Chimeric antigen receptor factory and method of use

Abstract

To provide a CAR T cell therapy against solid tumors such as renal cell carcinoma.SOLUTION: Provided herein is a chimeric antigen receptor of a CAR T cell targeting CAIX and CD70 as well as a cell comprising the same. Further the cell secretes a monoclonal antibody locally at a tumor site.SELECTED DRAWING: Figure 28

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 62 / 773,885, filed Nov. 30, 2018, and U.S. Provisional Application No. 62 / 826,462, filed Mar. 29, 2019, the entire contents of each of which are hereby incorporated by reference.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference into this application to more fully describe the state of the art known to those of skill in the art as of the date of the invention claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyrights whatsoever.

[0004] Incorporation by Reference of Sequence Listing The contents of the text file named "[ ]" created in [ ] are hereby incorporated by reference in their entirety.

[0005] Field of the Invention The present invention is directed to chimeric antigen receptors and cells comprising the same, which cells further secrete monoclonal antibodies locally at the tumor site.

Background Art

[0006] Background of the Invention Clear cell renal cell carcinoma (ccRCC) is a major type of RCC and is one of the ten most common cancers in both men and women. Other types of renal cancer include papillary renal cell carcinoma, chromophobe renal cell carcinoma, and other or unclassified types of renal cell carcinoma. See, e.g., Lancet 373(2009)1119-32 (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Lancet 373 (2009) 1119-32 [Overview of the project]

[0008] Other objectives and advantages of the present invention will be readily apparent from the following description.

[0009] Aspects of the present invention are directed toward engineered cells comprising a chimeric antigen receptor. In embodiments, the chimeric antigen receptor comprises extracