (SEQ ID NO: 5) and WT1_AS: TTCGCTGACAAGTTTTAC (SEQ ID NO: 6) for WT1 expression (expected product size: 188 base pairs).

[0408] To visualize the amplification products, 10 microliters of sample were mixed with 2 microliters of 6x loading buffer (New England Biolabs) and electrophoresed in 1.5% agarose gels, stained with ethidium bromide for DNA markers (New England Biolabs). The presence and intensity of the PCR product bands were determined on an ImageQuant LAS 4000 (GE Healthcare LifeSciences).

[0409] Quantitative PCR (qPCR)

[0410] Quantitative PCR was performed on an ABI7300 instrument (Applied Biosystems) using TaqMan Gene Expression Master Mix according to the manufacturer's instructions. The multiple cycle conditions for real-time PCR were 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. The multiple probes used for real-time PCR were purchased from Applied Biosystems; at the 5' end, they were coupled to the fluorochrome FAM. The following tests (primers and probes) were used: for TYR (cat#Hs00165976), for MAGE A4 (cat#Hs00751150), for WT1 (cat#Hs01103751). Beta-actin was used as a housekeeping gene for normalization (cat#Hs99999903).

[0411] Multiple peptides used in this study

[0412]

[0413]

[0414]

[0415] *Similar peptides based on alanine

[0416]

[0417]

[0418] *Similar peptides based on alanine

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] *Similar peptides based on alanine

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436] Embodiment 1:

[0437] Multiple TCR-like antibodies for HLA-A2 / tyrosinase

[0438] Isolation of multiple Abs with TCR-like specificity for HLA-A2 / tyrosine 369-377

[0439] Production of MHC-TyrD369-377 Complexes - Previous studies conducted by the present inventors have shown the use of large antibody phage libraries to generate recombinant antibodies with peptide specificity, HLA-A2-restricted specificity, for tumor and viral T cell epitopes. Such molecules are referred to as TCR-like antibodies. To produce antibodies with a specificity for the HLA-A2 / TyrD369-377 complex, a single-chain MHC construct was used to produce antibodies expressing the Tyrosinase peptide. 369–377 YMDGTMSQV, SEQ ID NO: 1). HHD mice were immunized by 5-6 injections of 50 μg / mouse of HLA-A2-peptide complexes. The first 2-3 injections were administered subcutaneously with QuilA adjuvant. Hybridoma clones were generated by fusion of spleen cells isolated from immunized mice with NSO myeloma cells (as described above by Weidanz et al. 2011, Int. Rev. Immunol. 30: 328-340), and screened and isolated by differential ELISA assays as described below. ELISA with purified HLA-A2-Tyr complexes and control HLA-A2 complexes showing other HLA-A2-restricted peptides (Table 15) were used to screen for specific clones, and isolated hybridoma clones were subcloned and sequenced. Two clones 906-11-D11 (referred to as D11, Fig.68) and 905-2-D7 (referred to as D7, Fig.69 ) is described.

[0440] Characterization of multiple TCR-like antibodies specific for HLA-A2 / tyrosinase 369-377

[0441] In order to determine the surface affinity of the multiple separated TCR-like antibodies, a surface plasmon resonance (SPR) binding analysis was used, and the multiple separated purified IgG-like TCR antibodies were indirectly immobilized on the surface plasmon resonance (SPR) sensor chip through anti-mouse or human IgG. The analyte was the purified single-chain recombinant HLA-A2 / tyrosinase complex at various concentrations. Figure 1 As shown, the multiple sensorgrams of SPR analysis show similar affinities for multiple TCR-like antibody clones MC1, D11 and D7 specific for the HLA-A2 / tyrosinase, with corresponding MC1 and D11 being 4.1 nanomolar concentrations and D7 being 3.8 nanomolar concentrations. The multiple results indicate that all three TCR-like antibody clones exhibit similar high affinity of 4 nanomolar concentrations for the specific HLA-A2 / peptide complex.

[0442] To investigate the superior peptide epitope specificity of the isolated TCR-like antibodies for the tyrosinase 369-377 peptide, alanine scanning was performed in which specific residues in the peptide were mutated to alanine, and the binding of the TCR-like antibodies to Ala mutant peptides was tested by loading the mutant peptides onto T2 antigen presenting cells. Binding was monitored by flow cytometry, and the extent of binding of the TCR-like antibodies to the mutant peptides was compared to that of T2 APCs loaded with the unmutated tyrosinase peptide as measured by mean fluorescence intensity (MFI). The various Ala mutant peptides (described in Figure 2 ) was monitored by flow cytometry using BB7.2, a monoclonal antibody against HLA-A2.

[0443] All Ala mutant peptides were efficiently loaded onto T2 cells compared to the native unmutated tyrosinase peptide (data not shown). Peptide loading efficiency was verified by using the ratio between the MFI of the HLA-A2-binding antibody BB7.2 and the MFI of unloaded T2 cells (>1). Figure 2As shown, all three TCR-like antibodies exhibited peptide-dependent binding, because specific mutations affected the binding, and when Ala was introduced at specific peptide positions, a reduction in the binding strength of the TCR-like antibodies was induced. The results show that in the case of HLA-A2 addition of various Ala mutant tyrosinase peptides, all three TCR-like antibodies exhibited peptide-specific and restricted binding, indicating that the antibodies are similar to TCR in their binding properties, and therefore, the antibodies bind to the MHC-peptide complex in an MHC-restricted and peptide-specific manner.

[0444] However, the difference in the superior specificity and peptide-dependent responses of the three TCR-like antibodies lies in the number of positions in the peptide that are sensitive to Ala mutations and affected binding sensitivity. When MC1 was bound to a single Ala-mutated peptide at one position #6, a significant decrease of 90% was shown, and both D11 and D7 showed a greater than 90% decrease for D11 at two positions #3, 6, and a greater than 90% reduction in binding at four positions #3, 4, 6, 7. A more modest but significant decrease of greater than 70% was further observed at three positions #1, 3, 6, 4, 6, 7 when MC1 was bound to multiple Ala-mutated peptides, although D11 and D7 showed a significant reduction of greater than 70% binding when five peptide residues were mutated to Ala (positions #1, 2, 3, 4, 6 for D11, and positions #2, 3, 4, 6, 7 for D7).

[0445] Overall, the alanine scanning analysis revealed that D11 and D7 were more affected and sensitive to multiple Ala mutations than MC1 by observing the ability of the various Ala-mutated Tyr peptides to correctly bind to the Tyr-specific TCR-like antibody. Figure 2 As shown in the data, D11 and D7 have higher peptide restriction and sensitivity in the binding properties of D11 and D7 compared to MC1; they are sensitive to Ala mutations in four of the nine peptide residues (excluding multiple anchor positions), while MC1 is only sensitive to three positions. D11 and D7 are sensitive to positions seven and five, respectively, in their binding properties. Specifically, D11 reduced the binding at position #7 by 68%, position #5 by 67%, and position #8 by 59%; D7 reduced the binding at position #5 by 66%, position #1 by 63%, and position #8 by 63%.

[0446] In conclusion, Ala scanning can be used as a means to determine the selectivity and excellent specificity of multiple TCR-like antibodies, and if a higher sensitivity to Ala mutation is shown, a higher specific and peptide-dependent binding can be observed. The strategy can be used to filter and screen the ideal TCR-like antibodies such as multiple MHC-restricted peptide-specific binders that exhibit higher and optimized selectivity and specificity properties.

[0447] Binding selectivity and specificity of multiple TCR-like antibodies for HLA-A2 / tyrosinase

[0448] In order to characterize the binding specificity of the multiple isolated TCR-like antibodies, the reactivity and specificity of the multiple purified IgGs were obtained by flow cytometry. Multiple specific or control peptides were added to multiple T2APCs and cultured with the Abs, followed by culture with PE-labeled anti-human or mouse Abs. Figure 3-7 As shown, the MC1( Figure 7 ), D11 and D7( Figures 3 to 6 ) IgG bound to T2 cells spiked with the tyrosinase peptide, but not significantly to cells spiked with control peptides (Table 15). For MC1, very low background binding was observed on control peptides with MCI ratios of 3-7 ( Figure 7 ), while D11 and D7 did not show any background binding ( Figures 3 to 6 ). The extent of presentation of the loaded peptides was monitored by binding of MAb BB7.2, which bound to all HLA-A2 peptide complexes. The results indicate that all three TCR-like antibodies exhibited specific binding to HLA-A2-restricted peptides, as they bound only to cells expressing the tyrosine, but not to other HLA-A2-restricted peptides.

[0449] In order to explore whether the multiple HLA-A2 / tyrosinase class TCR Abs can bind to multiple endogenously derived MHC-tyrosine salt complexes on the surface of multiple tumor cells, flow cytometry analysis was performed on multiple lines from multiple melanoma patients. Multiple cells were cultured with multiple anti-tyrosinase 369-377 / HLA-A2 class TCR antibodies and then cultured with PE-labeled anti-human or anti-mouse Abs. Figures 8 to 12As shown, the multiple TCR-like antibodies recognized multiple tyrosinase-positive and HLA-A2-positive cells with a very high intensity. As shown, it is shown that a large amount of HLA-A2-tyrosinase complexes are expressed on the surface of the multiple melanoma cells. The staining of the TCR-like antibodies is very uniform; intracellular staining of the multiple melanoma cells (e.g., 624.38, and 501A) with Abs against the tyrosine protein revealed that 95% of the multiple cells in each strain tested expressed the tyrosinase protein (data not shown). No reactivity was detected in tyrosinase-negative or HLA-A2-negative cells. Through extensive flow cytometric analysis of multiple cell lines from various tissue sources, the multiple cell lines were HLA-A2 positive and Ag (tyrosinase) negative, verifying the specificity of anti-tyrosinase / HLA-A2 class TCR Abs. The analysis is as shown in FIG. Figure 10-12 As shown, the reactivity of D11 and D7 was also detected in a panel of normal primary cells including multiple endothelial cells, fibroblasts, astrocytes, hepatocytes, kidney cells, cardiac myocytes, colon muscle and PBMC ( Figure 13-17 No binding was observed with the multiple HLA-A2+ and Tyr- normal primary cells, whereas background binding was observed when MC1 was tested on PBMC ( Fig.17 The analysis of the reactivity of D11 and D7 to multiple HLA-A2+ / tyrosinase+ melanoma cells and a broad panel of HLA-A2+ / tyrosinase- cells of various tissue origins including the multiple normal primary cells is as follows Figure 18-19 The reactivity of the D11 and D7 class TCR antibodies was extremely specific for multiple melanoma cells expressing HLA-A2 and the antigen tyrosinase.

[0450] The overall conclusion of the studies is that the TCR-like Abs are specific and that they only recognize the specific peptide-MHC complexes presented on the cell surface when the sufficient combination of HLA alleles and Ag is present. However, careful evaluation of the flow cytometry data showed that the results showed differential selectivity of MC1 when compared to D11 and D7. For example, analysis of MC1 binding to the HLA-A2+ and Tyr- cell lines HepG2, SW620, and Loucy, as shown in Figure 2 Fig. 9 As shown, background binding as measured by MFI was revealed, however, similar analysis of D11 and D7 on multiple cells revealed no binding ( Fig.10 and 12 ). By comparing the three TCR-like antibodies side by side on these and other cells ( Fig.12) revealed that MC1 exhibited significant binding to multiple melanoma cells with HLA-A2+ / tyr+, but had background binding on a variety of HLA-A2+ / Tyr- cells (SW620, Colo205, HepG2, Panc1, RPMI, DG75, Jeko1 and Loucy), while D11 and D7 did not exhibit any background binding to the multiple cells.

[0451] Therefore, it can be concluded that D11 and D7 are more specific and selective than MC1, and that combined flow cytometry studies and other analyses, for example, functional assays utilizing a large panel of cells from different tissue origins, expressing the appropriate HLA alleles and being antigen positive or negative, are useful tools for evaluating the selectivity of multiple TCR-class antibodies.

[0452] To further evaluate the superior specificity of the tyrosinase-specific TCR-like antibodies, their reactivity to peptides showing sequence similarity to the native tyrosinase was evaluated (Table 5).

[0453] Therefore, when alanine / glycine scanning data for a specific TCR-like antibody is available as described above, another round of screening of similar multiple peptides is performed. Based on alanine scanning, the contribution of each amino acid residue in the peptide antigen to the binding of TCRL is measured and evaluated. Multiple similar peptides that maintain the multiple key positions are identified by the above tools and are given higher priority. The multiple peptides are synthesized and evaluated for excellent specificity as described above.

[0454] The strategy described herein combines in silico analysis of peptide sequence similarity combined with mass spectrometry analysis of eluted HLA peptides, multiple peptide databases, and alanine scanning to provide a toolbox to fully control multiple peptide search parameters, more than other tools such as BLAST or ScanProsite provide. Multiple additional parameters are employed, including the range of peptide lengths allowed, the maximum number or differences allowed in sequences, and the requirement for HLA binding scores. The tool also accommodates the ability to define certain amino acids as equivalent. Most importantly is the ability to highlight multiple peptides that have been discovered by mass spectrometry or from database data or literature.

[0455] Using the above tools, three TCR-like antibodies with superior specificity were synthesized. A large panel of similar peptides was selected for evaluation according to the criteria described herein (Table 5). The multiple similar peptides have been added to multiple T2 APCs, and the reactivity of the multiple TCR-like antibodies has been tested. Fig. 20As shown, when MC1 was tested on a panel of similar peptides, it was observed that MC1 showed background binding to multiple peptides with sequence similarity to tyrosinase, such as KIAA0335 and KPNA1, compared to binding to the native tyrosinase peptide. Figures 21 to 28 As shown, the TCR-like antibodies D11 and D7 did not bind to any similar peptides from a large panel of the peptides analyzed, including no recognition of the KIAA0335 and KPNA1 peptides that showed background binding with MC1. The data demonstrate the superior selectivity and superior specificity of D11 and D7 compared to MC1, and demonstrate the usefulness of the similar peptide approach and the tools developed as described above as important tools to assess the selectivity and superior specificity ranking when evaluating a panel of TCR-like antibodies for the best and most ideal candidates for further evaluation.

[0456] In addition, after alanine scanning of multiple TCR-like antibodies, multiple additional similar peptides have been screened and tested. Since each amino acid in the TyrD peptide sequence is unlikely to contribute equally to the binding of Tyr TCRL, the peptide residues that are critical for Tyr TCRL binding are identified. A combination of multiple synthetic peptides was produced, in which each amino acid in the TyrD 9-mer was sequentially substituted with alanine. The ability of Tyr TCRL to bind to cells with one of the multiple peptides substituted with the multiple alanines was determined by FACS analysis, and the multiple binding results were compared with the binding results obtained with the non-mutated peptides. The residues at the positions where the alanine substitutions resulted in a huge decrease in binding compared to the non-mutated peptides were considered to be critical. A directional computer simulation was then performed to identify protein sequences containing only the multiple key position motifs. The multiple peptides were also used for specificity evaluation of multiple Tyr TCRLs (Tables 5S17 to S23). Similar peptides derived from the alanine scanning assays were synthesized and added to T2 APC cells and tested for reactivity with D11 and D7. Fig.28 As shown, no binding of the multiple peptides was observed, thereby further confirming and enhancing the excellent specificity and selectivity of the multiple TCR-like antibodies.

[0457] Example 1A

[0458] Characterization of multiple TCR-like antibodies for HLA-A2 / tyrosinase

[0459] Comparison of the superior specificity of multiple Abs with TCR-like specificity for HLA-A2 / tyrosinase 369-377

[0460] To characterize the binding specificity of the isolated TCR-like antibodies, the reactivity and specificity of the purified IgGs (with or without biotinylation) were evaluated by flow cytometry. T2 APCs or control peptides (Table 15) were added to the tyrosinase peptide and incubated with the Abs (D7, D11 or MC1), followed by incubation with PE-labeled streptavidin or PE-labeled anti-mouse Abs. Fig.38 As shown, multiple D11 and D7 TCRLs bound to T2 cells added with the tyrosinase peptide, but did not show binding to multiple cells added with the control peptide. In contrast, MC1 TCRL showed binding to multiple T2 cells added with the tyrosinase peptide and the unrelated peptide as a control.

[0461] To further evaluate the specificity of the multiple D7 and D11 class TCR antibodies, their reactivity with multiple peptides showing sequence similarity to the tyrosinase peptide was evaluated. The peptides are shown in Table 5.

[0462] like Fig.39 As shown in the Figures, TCRL has readily detectable binding to various peptides with sequence similarity to tyrosinase peptides such as KIAA0335 and KPNA1 (Table 14) and labeled S2, S4, S5, S9, S11, S13, S18, (S19, S22 and S23). Multiple D11 and D7 class TCR antibodies do not bind to any of the multiple peptides from the same group of multiple similar peptides. The multiple data confirm the superior selectivity and superior specificity of D11 and D7 TCRL compared to MC1 TCRL, and demonstrate the usefulness of the similar peptide method and multiple tools developed as described above to evaluate the selectivity and superior specificity ranking of multiple class TCR antibodies.

[0463] The present inventors investigated the binding specificity of multiple TCR-like Abs of HLA-A2 / tyrosinase to multiple MHC-tyrosinase peptide complexes endogenously displayed on the surface of multiple melanoma cell lines. Multiple cells were cultured with multiple TCR-like antibody Abs (with or without biotinylation) against tyrosinase 369-377 / HLA-A2, followed by culture with PE-labeled streptavidin or multiple anti-mouse Abs. A group of tumor cells and multiple normal primary cells that had been characterized for the expression of HLA-A2 (positive) and tyrosinase (positive or negative) were used to compare the binding of the multiple TCR-like antibodies. Fig.40AAs shown in Figures 1 to 3, the multiple TCR-like antibodies recognize multiple tyrosinase-positive and HLA-A2-positive cells. The multiple TCR-like antibodies were tested on multiple HLA-A2-positive cell lines from various sources, which do not show TyrRNA expression (Tyr-negative). Fig.40A As shown in Figures B to C, D11 and D7 TCRLs did not bind to any of the cells, while MC1 readily stained a variety of HLA-A2+ / Tyr- cells. D7 and D11 TCRLs did not show any binding to normal primary cells, while MC1 showed detectable binding to some of them ( Fig.40C ).

[0464] Overall, compared to MC1 TCRL, multiple D7 and D11 TCRLs demonstrated superior specificity and selectivity for recognizing tyrosinase peptides presented by HLA-A2.

[0465] Functional assays were used to further characterize the D7 and D11 class TCR antibodies. The variable regions of the TCRLs were fused to an anti-CD3 scFv that can be used to retarget effector T cells in a bispecific format to kill tumor target cells. Figures 41 to 44 As shown, multiple constructs of D7 and D11 CD3 bispecific TCR-like antibodies showed potent cytotoxicity against multiple melanoma 501A cells in vitro in the presence of multiple human PBMCs. Panc-1, a tyrosinase negative cell line, served as a negative control and showed no cytotoxicity. No cytotoxicity was detected against a panel of multiple HLA-A2+ / Tyr- normal human primary cells with multiple D7 and D11 TCRLs confirming their selectivity.

[0466] Example 1B

[0467] In vivo efficacy of D7 BS TCRL in the sc501A melanoma tumorigenesis model in multiple NOD / SCID mice

[0468] Fig.45 The in vivo efficacy of D7 BS TCRL in multiple NOD / SCID mouse models of sc501A melanoma tumor formation was shown. Clearly, administration of the bispecific antibody completely inhibited tumor formation over the 65-day experiment, as demonstrated by tumor volume. The results support the use of variable sequences of TCRL in multiple clinical settings described herein.

[0469] Example 2

[0470] Multiple TCR-like antibodies for HLA-A2 / WT1

[0471] Isolation and characterization of multiple Abs with TCR-like specificity for HLA-A2 / WT1

[0472] To generate the antibodies specific for the HLA-A2 / WT1 complex, recombinant peptide-HLA-A2 complexes were produced using a single-chain MHC construct expressing the WT1 peptide (RMFPNAPYL, SEQ ID NO: 151). Production of antibodies As described in the General Materials and Methods and Example 1 above, a class of TCR-specific clones designated B47 (also referred to as B47B6) was isolated and characterized ( Fig.70 ).

[0473] As a comparison of the binding selectivity of a class of TCR antibodies, a class of TCR antibodies called ESK1, Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T, Scott A, Whitten J, Maslak P, Casey E, TanT, Liu H, Zakhaleva V, Curcio M, Doubrovina E, O'Reilly RJ, Liu C, Scheinberg DA.

[0474] The binding affinity of B47 was evaluated by surface plasmon resonance (SPR) binding analysis, and the isolated purified IgG class TCR antibody was indirectly immobilized on the SPR sensor chip via anti-mouse IgG. The analyte was the purified single-chain recombinant HLA-A2 / WT1 complex used at various concentrations. Fig.29 As shown, the multiple sensorgrams of the SPR analysis showed an affinity of 4.4 nanomolar for the HLA-A2 / WT1 specific TCR-like antibody clone B47.

[0475] To characterize the binding specificity of the isolated TCR-like antibodies, the reactivity and specificity of the purified IgGs were evaluated by flow cytometry. T2 APCs were added to specific or control peptides (Table 15) and incubated with Abs, followed by PE-labeled anti-human or mouse Abs. Fig.30 and 31 As shown, B47 and ESK1 bound to T2 cells to which the WT1 peptide was added (Figure 30) but not to cells to which the control peptide was added ( Fig.31 ). A significant difference was observed between the binding strengths of B47 and ESK1.-4 Up to 10 -5 ESK1 was weakly bound to T2 cells with 10 -4 Multiple T2 cells with 10 molar concentrations of WT1 peptide (MFI 18 for ESK1 compared to 474 for B47) were observed at peptide concentrations of 10 -5 At molar concentrations, B47 still bound significantly (MFI 88), whereas binding of ESK1 was almost undetectable or very low ( Fig.30 The results showed that the affinity and binding sensitivity of B47 had significant differences, with ESK1 binding strength decreasing dramatically compared to B47 with a 10-fold decrease in peptide concentration. B47 and ESK1 did not bind to T2 APCs with control HLA-A2-restricted peptides ( Fig.31 ). The results indicate that both TCR-like antibodies exhibit specific binding to HLA-A2-restricted peptides, as they bind only to cells expressing the WT1, but not to other HLA-A2-restricted peptides.

[0476] To further investigate the superior specificity of the multiple WT1-class TCR antibodies, the above-described strategy was used to evaluate the binding to multiple similar peptides simulated in silico. Fig.32 and 33 As shown in Table 6, B47 did not bind to any similar peptides in a designed panel. Fig.32 As shown, ESK1 exhibited low background binding to two similar peptides. B47 was evaluated on additional multiple control peptides and multiple similar peptides ( Fig.34 ). Further analysis of the plurality of TCR-like antibodies was performed by flow cytometry using tumor cells that were HLA-A2 and expressed or did not express the WT1 antigen. Fig.35 As shown, the ESK1 WT1 class TCR-like antibody strongly bound to HLA-A2+ / WT+BV173 and SET2 cells, but B47 did not show any binding to the multiple cells at the level of sensitivity of the flow cytometric technique. To further investigate specificity, the reactivity of ESK1 and B47 was evaluated on cells that were HLA-A2 but did not express the WT1 gene as assessed by PCR. As shown, B47 did not bind to any of the multiple cells, while ESK1 bound to 501, A498, and SKMEL cells that were found to be WT1 negative. Other WT1 negative cells were not bound by ESK1. The level of HLA-A2 expression was monitored by MAb BB7.2, which recognizes all HLA-A2 / peptide molecules on the cell surface. Fig.36A summary of the binding data for the B47 WT-specific TCR-like antibodies is shown.

[0477] To further investigate the conflicting data regarding the binding of ESK1 and B47 to HLA-A2+ / WT1+ BV173 and SET2 cells, i.e., binding could be significantly detected by ESK1, but not B47, we employed direct biochemical approaches to assess actual WT1 expression on the cells. We employed a strategy of elution of HLA peptides from various tissues and BV173 and SET2 cells, followed by MS analysis of the eluted peptides. The data from the experiments indicated that the WT1 peptide was not detected in any of the clinical tissues or cell lines in which the MS was run. In-depth analysis of the BV173 or SET-2 cell lines (mRNA WT1-positive) failed to detect the peptide (Orbitrap or Q Exactive MS instruments). The WT1 peptide was detected by Orbitrap MS followed by direct elution from T2 peptide-spiked cells that were spiked with 10% of various WT1 peptide concentrations. -5 , 10 -7 , 10 -9 molar concentration, and the MS was added with peptide concentration of 10 -5 and 10 -7 The peptide was detected in multiple eluates of multiple T2 APC with different molar concentrations, and the peptide was detected by the MS from the eluate containing 10 -7 Multiple T2 APCs at molar concentrations of the peptide detected the peptide corresponding to actual expression of approximately 250 sites / cell (using the Orbitrap MS).

[0478] The data illustrate the utility of the described binding tools for peptide-spiked cells displaying similar peptides and cells of various histological origins to assess the specificity and selectivity of TCR-like antibodies.

[0479] To further investigate epitope specificity, alanine scanning mutagenesis was performed on the WT1 peptide sequence. Fig.37As shown, it is shown that only mutations in position 1 of the WT1 peptide affect the binding strength of ESK1, indicating that the binding selectivity and superior specificity of ESK1 are limited compared to B47, as is the observed specificity pattern, as observed for multiple similar peptides, and for multiple cells of HLA-A2+ / WT1- / . The multiple data indicate that the selectivity and superior specificity of B47 are excellent compared to ESK1, and the toolbox described herein is a valuable tool to evaluate the selectivity and superior specificity of multiple TCR-like antibodies during screening, phenotyping, and preclinical development of multiple TCR-like antibodies.

[0480] Example 2A

[0481] Multiple TCR-like antibodies for HLA-A2 / WT1

[0482] Comparison of the superior specificity of multiple Abs with TCR-like specificity for HLA-A2 / WT1

[0483] The selectivity of multiple TCR-like antibodies B47 and ESK1 was compared (Dao et al., Science Translational Medicine, 2013 Mar 13;5(176):176ra33).

[0484] T2 APCs were added with specific (WT1, SEQ ID NO: 141) or control peptides (Table 15) and incubated with B47 and ESK1 antibodies, followed by PE-labeled streptavidin or anti-mouse Abs. Both B47 and ESK1 TCRLs bound to T2 cells added with the WT1 peptide, but not to cells added with control peptides ( Fig.46 A panel of similar peptides (Table 6) was synthesized to further characterize the specificity of the WT1 TCRL. The B47 TCRL did not bind to T2 cells spiked with any of the similar peptides, whereas ESK1 TCRL showed detectable binding to several of the similar peptides ( Fig.47 ). ESK1 TCRL showed binding to a similar peptide derived from HDAC2 (histone deacetylase 2, Table 14), which is commonly expressed in many normal cells. , WT1-S10 (SEQ ID NO: 151) was expressed in multiple normal tissues, as confirmed by mass spectrometry in brain, cerebral cortex, heart, kidney, liver, lung, and other normal tissues (Table 14).

[0485] Further characterization of the binding of B47 to multiple ESK1 TCRLs by SPR showed that the affinity of B47 (5 nanomolar) was stronger than that of ESK1 (200 nanomolar), mainly due to the faster dissociation rate of ESK1 with multiple MHC-WT1 peptide complexes ( Fig.48 ).

[0486] Additional alanine scanning mutagenesis of the WT1 peptide was performed to improve the peptide epitope specificity of multiple B47-class TCR antibodies ( Fig.49 ). The multiple mutant peptides were added to multiple T2 cells and the binding analysis was performed as described above. The loading of the various Ala mutations was monitored by flow cytometry using the BB7.2 monoclonal antibody against HLA-A2.

[0487] like Fig.49 As shown, multiple substitutions of Ala at some positions significantly affected B47 binding to the multiple mutant peptides. B47 TCRL showed greater sensitivity to multiple positional substitutions (compared to ESK1, Fig.37 ). When four residues in the peptide were mutated to alanine (positions 1, 3, 4, and 7), the B47 class TCR antibody lost >73% of its binding to the presented peptide. The sensitivity of the fifth position can be attributed to position number 5. For both B47 and ESK1TCRLs, position 2 is critical as it is thought to serve as an anchor position for the peptides in the HLA-A2 peptide binding groove.

[0488] Further characterization and comparison between B47 and ESK1 TCRL were performed on multiple tumor cell lines and primary cells from various sources. Fig.50As shown, B47 does not bind to a group of cells, and the cells of the group are all HLA-A2 positive and WT1 mRNA positive or negative multiple cells. In contrast, ESK1 TCRL binds to a number of tumors and normal primary cells (all HLA-A2+). For example, JVM2 and IM9 (both HLA-A2 positive and WT1 negative) and multiple normal primary astrocytes show binding. Cytotoxicity assays using TCRL-aCD3 bispecific constructs and multiple human PBMCs show that B47 TCRL does not induce the death of multiple cells of HLA-A2+ / WT1+ or HLA-A2+ / WT1-, while ESK1 TCRL-aCD3 is cytotoxic to a number of cells, including WT-1 negative. Therefore, B47 TCRL in the form of the bispecific both shows superior specificity in combination and functional activity, compared to ESK1, which binds to and retargets CD3 T cells to some cells, including multiple normal primary cells, regardless of the expression of WT-1.

[0489] Example 3

[0490] Multiple TCR-like antibodies specific for HLA-A2 / MAGE-A4

[0491] Example 3A

[0492] Isolation and characterization of TCRL specific for HLA-A2 / MAGE-A4

[0493] To characterize the binding specificity of the isolated TCR-like antibodies, the reactivity and specificity of the purified IgGs were evaluated by flow cytometry. MAGE-A4 peptide or control peptides (Table 15) were added to T2 APCs and cultured with the TCRL Ab C106B, followed by PE-labeled streptavidin or PE-labeled anti-mouse Abs. Fig.52 As shown, C106B9 bound to T2 cells to which MAGE-A4 peptide was added, but did not bind to cells to which control peptide was added.

[0494] To further evaluate the specificity of the C106B9 class TCR antibodies, their reactivity with multiple peptides showing sequence similarity to the MAGE-A4 peptide was evaluated. The peptides are shown in Table 8.

[0495] like Fig.53As shown, C106B9 TCRL class TCR antibody does not bind to any of the peptides from the same group of similar peptides. The data demonstrate the high selectivity and high specificity of C106B9, and demonstrate the usefulness of the similar peptide method and the tools developed as described above to evaluate the selectivity and excellent specificity of multiple TCRLs.

[0496] In order to determine the surface affinity of the multiple separated TCR-like antibodies, a surface plasmon resonance (SPR) binding analysis was used, and the separated purified IgG-like TCR antibodies were indirectly immobilized on the surface plasmon resonance (SPR) sensor chip through anti-mouse IgG. The analyte was the purified single-chain recombinant HLA-A2 / MAGE-A4 complex at various concentrations. Fig.54 As shown, the multiple sensorgrams of SPR analysis showed similar affinities for the multiple TCR-like antibody clone C106B9 specific for the HLA-A2 / MAGE-A4 with a corresponding affinity of 8.8 nanomolar concentration.

[0497] To investigate the superior peptide epitope specificity of the isolated TCR-like antibodies for the MAGE-A4 peptide, alanine scanning was performed in which specific residues in the peptide were mutated to alanine, and the binding of the TCR-like antibodies to Ala mutant peptides was tested by loading the mutant peptides onto T2 antigen presenting cells (Table 9). Binding was monitored by flow cytometry, and the extent of binding of the TCR-like antibodies to the mutant peptides was compared to that of T2 APCs loaded with the unmutated MAGE-A4 peptide as measured by mean fluorescence intensity (MFI). The various Ala mutant peptides (described in Figure 2 ) was monitored by flow cytometry using BB7.2, a monoclonal antibody against HLA-A2.

[0498] All Ala mutant peptides were efficiently added to multiple T2 cells compared to the native unmutated MAGE-A4 (data not shown). Fig.55As shown, the TCR-like antibody exhibited peptide-dependent binding because multiple specific mutations affected the binding and when Ala was introduced at multiple specific peptide positions, a reduction in the binding strength of the TCR-like antibody was induced. The multiple results showed that in the case of HLA-A2 adding various Ala-mutated MAGE-A4 peptides, the MAGE-A4-like TCR antibody exhibited peptide-specific and restricted binding, indicating that the antibody is similar to TCR in the binding properties of the antibody, and therefore, the antibody binds to the MHC-peptide complex in an MHC-restricted and peptide-specific manner.

[0499] The C106B9 class TCR antibody exhibited a significant decrease of 90% in binding to the Ala mutant peptide at four positions #4, 5, 6 and 7. The sensitivity of the fifth position was attributed to position number 2 (33% decrease).

[0500] In general, the alanine scanning analysis revealed that Ala scanning can be used as a means to determine the selectivity and excellent specificity of multiple TCR-like antibodies, and if a higher sensitivity is shown to Ala mutations, a higher specific and peptide-dependent binding can be observed. The strategy can be used to filter and screen the ideal TCR-like antibodies such as multiple MHC-restricted peptide-specific binders that exhibit higher and optimized selectivity and specificity properties.

[0501] The present inventors investigated the binding specificity of HLA-A2 / MAGE-A4 TCR-like Abs to multiple MHC-MAGE-A4 peptide complexes endogenously displayed on the surface of multiple melanoma cell lines. Multiple cells were cultured with multiple anti-MAGE-A4-HLA-A2 TCR-like antibody Abs, followed by PE-labeled streptavidin or multiple anti-mouse Abs. A group of tumor cells and multiple normal primary cells were characterized for the expression of HLA-A2 (positive) and MAGE-A4 (positive or negative) and were used to compare the binding of the multiple TCR-like antibodies. Fig.56 As shown, the TCR-like antibody recognized multiple cells with low intensity of MAGE-A4-positive and HLA-A2-positive. The TCR-like antibody was tested on multiple HLA-A2-positive cell lines of various sources, which did not show MAGE-A4 RNA expression (MAGE-A4-negative), and the killing ability of these cells with a MAGE-A4 / HLA-A2 TCRL-bispecific construct was also tested. Fig.56 As shown, C106B9 did not bind to any of the multiple cells.

[0502] Multiple functional analyses were used to further characterize the C106B9 class TCR antibody. Multiple variable regions of multiple TCRLs were fused to an anti-CD3 scFv, which can retarget multiple effector T cells in a bispecific format to kill tumor target cells. Fig.57 As shown, multiple constructs of the C106B9 bispecific TCR-like antibody showed potent cytotoxicity against multiple MAGE-A4 positive cells in vitro in the presence of multiple human PBMCs. TCCSUP and OVCAR, MAGE-A4 negative cell lines served as negative controls and showed no cytotoxicity. As further described in Fig.58 As shown, no cytotoxicity was detected using C106B9 TCRL against a panel of multiple HLA-A2+ / MAGE-A4- normal human primary cells confirming their selectivity.

[0503] Example 3B

[0504] In vivo efficacy of C106B9 BS TCRL in the scA375 melanoma tumorigenesis model in multiple NOD / SCID mice

[0505] Fig.59 The in vivo efficacy of C106B9 BS TCRL in multiple NOD / SCID mice in the scA375 melanoma tumor formation model was shown. Clearly, administration of the bispecific antibody completely inhibited tumor formation over the 35-day experiment, as demonstrated by tumor volume. The results support the use of variable sequences of TCRL in multiple clinical settings described herein.

[0506] Example 4

[0507] Multiple TCR-like antibodies specific for HLA-A2 / MAGE-A9

[0508] Isolation and characterization of TCRL specific for HLA-A2 / MAGE-A9

[0509] To characterize the binding specificity of the multiple isolated TCR-like antibodies, the reactivity and specificity of the multiple purified IgGs were evaluated by flow cytometry. MAGE-A9 peptide or multiple control peptides were added to multiple T2 APCs and cultured with the TCRL Ab F184C7, followed by culture with PE-labeled streptavidin or multiple PE-labeled anti-mouse Abs. Fig.60 As shown, F184C7 bound to T2 cells to which MAGE-A9 peptide was added, but did not bind to cells to which control peptide was added.

[0510] To further evaluate the specificity of the F184C7 class TCR antibodies, their reactivity with multiple peptides showing sequence similarity to the MAGE-A9 peptide was evaluated. The peptides are shown in Table 10.

[0511] like Fig.61 As shown, the F184C7 TCRL class TCR antibody does not bind to any of the peptides from the same group of similar peptides. The data demonstrate the high selectivity and specificity of F184C7, and demonstrate the usefulness of the similar peptide method and the tools developed as described above to evaluate the selectivity and excellent specificity of multiple TCRLs.

[0512] To investigate the superior peptide epitope specificity of the isolated TCR-like antibodies for the MAGE-A9 peptide, alanine scanning was performed in which specific residues in the peptide were mutated to alanine, and the binding of the TCR-like antibodies to Ala mutant peptides was tested by loading the mutant peptides onto T2 antigen presenting cells (Table 11). Binding was monitored by flow cytometry, and the extent of binding of the TCR-like antibodies to the mutant peptides was compared to that of T2 APCs loaded with the unmutated MAGE-A4 peptide as measured by mean fluorescence intensity (MFI). The various Ala mutant peptides (described in Figure 2 ) was monitored by flow cytometry using BB7.2, a monoclonal antibody against HLA-A2.

[0513] All Ala mutant peptides were efficiently added to multiple T2 cells compared to the native unmutated MAGE-A9 (data not shown). Fig.62 As shown, the TCR-like antibody exhibited peptide-dependent binding because multiple specific mutations affected the binding and when Ala was introduced at multiple specific peptide positions, a reduction in the binding strength of the TCR-like antibody was induced. The multiple results showed that in the case of HLA-A2 adding various Ala-mutated MAGE-A9 peptides, the TCR-like antibody exhibited peptide-specific and restricted binding, indicating that the antibody is similar to TCR in the binding properties of the antibody, and therefore, the antibody binds to the MHC-peptide complex in an MHC-restricted and peptide-specific manner.

[0514] The F184C7 class TCR antibody exhibited a significant decrease of 90% in binding to the Ala mutant peptide at five positions #3, 5, 6, 7 and 8.

[0515] In general, the alanine scanning analysis revealed that Ala scanning can be used as a means to determine the selectivity and excellent specificity of multiple TCR-like antibodies, and if a higher sensitivity is shown to Ala mutations, a higher specific and peptide-dependent binding can be observed. The strategy can be used to filter and screen the ideal TCR-like antibodies such as multiple MHC-restricted peptide-specific binders that exhibit higher and optimized selectivity and specificity properties.

[0516] The present inventors investigated the binding specificity of the HLA-A2 / MAGE-A9 class TCR Ab to a panel of normal primary cells of various origins that do not express MAGE-A9 RNA. Fig.63 As shown, F184C7TCRL did not bind to any of the plurality of cells. The positive control group was a plurality of T2 cells to which the MAGE-A9 peptide was added, and the plurality of T2 cells were strongly bound by the F184C7 peptide.

[0517] Example 5

[0518] Multiple TCR-like antibodies specific for HLA-A2 / PAP

[0519] Isolation and characterization of TCRL specific for HLA-A2 / PAP

[0520] To characterize the binding specificity of the isolated TCR-like antibodies, the reactivity and specificity of the purified IgGs were evaluated by flow cytometry. PAP peptide or control peptides were added to T2 APCs and cultured with the TCRL Ab D10A3, followed by culture with PE-labeled streptavidin or PE-labeled anti-mouse Abs. Fig.64 As shown, D10A3 bound to T2 cells to which PAP peptide was added, but did not bind to cells to which control peptide was added.

[0521] To further evaluate the specificity of the D10A3 class TCR antibodies, their reactivity with a number of peptides showing sequence similarity to the PAP peptide was evaluated. The peptides are shown in Table 12.

[0522] like Fig.65 As shown, the D10A3 TCRL class TCR antibody does not bind to any of the peptides from the same group of similar peptides. The data demonstrate the high selectivity and specificity of D10A3 and the usefulness of the similar peptide approach and the tools developed as described above to assess the selectivity and excellent specificity of TCRLs.

[0523] To investigate the superior peptide epitope specificity of the isolated TCR-like antibodies for the PAP peptide, alanine scanning was performed in which specific residues in the peptide were mutated to alanine and the binding of the TCR-like antibodies to Ala mutant peptides was tested by loading the mutant peptides onto T2 antigen presenting cells (Table 13). Binding was monitored by flow cytometry and the extent of binding of the TCR-like antibodies to the mutant peptides was compared to that of T2 APCs loaded with the unmutated PAP peptide as measured by mean fluorescence intensity (MFI). The various Ala mutant peptides (described in Figure 2 ) was monitored by flow cytometry using BB7.2, a monoclonal antibody against HLA-A2.

[0524] All Ala mutant peptides were efficiently added to multiple T2 cells compared to the native unmutated PAP (data not shown). Fig.66 As shown, the TCR-like antibody exhibited peptide-dependent binding because multiple specific mutations affected the binding and when Ala was introduced at multiple specific peptide positions, a reduction in the binding strength of the TCR-like antibody was induced. The multiple results showed that in the case of HLA-A2 adding various Ala mutant PAP peptides, the TCR-like antibody exhibited peptide-specific and restricted binding, indicating that the antibody is similar to TCR in the binding properties of the antibody, and therefore, the antibody binds to the MHC-peptide complex in an MHC-restricted and peptide-specific manner.

[0525] The D10A3 class TCR antibody exhibited a significant decrease of 90% at three positions #3, 6, and 8 in binding to the Ala mutant peptide. A 70% reduction in binding to an Ala mutant peptide at position #4 was also observed. A fifth position of sensitivity was attributed to position number 7 (45% reduction).

[0526] The present inventors investigated the binding specificity of the HLA-A2 / PAP class TCR Ab to a panel of normal primary cells of various origins that do not express PAP RNA. Fig.67 As shown, D10A3 TCRL did not bind to any of the plurality of cells. The positive control group was a plurality of T2 cells to which the PAP peptide was added, and the plurality of T2 cells were strongly bound by the D10A3 peptide.

[0527] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many alternatives, modifications and variations are apparent to those skilled in the art. Therefore, the present invention is intended to cover all such alternatives, modifications and variations that fall within the spirit and scope of the claims.

[0528] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent that the individual publications, patents or patent applications are expressly and individually marked as being incorporated herein by reference. In addition, the citation or identification of any reference in this application should not be construed as an admission that the reference is available as prior art for the present invention. To the same extent, the section headings used should not be considered as necessary limitations. SEQUENCE LISTING <110> Adiset Biotech Pere Rouge, Mira Dankebeck, Galit Rhett, Yoram Bell, Elan Sinnick, Karen Tebbur, Elbaz, Yere Sheparbet, Seri, Hyères Erel Segal, Leut Oren, Raymont Alisek Fiss, Droor Himmel <120> Affinity entities with TCR-like antibody binding domains and uses thereof <130> 66429 <150> US 62 / 172,264 <151> 2015-06-08 <150> NL N2014935 <151> 2015-06-08 <160> 384 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Short peptide <400> 1 Tyr Met Asp Gly Thr Met Ser Gln Val 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(9) <223> 短胜肽 <400> 2 Tyr Leu Leu Pro Ala Ile Val His Ile 1 5 <210> 3 <211> 19 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(19) <223> 短胜肽 <400> 3 Thr Thr Ala Gly Cys Ala Ala Ala Gly Cys Ala Thr Ala Cys Cys Ala 1 5 10 15 Thr Cys Ala <210> 4 <211> 19 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(19) <223> 短胜肽 <400> 4 Cys Cys Ala Gly Ala Cys Ala Ala Ala Gly Ala Gly Gly Thr Cys Ala 1 5 10 15 Thr Ala Ala <210> 5 <211> 18 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(18) <223> Short peptide <400> 5 Ala Gly Gly Cys Thr Gly Cys Ala Ala Thr Ala Ala Gly Ala Gly Ala 1 5 10 15 Thr Ala <210> 6 <211> 18 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(18) <223> Short peptide <400> 6 Thr Thr Cys Gly Cys Thr Gly Ala Cys Ala Ala Gly Thr Thr Thr Thr 1 5 10 15 Ala Cys <210> 7 <211> 10 <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Short peptide <400> 7 Glu Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 <210> 8 <211> 163 <212> PRT <213> homo sapiens <400> 8 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 100 105 110 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 115 120 125 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 130 135 140 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 145 150 155 160 Pro Pro Arg <210> 9 <211> 164 <212> PRT <213> homo sapiens <400> 9 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 100 105 110 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 145 150 155 160 Leu Pro Pro Arg <210> 10 <211> 123 <212> PRT <213> Homo sapiens <400> 10 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Trp Lys His Leu Cys Pro Ser Pro Leu 35 40 45 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 50 55 60 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 65 70 75 80 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 85 90 95 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 100 105 110 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 115 120 <210> 11 <211> 101 <212> PRT <213> homo sapiens <400> 11 Put Leu Arg Leu Leu Leu Ala Leu Donkey Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys 20 25 30 Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 35 40 45 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 50 55 60 Ser Arg Leu Leu His Ser Asp Tyr Met Donkey Met Thr Pro Arg Arg Pro 65 70 75 80 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 85 90 95 Ala Ala Tyr Arg Expensive 100 <210> 12 <211> 220 <212> PRT <213> homo sapiens <400> 12 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 <210> 13 <211> 255 <212> PRT <213> homo sapiens <400> 13 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 14 <211> 199 <212> PRT <213> homo sapiens <400> 14 Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45 Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu 65 70 75 80 Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu 115 120 125 His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140 Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu 145 150 155 160 Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170 175 Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser 180 185 190 Arg Leu Thr Asp Val Thr Leu 195 <210> 15 <211> 277 <212> PRT <213> homo sapiens <400> 15 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys Ile 275 <210> 16 <211> 184 <212> PRT <213> Homo sapiens <400> 16 Met Ala Pro Leu Leu Pro Ile Arg Thr Leu Pro Leu Ile Leu Ile Leu 1 5 10 15 Leu Ala Leu Leu Ser Pro Gly Ala Ala Asp Phe Asn Ile Ser Ser Leu 20 25 30 Ser Gly Leu Leu Ser Pro Ala Leu Thr Glu Ser Leu Leu Val Ala Leu 35 40 45 Pro Pro Cys His Leu Thr Gly Gly Asn Ala Thr Leu Met Val Arg Arg 50 55 60 Ala Asn Asp Ser Lys Val Val Thr Ser Ser Phe Val Val Pro Pro Cys 65 70 75 80 Arg Gly Arg Arg Glu Leu Val Ser Val Val Asp Ser Gly Ala Gly Phe 85 90 95 Thr Val Thr Arg Leu Ser Ala Tyr Gln Val Thr Asn Leu Val Pro Gly 100 105 110 Thr Lys Phe Tyr Ile Ser Tyr Leu Val Lys Lys Gly Thr Ala Thr Glu 115 120 125 Ser Ser Arg Glu Ile Pro Met Ser Thr Leu Pro Arg Arg Asn Met Glu 130 135 140 Ser Ile Gly Leu Gly Met Ala Arg Thr Gly Gly Met Val Val Ile Thr 145 150 155 160 Val Leu Leu Ser Val Ala Met Phe Leu Leu Val Leu Gly Phe Ile Ile 165 170 175 Ala Leu Ala Leu Gly Ser Arg Lys 180 <210> 17 <211> 273 <212> PRT <213> Homo sapiens <400> 17 Met Ala Ser Ala Ala Ala Ala Glu Ala Glu Lys Gly Ser Pro Val Val 1 5 10 15 Val Gly Leu Leu Val Val Gly Asn Ile Ile Ile Leu Leu Ser Gly Leu 20 25 30 Ser Leu Phe Ala Glu Thr Ile Trp Val Thr Ala Asp Gln Tyr Arg Val 35 40 45 Tyr Pro Leu Met Gly Val Ser Gly Lys Asp Asp Val Phe Ala Gly Ala 50 55 60 Trp Ile Ala Ile Phe Cys Gly Phe Ser Phe Phe Met Val Ala Ser Phe 65 70 75 80 Gly Val Gly Ala Ala Leu Cys Arg Arg Arg Ser Met Val Leu Thr Tyr 85 90 95 Leu Val Leu Met Leu Ile Val Tyr Ile Phe Glu Cys Ala Ser Cys Ile 100 105 110 Thr Ser Tyr Thr His Arg Asp Tyr Met Val Ser Asn Pro Ser Leu Ile 115 120 125 Thr Lys Gln Met Leu Thr Phe Tyr Ser Ala Asp Thr Asp Gln Gly Gln 130 135 140 Glu Leu Thr Arg Leu Trp Asp Arg Val Met Ile Glu Gln Glu Cys Cys 145 150 155 160 Gly Thr Ser Gly Pro Met Asp Trp Val Asn Phe Thr Ser Ala Phe Arg 165 170 175 Ala Ala Thr Pro Glu Val Val Phe Pro Trp Pro Pro Leu Cys Cys Arg 180 185 190 Arg Thr Gly Asn Phe Ile Pro Leu Asn Glu Glu Gly Cys Arg Leu Gly 195 200 205 His Met Asp Tyr Leu Phe Thr Lys Ala Gly Val Gln Trp His Asn Leu 210 215 220 Ser Ser Leu Gln Arg Leu Pro Pro Gly Phe Lys Gly Phe Ser His Leu 225 230 235 240 Ser Phe Gln Ser Ser Trp Asp Tyr Arg Ala Ala Ser Asn Thr Ser Ala 245 250 255 Thr Pro Ser Thr Ala Thr Arg Gly Val Ser Arg Gly Leu Gly Leu Pro 260 265 270 Ser <210> 18 <211> 258 <212> PRT <213> homo sapiens <400> 18 Met Ala Ser Ala Ala Ala Ala Glu Ala Glu Lys Gly Ser Pro Val Val 1 5 10 15 Val Gly Leu Leu Val Val Gly Asn Ile Ile Ile Leu Leu Ser Gly Leu 20 25 30 Ser Leu Phe Ala Glu Thr Ile Trp Val Thr Ala Asp Gln Tyr Arg Val 35 40 45 Tyr Pro Leu Met Gly Val Ser Gly Lys Asp Asp Val Phe Ala Gly Ala 50 55 60 Trp Ile Ala Ile Phe Cys Gly Phe Ser Phe Phe Met Val Ala Ser Phe 65 70 75 80 Gly Val Gly Ala Ala Leu Cys Arg Arg Arg Ser Met Val Leu Thr Tyr 85 90 95 Leu Val Leu Met Leu Ile Val Tyr Ile Phe Glu Cys Ala Ser Cys Ile 100 105 110 Thr Ser Tyr Thr His Arg Asp Tyr Met Val Ser Asn Pro Ser Leu Ile 115 120 125 Thr Lys Gln Met Leu Thr Phe Tyr Ser Ala Asp Thr Asp Gln Gly Gln 130 135 140 Glu Leu Thr Arg Leu Trp Asp Arg Val Met Ile Glu Gln Glu Cys Cys 145 150 155 160 Gly Thr Ser Gly Pro Met Asp Trp Val Asn Phe Thr Ser Ala Phe Arg 165 170 175 Ala Ala Thr Pro Glu Val Val Phe Pro Trp Pro Pro Leu Cys Cys Arg 180 185 190 Arg Thr Gly Asn Phe Ile Pro Leu Asn Glu Glu Gly Cys Arg Leu Gly 195 200 205 His Met Asp Tyr Leu Phe Thr Lys Gly Cys Phe Glu His Ile Gly His 210 215 220 Ala Ile Asp Ser Tyr Thr Trp Gly Ile Ser Trp Phe Gly Phe Ala Ile 225 230 235 240 Leu Met Trp Thr Leu Pro Val Met Leu Ile Ala Met Tyr Phe Tyr Thr 245 250 255 Met Leu <210> 19 <211> 245 <212> PRT <213> homo sapiens <400> 19 Met Lys Gln Ser Phe Pro Leu Phe Leu Thr Pro Ser Pro Trp Lys Thr 1 5 10 15 Thr Val Leu Leu Leu Tyr Met Arg Ile Cys Tyr Val Pro Ser Tyr Lys 20 25 30 Trp Asn Tyr Ser Ile Gly Leu Ile Tyr Leu Gly Ile Val Ser Glu Leu 35 40 45 Pro His Met Val Gly Ile Gly Gln Asn Ser Ser Phe Asn Ser Trp Met 50 55 60 Glu Ser Gln Phe Leu His Pro Ser Met Glu Pro Gly Gln Trp Leu Pro 65 70 75 80 Tyr Ile Thr Ile Phe Arg Phe Thr His Ile Ile Arg Cys Val Arg Ile 85 90 95 Ser Phe Leu Phe Asn Ile Pro Trp Tyr Gly Tyr Pro His Phe Val Cys 100 105 110 His Ser Ser Val Ser Gly His Leu Gly Tyr Phe Tyr Leu Leu Leu Leu 115 120 125 Trp Leu Val Cys Cys Glu His Arg Cys Thr Asn Ile Cys Ser Arg Gln 130 135 140 Thr Ser Phe Lys Arg Leu Phe Leu Lys Lys Tyr Val Ser Tyr Asn Ile 145 150 155 160 Phe Leu Leu Cys Val Glu Ser Asp Ile Ser Ile Asp Leu Glu Gly Tyr 165 170 175 Gly Met Gly Cys Thr Asn Ile Cys Ser Arg Gln Thr Ser Phe Lys Arg 180 185 190 Leu Phe Lys Arg Lys Tyr Arg Cys Leu Leu Asn Met Phe Leu Val Met 195 200 205 Asn Val Glu Ser Gly Thr Asn Arg Tyr Met Glu Val Arg Arg Ala Trp 210 215 220 Arg Gly Ser Lys Trp Glu Asp Glu Glu Asn Trp Leu Gly Ile Asp Val 225 230 235 240 Tyr Phe Glu Asp Arg 245 <210> 20 <211> 114 <212> PRT <213> homo sapiens <400> 20 Met Ala Gly Leu Ala Leu Gln Pro Gly Thr Ala Leu Leu Cys Tyr Ser 1 5 10 15 Cys Lys Ala Gln Val Ser Asn Glu Asp Cys Leu Gln Val Glu Asn Cys 20 25 30 Thr Gln Leu Gly Glu Gln Cys Trp Thr Ala Arg Ile Arg Ala Val Gly 35 40 45 Leu Leu Thr Val Ile Ser Lys Gly Cys Ser Leu Asn Cys Val Asp Asp 50 55 60 Ser Gln Asp Tyr Tyr Val Gly Lys Lys Asn Ile Thr Cys Cys Asp Thr 65 70 75 80 Asp Leu Cys Asn Ala Ser Gly Ala His Ala Leu Gln Pro Ala Ala Ala 85 90 95 Ile Leu Ala Leu Leu Pro Ala Leu Gly Leu Leu Leu Trp Gly Pro Gly 100 105 110 Gln Leu <210> 21 <211> 386 <212> PRT <213> homo sapiens <400> 21 Met Arg Ala Ala Pro Leu Leu Leu Ala Arg Ala Ala Ser Leu Ser Leu 1 5 10 15 Gly Phe Leu Phe Leu Leu Phe Phe Trp Leu Asp Arg Ser Val Leu Ala 20 25 30 Lys Glu Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser 35 40 45 Pro Ile Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro 50 55 60 Gln Gly Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu 65 70 75 80 Leu Gly Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser 85 90 95 Tyr Lys His Glu Gln Val Tyr Ile Arg Ser Thr Asp Val Asp Arg Thr 100 105 110 Leu Met Ser Ala Met Thr Asn Leu Ala Ala Leu Phe Pro Pro Glu Gly 115 120 125 Val Ser Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His 130 135 140 Thr Val Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn 145 150 155 160 Cys Pro Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu 165 170 175 Phe Gln Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly 180 185 190 Lys Leu Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys 195 200 205 Val Tyr Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro 210 215 220 Ser Trp Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu 225 230 235 240 Leu Ser Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser 245 250 255 Arg Leu Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys 260 265 270 Arg Ala Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala 275 280 285 His Asp Thr Thr Val Ser Gly Leu Gln Met Ala Leu Asp Val Tyr Asn 290 295 300 Gly Leu Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe 305 310 315 320 Glu Lys Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln 325 330 335 His Glu Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro 340 345 350 Leu Glu Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp 355 360 365 Ser Thr Glu Cys Met Thr Thr Asn Ser His Gln Gly Thr Glu Asp Ser 370 375 380 Thr Asp 385 <210> 22 <211> 418 <212> PRT <213> homo sapiens <400> 22 Met Arg Ala Ala Pro Leu Leu Leu Ala Arg Ala Ala Ser Leu Ser Leu 1 5 10 15 Gly Phe Leu Phe Leu Leu Phe Phe Trp Leu Asp Arg Ser Val Leu Ala 20 25 30 Lys Glu Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser 35 40 45 Pro Ile Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro 50 55 60 Gln Gly Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu 65 70 75 80 Leu Gly Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser 85 90 95 Tyr Lys His Glu Gln Val Tyr Ile Arg Ser Thr Asp Val Asp Arg Thr 100 105 110 Leu Met Ser Ala Met Thr Asn Leu Ala Ala Leu Phe Pro Pro Glu Gly 115 120 125 Val Ser Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His 130 135 140 Thr Val Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn 145 150 155 160 Cys Pro Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu 165 170 175 Phe Gln Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly 180 185 190 Lys Leu Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys 195 200 205 Val Tyr Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro 210 215 220 Ser Trp Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu 225 230 235 240 Leu Ser Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser 245 250 255 Arg Leu Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys 260 265 270 Arg Ala Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala 275 280 285 His Asp Thr Thr Val Ser Gly Leu Gln Met Ala Leu Asp Val Tyr Asn 290 295 300 Gly Leu Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe 305 310 315 320 Glu Lys Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln 325 330 335 His Glu Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro 340 345 350 Leu Glu Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp 355 360 365 Ser Thr Glu Cys Met Thr Thr Asn Ser His Gln Val Leu Lys Val Ile 370 375 380 Phe Ala Val Ala Phe Cys Leu Ile Ser Ala Val Leu Met Val Leu Leu 385 390 395 400 Phe Ile His Ile Arg Arg Gly Leu Cys Trp Gln Arg Glu Ser Tyr Gly 405 410 415 Asn Ile <210> 23 <211> 353 <212> PRT <213> Homo sapiens <400> 23 Met Arg Ala Ala Pro Leu Leu Leu Ala Arg Ala Ala Ser Leu Ser Leu 1 5 10 15 Gly Phe Leu Phe Leu Leu Phe Phe Trp Leu Asp Arg Ser Val Leu Ala 20 25 30 Lys Glu Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser 35 40 45 Pro Ile Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro 50 55 60 Gln Gly Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu 65 70 75 80 Leu Gly Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser 85 90 95 Tyr Lys His Glu Gln Val Tyr Ile Arg Ser Thr Asp Val Asp Arg Thr 100 105 110 Leu Met Ser Ala Met Thr Asn Leu Ala Ala Leu Phe Pro Pro Glu Gly 115 120 125 Val Ser Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His 130 135 140 Thr Val Pro Leu Ser Glu Asp Gln Asp Phe Ile Ala Thr Leu Gly Lys 145 150 155 160 Leu Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys Val 165 170 175 Tyr Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro Ser 180 185 190 Trp Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu Leu 195 200 205 Ser Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser Arg 210 215 220 Leu Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys Arg 225 230 235 240 Ala Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala His 245 250 255 Asp Thr Thr Val Ser Gly Leu Gln Met Ala Leu Asp Val Tyr Asn Gly 260 265 270 Leu Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe Glu 275 280 285 Lys Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln His 290 295 300 Glu Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro Leu 305 310 315 320 Glu Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp Ser 325 330 335 Thr Glu Cys Met Thr Thr Asn Ser His Gln Gly Thr Glu Asp Ser Thr 340 345 350 Asp <210> 24 <211> 335 <212> PRT <213> homo sapiens <400> 24 Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys 1 5 10 15 Ala Pro Ser Leu Leu Val Ala Leu Asp Ile Cys Ser Lys Asn Pro Cys 20 25 30 His Asn Gly Gly Leu Cys Glu Glu Ile Ser Gln Glu Val Arg Gly Asp 35 40 45 Val Phe Pro Ser Tyr Thr Cys Thr Cys Leu Lys Gly Tyr Ala Gly Asn 50 55 60 His Cys Glu Thr Lys Cys Val Glu Pro Leu Gly Leu Glu Asn Gly Asn 65 70 75 80 Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu 85 90 95 Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly 100 105 110 Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile 115 120 125 Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln 130 135 140 Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val 145 150 155 160 Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn 165 170 175 Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 180 185 190 Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr 195 200 205 Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 210 215 220 Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser 225 230 235 240 Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly 245 250 255 Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 260 265 270 Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp 275 280 285 Leu Gln Ile Phe Pro Gly Asn Trp Asp Asn His Ser His Lys Lys Asn 290 295 300 Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg Ile Leu Pro Val 305 310 315 320 Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu Leu Gly Cys 325 330 335 <210> 25 <211> 264 <212> PRT <213> homo sapiens <400> 25 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn 50 55 60 Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg 65 70 75 80 Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu 85 90 95 Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu 100 105 110 Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr 115 120 125 Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr 130 135 140 Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln 145 150 155 160 Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val 165 170 175 Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val 180 185 190 Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala 195 200 205 Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 210 215 220 Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys 225 230 235 240 Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala 245 250 255 Val Ala Ala Thr Ser Ala Asn Leu 260 <210> 26 <211> 203 <212> PRT <213> homo sapiens <400> 26 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Ile Pro Ala Pro Thr Thr Thr Lys Ser Cys Arg 50 55 60 Glu Thr Phe Leu Lys Cys Phe Cys Arg Phe Ile Asn Lys Gly Val Phe 65 70 75 80 Trp Ala Ser Pro Ile Leu Ser Ser Val Ser Asp Val Pro Phe Pro Phe 85 90 95 Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu 100 105 110 Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala 115 120 125 Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile 130 135 140 Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr 145 150 155 160 His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro 165 170 175 Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr 180 185 190 Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 195 200 <210> 27 <211> 159 <212> PRT <213> homo sapiens <400> 27 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn 50 55 60 Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg 65 70 75 80 Asp Ile Ser Glu Met Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly 85 90 95 Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu 100 105 110 Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr 115 120 125 Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser 130 135 140 Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 145 150 155 <210> 28 <211> 475 <212> PRT <213> homo sapiens <400> 28 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His 50 55 60 Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu 65 70 75 80 Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln 85 90 95 Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr 100 105 110 Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro 115 120 125 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 130 135 140 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 145 150 155 160 Ala Pro Asp Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His 165 170 175 Asn Val Thr Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu 180 185 190 Val His Asn Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys 195 200 205 Ser Thr Pro Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr 210 215 220 Leu Ala Ser His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser 225 230 235 240 Thr Val Pro Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu 245 250 255 Ser Thr Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu 260 265 270 Gln Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu 275 280 285 Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly 290 295 300 Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val 305 310 315 320 Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp 325 330 335 Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr 340 345 350 Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe 355 360 365 Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu 370 375 380 Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala 385 390 395 400 Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile 405 410 415 Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr 420 425 430 His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro 435 440 445 Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr 450 455 460 Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 465 470 475 <210> 29 <211> 282 <212> PRT <213> homo sapiens <400> 29 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Leu Ser 50 55 60 Thr Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln 65 70 75 80 Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu 85 90 95 Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly 100 105 110 Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val 115 120 125 Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val 130 135 140 Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn 145 150 155 160 Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser 165 170 175 Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val 180 185 190 Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu 195 200 205 Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe 210 215 220 Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His 225 230 235 240 Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr 245 250 255 Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn 260 265 270 Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 275 280 <210> 30 <211> 238 <212> PRT <213> homo sapiens <400> 30 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Ile Tyr 50 55 60 Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly 65 70 75 80 Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn 85 90 95 Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala 100 105 110 Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro 115 120 125 Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile 130 135 140 Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr 145 150 155 160 Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln 165 170 175 Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr 180 185 190 Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp 195 200 205 Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu 210 215 220 Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 225 230 235 <210> 31 <211> 241 <212> PRT <213> homo sapiens <400> 31 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Leu 50 55 60 Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe 65 70 75 80 Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly 85 90 95 Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr 100 105 110 Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser 115 120 125 Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly 130 135 140 Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala 145 150 155 160 Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn 165 170 175 Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met 180 185 190 Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser 195 200 205 Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly 210 215 220 Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn 225 230 235 240 Leu <210> 32 <211> 198 <212> PRT <213> homo sapiens <400> 32 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Leu Ser Thr Gly Val Ser Phe Phe Phe Leu Ser 50 55 60 Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu Glu Asp Pro Ser 65 70 75 80 Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu 85 90 95 Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe 100 105 110 Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly 115 120 125 Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr 130 135 140 Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Gly Cys 145 150 155 160 Leu Ser Val Pro Pro Lys Glu Leu Arg Ala Ala Gly His Leu Ser Ser 165 170 175 Pro Gly Tyr Leu Pro Ser Tyr Glu Arg Val Pro His Leu Pro His Pro 180 185 190 Trp Ala Leu Cys Ala Pro 195 <210> 33 <211> 189 <212> PRT <213> homo sapiens <400> 33 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn 50 55 60 Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg 65 70 75 80 Asp Ile Ser Glu Met Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala 85 90 95 Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu 100 105 110 Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu 115 120 125 Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro 130 135 140 Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg 145 150 155 160 Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser 165 170 175 Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 180 185 <210> 34 <211> 177 <212> PRT <213> homo sapiens <400> 34 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Leu Ser 50 55 60 Thr Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln 65 70 75 80 Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu 85 90 95 Gln Arg Asp Ile Ser Glu Met Ala Val Cys Gln Cys Arg Arg Lys Asn 100 105 110 Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met 115 120 125 Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser 130 135 140 Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly 145 150 155 160 Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn 165 170 175 Leu <210> 35 <211> 255 <212> PRT <213> homo sapiens <400> 35 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp 50 55 60 Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile 65 70 75 80 Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro 85 90 95 Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile 100 105 110 Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala 115 120 125 Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val 130 135 140 Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly 145 150 155 160 Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val 165 170 175 Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly 180 185 190 Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu 195 200 205 Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr 210 215 220 Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser 225 230 235 240 Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 245 250 255 <210> 36 <211> 150 <212> PRT <213> homo sapiens <400> 36 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp 50 55 60 Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Ala Val Cys Gln 65 70 75 80 Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp 85 90 95 Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 100 105 110 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 115 120 125 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 130 135 140 Ala Thr Ser Ala Asn Leu 145 150 <210> 37 <211> 158 <212> PRT <213> homo sapiens <400> 37 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Ile Pro Ala Pro Thr Thr Thr Lys Ser Cys Arg 50 55 60 Glu Thr Phe Leu Lys Cys Phe Cys Arg Phe Ile Asn Lys Gly Val Phe 65 70 75 80 Trp Ala Ser Pro Ile Leu Ser Ser Val Trp Gly Trp Gly Ala Arg Leu 85 90 95 Gly His Arg Ala Ala Gly Ala Gly Leu Cys Ser Gly Cys Ala Gly His 100 105 110 Cys Leu Ser His Cys Leu Gly Cys Leu Ser Val Pro Pro Lys Glu Leu 115 120 125 Arg Ala Ala Gly His Leu Ser Ser Pro Gly Tyr Leu Pro Ser Tyr Glu 130 135 140 Arg Val Pro His Leu Pro His Pro Trp Ala Leu Cys Ala Pro 145 150 155 <210> 38 <211> 484 <212> PRT <213> homo sapiens <400> 38 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val Ser 50 55 60 Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser Ser 65 70 75 80 Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala Thr Glu Pro Ala 85 90 95 Ser Gly Ser Ala Ala Thr Trp Gly Gln Asp Val Thr Ser Val Pro Val 100 105 110 Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala His Asp Val Thr 115 120 125 Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala 130 135 140 His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr 145 150 155 160 Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala 165 170 175 Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ala Ser Gly 180 185 190 Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly Thr Ser Ala 195 200 205 Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe Ser Ile Pro 210 215 220 Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His Ser Thr Lys 225 230 235 240 Thr Asp Ala Ser Ser Thr His His Ser Thr Val Pro Pro Leu Thr Ser 245 250 255 Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val Ser Phe Phe 260 265 270 Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu Glu 275 280 285 Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu 290 295 300 Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn 305 310 315 320 Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe 325 330 335 Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln 340 345 350 Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val 355 360 365 Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly 370 375 380 Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val 385 390 395 400 Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg 405 410 415 Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr 420 425 430 His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val 435 440 445 Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly 450 455 460 Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr 465 470 475 480 Ser Ala Asn Leu <210> 39 <211> 219 <212> PRT <213> homo sapiens <400> 39 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn 50 55 60 Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg 65 70 75 80 Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu 85 90 95 Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu 100 105 110 Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr 115 120 125 Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr 130 135 140 Ile Ser Asp Val Ser Val Trp Gly Trp Gly Ala Arg Leu Gly His Arg 145 150 155 160 Ala Ala Gly Ala Gly Leu Cys Ser Gly Cys Ala Gly His Cys Leu Ser 165 170 175 His Cys Leu Gly Cys Leu Ser Val Pro Pro Lys Glu Leu Arg Ala Ala 180 185 190 Gly His Leu Ser Ser Pro Gly Tyr Leu Pro Ser Tyr Glu Arg Val Pro 195 200 205 His Leu Pro His Pro Trp Ala Leu Cys Ala Pro 210 215 <210> 40 <211> 217 <212> PRT <213> homo sapiens <400> 40 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Gly Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser 20 25 30 Val Pro Ser Ser Thr Glu Lys Asn Ala Ile Tyr Lys Gln Gly Gly Phe 35 40 45 Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln 50 55 60 Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu 65 70 75 80 Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu 85 90 95 Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala 100 105 110 Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu 115 120 125 Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala 130 135 140 Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro 145 150 155 160 Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr 165 170 175 His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu 180 185 190 Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro 195 200 205 Ala Val Ala Ala Thr Ser Ala Asn Leu 210 215 <210> 41 <211> 239 <212> PRT <213> homo sapiens <400> 41 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Ile Pro 50 55 60 Ala Pro Thr Thr Thr Lys Ser Cys Arg Glu Thr Phe Leu Lys Trp Pro 65 70 75 80 Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile 85 90 95 Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala 100 105 110 Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val 115 120 125 Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly 130 135 140 Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val 145 150 155 160 Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly 165 170 175 Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu 180 185 190 Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr 195 200 205 Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser 210 215 220 Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 225 230 235 <210> 42 <211> 230 <212> PRT <213> homo sapiens <400> 42 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Ile Pro Ala Pro Thr Thr Thr Lys Ser Cys Arg 50 55 60 Glu Thr Phe Leu Lys Trp Pro Gly Ser Val Val Val Gln Leu Thr Leu 65 70 75 80 Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe 85 90 95 Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser 100 105 110 Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly 115 120 125 Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val 130 135 140 Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln 145 150 155 160 Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp 165 170 175 Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 180 185 190 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 195 200 205 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 210 215 220 Ala Thr Ser Ala Asn Leu 225 230 <210> 43 <211> 212 <212> PRT <213> homo sapiens <400> 43 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Ile Pro 50 55 60 Ala Pro Thr Thr Thr Lys Ser Cys Arg Glu Thr Phe Leu Lys Cys Phe 65 70 75 80 Cys Arg Phe Ile Asn Lys Gly Val Phe Trp Ala Ser Pro Ile Leu Ser 85 90 95 Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly 100 105 110 Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val 115 120 125 Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg 130 135 140 Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr 145 150 155 160 His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val 165 170 175 Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly 180 185 190 Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr 195 200 205 Ser Ala Asn Leu 210 <210> 44 <211> 273 <212> PRT <213> homo sapiens <400> 44 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Leu Ser Thr Gly Val Ser Phe Phe Phe Leu Ser 50 55 60 Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu Glu Asp Pro Ser 65 70 75 80 Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu 85 90 95 Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe 100 105 110 Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly 115 120 125 Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr 130 135 140 Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser 145 150 155 160 Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly 165 170 175 Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala 180 185 190 Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn 195 200 205 Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met 210 215 220 Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser 225 230 235 240 Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly 245 250 255 Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn 260 265 270 Leu <210> 45 <211> 575 <212> PRT <213> homo sapiens <400> 45 Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly 1 5 10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Gly Ser Gln Val Trp Gly Gly 20 25 30 Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe Pro Asp 35 40 45 Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys Arg Ser Phe Val 50 55 60 Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Val Leu Gly Gly Pro 65 70 75 80 Val Ser Gly Leu Ser Ile Gly Thr Gly Arg Ala Met Leu Gly Thr His 85 90 95 Thr Met Glu Val Thr Val Tyr His Arg Arg Gly Ser Arg Ser Tyr Val 100 105 110 Pro Leu Ala His Ser Ser Ser Ala Phe Thr Ile Thr Asp Gln Val Pro 115 120 125 Phe Ser Val Ser Val Ser Gln Leu Arg Ala Leu Asp Gly Gly Asn Lys 130 135 140 His Phe Leu Arg Asn Gln Pro Leu Thr Phe Ala Leu Gln Leu His Asp 145 150 155 160 Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu Ser Tyr Thr Trp Asp Phe 165 170 175 Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg Ala Leu Val Val Thr His 180 185 190 Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala Gln Val Val Leu Gln Ala 195 200 205 Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser Pro Val Pro Gly Thr Thr 210 215 220 Asp Gly His Arg Pro Thr Ala Glu Ala Pro Asn Thr Thr Ala Gly Gln 225 230 235 240 Val Pro Thr Thr Glu Val Val Gly Thr Thr Pro Gly Gln Ala Pro Thr 245 250 255 Ala Glu Pro Ser Gly Thr Thr Ser Val Gln Val Pro Thr Thr Glu Val 260 265 270 Ile Ser Thr Ala Pro Val Gln Met Pro Thr Ala Glu Ser Thr Gly Met 275 280 285 Thr Pro Glu Lys Val Pro Val Ser Glu Val Met Gly Thr Thr Leu Ala 290 295 300 Glu Met Ser Thr Pro Glu Ala Thr Gly Met Thr Pro Ala Glu Val Ser 305 310 315 320 Ile Val Val Leu Ser Gly Thr Thr Ala Ala Gln Val Thr Thr Thr Glu 325 330 335 Trp Val Glu Thr Thr Ala Arg Glu Leu Pro Ile Pro Glu Pro Glu Gly 340 345 350 Pro Asp Ala Ser Ser Ile Met Ser Thr Glu Ser Ile Thr Gly Ser Leu 355 360 365 Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu Val Lys Arg Gln 370 375 380 Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val Thr 385 390 395 400 Leu Asp Ile Val Gln Gly Ile Glu Ser Ala Glu Ile Leu Gln Ala Val 405 410 415 Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu Thr Val Ser Cys Gln Gly 420 425 430 Gly Leu Pro Lys Glu Ala Cys Met Glu Ile Ser Ser Pro Gly Cys Gln 435 440 445 Pro Pro Ala Gln Arg Leu Cys Gln Pro Val Leu Pro Ser Pro Ala Cys 450 455 460 Gln Leu Val Leu His Gln Ile Leu Lys Gly Gly Ser Gly Thr Tyr Cys 465 470 475 480 Leu Asn Val Ser Leu Ala Asp Thr Asn Ser Leu Ala Val Val Ser Thr 485 490 495 Gln Leu Ile Met Pro Gly Gln Glu Ala Gly Leu Gly Gln Val Pro Leu 500 505 510 Ile Val Gly Ile Leu Leu Val Leu Met Ala Val Val Leu Ala Ser Leu 515 520 525 Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp Phe Ser Val Pro Gln Leu 530 535 540 Pro His Ser Ser Ser His Trp Leu Arg Leu Pro Arg Ile Phe Cys Ser 545 550 555 560 Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu Ser Gly Gln Gln Val 565 570 575 <210> 46 <211> 661 <212> PRT <213> Homo sapiens <400> 46 Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly 1 5 10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp 20 25 30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu 35 40 45 Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly 50 55 60 Gln Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala 65 70 75 80 Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 85 90 95 Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100 105 110 Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115 120 125 Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser 130 135 140 Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp 145 150 155 160 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg 165 170 175 Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg 180 185 190 Gly Ser Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr 195 200 205 Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu Arg Ala 210 215 220 Leu Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe 225 230 235 240 Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245 250 255 Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg 260 265 270 Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala 275 280 285 Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser 290 295 300 Pro Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro 305 310 315 320 Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 325 330 335 Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 340 345 350 Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355 360 365 Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375 380 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met 385 390 395 400 Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405 410 415 Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro 420 425 430 Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu 435 440 445 Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu 450 455 460 Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr 465 470 475 480 Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 485 490 495 Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505 510 Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile 515 520 525 Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530 535 540 Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly 545 550 555 560 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser 565 570 575 Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Gly Gln Glu Ala Gly 580 585 590 Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu Leu Val Leu Met Ala 595 600 605 Val Val Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610 615 620 Phe Ser Val Pro Gln Leu Pro His Ser Ser Ser His Trp Leu Arg Leu 625 630 635 640 Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 645 650 655 Ser Gly Gln Gln Val 660 <210> 47 <211> 668 <212> PRT <213> homo sapiens <400> 47 Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly 1 5 10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp 20 25 30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu 35 40 45 Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly 50 55 60 Gln Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala 65 70 75 80 Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 85 90 95 Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100 105 110 Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115 120 125 Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser 130 135 140 Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp 145 150 155 160 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg 165 170 175 Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg 180 185 190 Gly Ser Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr 195 200 205 Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu Arg Ala 210 215 220 Leu Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe 225 230 235 240 Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245 250 255 Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg 260 265 270 Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala 275 280 285 Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser 290 295 300 Pro Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro 305 310 315 320 Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 325 330 335 Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 340 345 350 Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355 360 365 Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375 380 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met 385 390 395 400 Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405 410 415 Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro 420 425 430 Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu 435 440 445 Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu 450 455 460 Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr 465 470 475 480 Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 485 490 495 Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505 510 Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile 515 520 525 Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530 535 540 Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly 545 550 555 560 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser 565 570 575 Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Val Pro Gly Ile Leu 580 585 590 Leu Thr Gly Gln Glu Ala Gly Leu Gly Gln Val Pro Leu Ile Val Gly 595 600 605 Ile Leu Leu Val Leu Met Ala Val Val Leu Ala Ser Leu Ile Tyr Arg 610 615 620 Arg Arg Leu Met Lys Gln Asp Phe Ser Val Pro Gln Leu Pro His Ser 625 630 635 640 Ser Ser His Trp Leu Arg Leu Pro Arg Ile Phe Cys Ser Cys Pro Ile 645 650 655 Gly Glu Asn Ser Pro Leu Leu Ser Gly Gln Gln Val 660 665 <210> 48 <211> 118 <212> PRT <213> homo sapiens <400> 48 Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly 1 5 10 15 His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile 20 25 30 Leu Thr Val Ile Leu Gly Val Leu Leu Leu Ile Gly Cys Trp Tyr Cys 35 40 45 Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val 50 55 60 Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp 65 70 75 80 His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val 85 90 95 Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser 100 105 110 Pro Pro Pro Tyr Ser Pro 115 <210> 49 <211> 1069 <212> PRT <213> Homo sapiens <400> 49 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr 885 890 895 Phe Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe 900 905 910 Gly Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val 915 920 925 Asn Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu 930 935 940 Gln Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln 945 950 955 960 Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr 965 970 975 Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser 980 985 990 Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln 995 1000 1005 Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg 1010 1015 1020 Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr 1025 1030 1035 Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu 1040 1045 1050 Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu 1055 1060 1065 Asp <210> 50 <211> 1132 <212> PRT <213> Homo sapiens <400> 50 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln 1010 1015 1020 Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp 1025 1030 1035 Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly 1040 1045 1050 Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu 1055 1060 1065 Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr 1070 1075 1080 Arg His Arg Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr 1085 1090 1095 Ala Gln Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr 1100 1105 1110 Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys 1115 1120 1125 Thr Ile Leu Asp 1130 <210> 51 <211> 353 <212> PRT <213> homo sapiens <400> 51 Met Ala His Phe Pro Gly Phe Gly Gln Ser Leu Leu Phe Gly Tyr Pro 1 5 10 15 Val Tyr Val Phe Gly Asp Cys Val Gln Gly Asp Trp Cys Pro Ile Ser 20 25 30 Gly Gly Leu Cys Ser Ala Arg Leu His Arg His Ala Leu Leu Ala Thr 35 40 45 Cys Pro Glu His Gln Ile Thr Trp Asp Pro Ile Asp Gly Arg Val Ile 50 55 60 Gly Ser Ala Leu Gln Phe Leu Ile Pro Arg Leu Pro Ser Phe Pro Thr 65 70 75 80 Gln Arg Thr Ser Lys Thr Leu Lys Val Leu Thr Pro Pro Ile Thr His 85 90 95 Thr Thr Pro Asn Ile Pro Pro Ser Phe Leu Gln Ala Met Arg Lys Tyr 100 105 110 Ser Pro Phe Arg Asn Gly Tyr Met Glu Pro Thr Leu Gly Gln His Leu 115 120 125 Pro Thr Leu Ser Phe Pro Asp Pro Gly Leu Arg Pro Gln Asn Leu Tyr 130 135 140 Thr Leu Trp Gly Gly Ser Val Val Cys Met Tyr Leu Tyr Gln Leu Ser 145 150 155 160 Pro Pro Ile Thr Trp Pro Leu Leu Pro His Val Ile Phe Cys His Pro 165 170 175 Gly Gln Leu Gly Ala Phe Leu Thr Asn Val Pro Tyr Lys Arg Ile Glu 180 185 190 Lys Leu Leu Tyr Lys Ile Ser Leu Thr Thr Gly Ala Leu Ile Ile Leu 195 200 205 Pro Glu Asp Cys Leu Pro Thr Thr Leu Phe Gln Pro Ala Arg Ala Pro 210 215 220 Val Thr Leu Thr Ala Trp Gln Asn Gly Leu Leu Pro Phe His Ser Thr 225 230 235 240 Leu Thr Thr Pro Gly Leu Ile Trp Thr Phe Thr Asp Gly Thr Pro Met 245 250 255 Ile Ser Gly Pro Cys Pro Lys Asp Gly Gln Pro Ser Leu Val Leu Gln 260 265 270 Ser Ser Ser Phe Ile Phe His Lys Phe Gln Thr Lys Ala Tyr His Pro 275 280 285 Ser Phe Leu Leu Ser His Gly Leu Ile Gln Tyr Ser Ser Phe His Asn 290 295 300 Leu His Leu Leu Phe Glu Glu Tyr Thr Asn Ile Pro Ile Ser Leu Leu 305 310 315 320 Phe Asn Glu Lys Glu Ala Asp Asp Asn Asp His Glu Pro Gln Ile Ser 325 330 335 Pro Gly Gly Leu Glu Pro Leu Ser Glu Lys His Phe Arg Glu Thr Glu 340 345 350 Val <210> 52 <211> 345 <212> PRT <213> homo sapiens <400> 52 Met Ala His Phe Pro Gly Phe Gly Gln Ser Leu Leu Tyr Gly Tyr Pro 1 5 10 15 Val Tyr Val Phe Gly Asp Cys Val Gln Ala Asp Trp Cys Pro Ile Ser 20 25 30 Gly Gly Leu Cys Ser Pro Arg Leu His Arg His Ala Leu Leu Ala Thr 35 40 45 Cys Pro Glu His Gln Ile Thr Trp Asp Pro Ile Asp Gly Arg Val Val 50 55 60 Gly Ser Pro Leu Gln Tyr Leu Ile Pro Arg Leu Pro Ser Phe Pro Thr 65 70 75 80 Gln Arg Thr Ser Lys Thr Leu Lys Val Leu Thr Pro Pro Thr Thr Pro 85 90 95 Val Thr Pro Lys Val Pro Pro Ser Phe Phe Gln Ser Val Arg Arg His 100 105 110 Ser Pro Tyr Arg Asn Gly Cys Leu Glu Thr Thr Leu Gly Glu Gln Leu 115 120 125 Pro Ser Leu Ala Phe Pro Glu Pro Gly Leu Arg Pro Gln Asn Val Tyr 130 135 140 Thr Ile Trp Gly Lys Thr Ile Val Cys Leu Tyr Ile Tyr Gln Leu Ser 145 150 155 160 Pro Pro Met Thr Trp Pro Leu Ile Pro His Val Ile Phe Cys Asn Pro 165 170 175 Arg Gln Leu Gly Ala Phe Leu Ser Asn Val Pro Pro Lys Arg Leu Glu 180 185 190 Glu Leu Leu Tyr Lys Leu Tyr Leu His Thr Gly Ala Ile Ile Ile Leu 195 200 205 Pro Glu Asp Ala Leu Pro Thr Thr Leu Phe Gln Pro Val Arg Ala Pro 210 215 220 Cys Val Gln Thr Thr Trp Asn Thr Gly Leu Leu Pro Tyr Gln Pro Asn 225 230 235 240 Leu Thr Thr Pro Gly Leu Ile Trp Thr Phe Asn Asp Gly Ser Pro Met 245 250 255 Ile Ser Gly Pro Cys Pro Lys Ala Gly Gln Pro Ser Leu Val Val Gln 260 265 270 Ser Ser Leu Leu Ile Phe Glu Arg Phe Gln Thr Lys Ala Tyr His Pro 275 280 285 Ser Tyr Leu Leu Ser His Gln Leu Ile Gln Tyr Ser Ser Phe His His 290 295 300 Leu Tyr Leu Leu Phe Asp Glu Tyr Thr Thr Ile Pro Phe Ser Leu Leu 305 310 315 320 Phe Lys Glu Lys Glu Gly Asp Asp Arg Asp Asn Asp Pro Leu Pro Gly 325 330 335 Ala Thr Ala Ser Pro Gln Gly Gln Asn 340 345 <210> 53 <211> 180 <212> PRT <213> homo sapiens <400> 53 Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 10 15 Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30 Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45 Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60 His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala 65 70 75 80 Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95 Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110 Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125 Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met 145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 165 170 175 Gly Gln Arg Arg 180 <210> 54 <211> 309 <212> PRT <213> Homo sapiens <400> 54 Met Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu 1 5 10 15 Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Thr 20 25 30 Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr 35 40 45 Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe 50 55 60 Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser 65 70 75 80 Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser 85 90 95 Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe 100 105 110 Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met 115 120 125 Leu Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe 130 135 140 Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys 145 150 155 160 Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly 165 170 175 Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr 180 185 190 Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His 195 200 205 Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr 210 215 220 Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr 225 230 235 240 Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp 245 250 255 Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala 260 265 270 Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala 275 280 285 Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu 290 295 300 Glu Glu Glu Gly Val 305 <210> 55 <211> 314 <212> PRT <213> homo sapiens <400> 55 Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu 1 5 10 15 Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30 Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45 Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60 Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp 65 70 75 80 Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95 Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 100 105 110 Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120 125 Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 130 135 140 Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu 145 150 155 160 Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr 165 170 175 Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180 185 190 Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200 205 Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215 220 Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly 225 230 235 240 Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu 245 250 255 Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 260 265 270 Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His 275 280 285 His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300 His Glu Trp Val Leu Arg Glu Gly Glu Glu 305 310 <210> 56 <211> 1225 <212> PRT <213> homo sapiens <400> 56 Met Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu 1 5 10 15 Arg His Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu 20 25 30 Thr Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln 35 40 45 Glu Val Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val 50 55 60 Pro Leu Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp 65 70 75 80 Asn Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr 85 90 95 Thr Pro Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu 100 105 110 Arg Ser Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn 115 120 125 Pro Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His 130 135 140 Lys Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg 145 150 155 160 Ala Cys His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly 165 170 175 Glu Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly 180 185 190 Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu 195 200 205 Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala 210 215 220 Cys Leu His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala 225 230 235 240 Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu 245 250 255 Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn 260 265 270 Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His 275 280 285 Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys 290 295 300 Ser Lys Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu 305 310 315 320 Arg Glu Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly 325 330 335 Cys Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp 340 345 350 Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln 355 360 365 Val Phe Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala 370 375 380 Trp Pro Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val 385 390 395 400 Ile Arg Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln 405 410 415 Gly Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly 420 425 430 Ser Gly Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His 435 440 445 Thr Val Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu 450 455 460 His Thr Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala 465 470 475 480 Cys His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr 485 490 495 Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu 500 505 510 Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg 515 520 525 His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val 530 535 540 Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr 545 550 555 560 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro 565 570 575 Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala 580 585 590 Cys Gln Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp 595 600 605 Asp Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile 610 615 620 Ile Ser Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val 625 630 635 640 Phe Gly Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr 645 650 655 Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro 660 665 670 Ser Gly Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr 675 680 685 Glu Leu Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val 690 695 700 Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val 705 710 715 720 Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu 725 730 735 Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val 740 745 750 Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr 755 760 765 Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg 770 775 780 Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala 785 790 795 800 Lys Gly Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu 805 810 815 Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr 820 825 830 Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His 835 840 845 Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile 850 855 860 Leu Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val 865 870 875 880 Thr Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile 885 890 895 Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro 900 905 910 Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys 915 920 925 Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser 930 935 940 Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln 945 950 955 960 Asn Glu Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg 965 970 975 Ser Leu Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu 980 985 990 Tyr Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly 995 1000 1005 Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg 1010 1015 1020 Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1025 1030 1035 Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1040 1045 1050 Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1055 1060 1065 Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser 1070 1075 1080 Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val 1085 1090 1095 Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro 1100 1105 1110 Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1115 1120 1125 Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1130 1135 1140 Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1145 1150 1155 Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1160 1165 1170 Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1175 1180 1185 Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro 1190 1195 1200 Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1205 1210 1215 Leu Gly Leu Asp Val Pro Val 1220 1225 <210> 57 <211> 1240 <212> PRT <213> homo sapiens <400> 57 Met Pro Arg Gly Ser Trp Lys Pro Gln Val Cys Thr Gly Thr Asp Met 1 5 10 15 Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg 20 25 30 His Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr 35 40 45 Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu 50 55 60 Val Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro 65 70 75 80 Leu Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn 85 90 95 Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr 100 105 110 Pro Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg 115 120 125 Ser Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro 130 135 140 Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys 145 150 155 160 Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala 165 170 175 Cys His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu 180 185 190 Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly 195 200 205 Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln 210 215 220 Cys Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys 225 230 235 240 Leu His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu 245 250 255 Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly 260 265 270 Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr 275 280 285 Leu Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn 290 295 300 Gln Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser 305 310 315 320 Lys Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg 325 330 335 Glu Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys 340 345 350 Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly 355 360 365 Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val 370 375 380 Phe Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp 385 390 395 400 Pro Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile 405 410 415 Arg Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly 420 425 430 Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser 435 440 445 Gly Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr 450 455 460 Val Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His 465 470 475 480 Thr Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys 485 490 495 His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln 500 505 510 Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu 515 520 525 Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His 530 535 540 Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr 545 550 555 560 Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys 565 570 575 Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp 580 585 590 Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys 595 600 605 Gln Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp 610 615 620 Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile 625 630 635 640 See Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe 645 650 655 Gly Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met 660 665 670 Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser 675 680 685 Gly Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu 690 695 700 Leu Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr 705 710 715 720 Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala 725 730 735 Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile 740 745 750 Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser 755 760 765 Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln 770 775 780 Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly 785 790 795 800 Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys 805 810 815 Gly Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala 820 825 830 Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp 835 840 845 Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala 850 855 860 Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu 865 870 875 880 Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr 885 890 895 Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro 900 905 910 Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln 915 920 925 Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp 930 935 940 Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu 945 950 955 960 Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn 965 970 975 Glu Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser 980 985 990 Leu Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr 995 1000 1005 Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly 1010 1015 1020 Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg 1025 1030 1035 Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1040 1045 1050 Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1055 1060 1065 Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1070 1075 1080 Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser 1085 1090 1095 Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val 1100 1105 1110 Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro 1115 1120 1125 Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1130 1135 1140 Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1145 1150 1155 Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1160 1165 1170 Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1175 1180 1185 Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1190 1195 1200 Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro 1205 1210 1215 Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1220 1225 1230 Leu Gly Leu Asp Val Pro Val 1235 1240 <210> 58 <211> 1055 <212> PRT <213> Homo sapiens <400> 58 Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385 390 395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val 465 470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630 635 640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685 Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700 Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 705 710 715 720 Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825 830 Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835 840 845 Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860 Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 865 870 875 880 Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg 885 890 895 Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910 Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925 Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940 Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 945 950 955 960 Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965 970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr 1010 1015 1020 Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly 1025 1030 1035 Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg 1040 1045 1050 Asn Met 1055 <210> 59 <211> 603 <212> PRT <213> homo sapiens <400> 59 Met Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu 1 5 10 15 Arg His Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu 20 25 30 Thr Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln 35 40 45 Glu Val Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val 50 55 60 Pro Leu Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp 65 70 75 80 Asn Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr 85 90 95 Thr Pro Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu 100 105 110 Arg Ser Leu Th...

Claims

1. An affinity binding entity, characterized in that The affinity binding entity comprises an antigen binding region, wherein the antigen binding region comprises: (i) multiple complementarity determining region (CDR) sequences of an immunoglobulin heavy chain, arranged from N-terminus to C-terminus as follows: CDR1 heavy chain (HC) SEQ ID NO: 341GYWIE CDR2 HC SEQ ID NO:342EILPSGGTNYNEKFKG CDR3 HC SEQ ID NO:343DSNSFTY; and (ii) a plurality of CDR sequences of an immunoglobulin light chain, which are arranged from N-terminus to C-terminus as follows: CDR1 light chain (LC) SEQ ID NO: 335 SVSSSVDYIH CDR2 LC SEQ ID NO:336 STSILAS CDR3 LC SEQ ID NO:337 QQRSSYPPT The affinity binding entity is capable of binding HLA-A2 / MAGE-A4 in a major histocompatibility complex-defined manner 230-239 , wherein the affinity binding entity is selected from the group consisting of an antibody, a T cell receptor (TCR) and a chimeric antigen receptor.

2. The affinity binding entity according to claim 1, characterized in that The affinity binding entity comprises a therapeutic moiety.

3. The affinity binding entity according to claim 1, characterized in that The affinity binding entity comprises a detectable moiety.

4. The affinity binding entity according to any one of claims 1 to 3, characterized in that The antibody is a single-chain Fv (scFv), a bispecific antibody or a full-length antibody.

5. The affinity binding entity according to any one of claims 1 to 3, characterized in that The antibody is a humanized antibody.

6. An expression vector, characterized in that: The expression vector comprises a polynucleotide comprising a nucleic acid sequence encoding the affinity binding entity of any one of claims 1 to 5, wherein the polynucleotide is operably linked to a cis-acting regulatory component.

7. A cell, characterized in that The cell comprises the expression vector of claim 6.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: the affinity binding entity according to any one of claims 1 to 5, the expression vector according to claim 6 or the cell according to claim 7.

9. Use of the affinity binding entity of any one of claims 1 to 5, the expression vector of claim 6 or the cell of claim 7 in the preparation of a medicament for treating cancer, wherein the cancer is selected from melanoma, T-cell leukemia / lymphoma, testicular cancer, head and neck cancer, esophageal cancer, osteosarcoma, multiple myeloma and B-leukemia.

10. Use of the affinity binding entity of any one of claims 1 to 5, the expression vector of claim 6 or the cell of claim 7 in the preparation of a medicament for treating cancer, wherein the cancer is selected from melanoma, osteosarcoma, multiple myeloma and B-leukemia.

11. Use of the antibody of any one of claims 1-5 in the preparation of a kit for detecting the presence of cancer cells, wherein the cells are contacted with the affinity binding entity under conditions that allow the formation of immune complexes, wherein the presence of the immune complexes or the level of the immune complexes is used to indicate the cancer cells.

12. Use of the antibody of any one of claims 1-5 in the preparation of a kit for detecting the presence of cancer cells from a biological sample, wherein the sample is contacted with the affinity binding entity under conditions that allow the formation of immune complexes, wherein the presence of the immune complexes or the level of the immune complexes is indicative of the cancer cells.

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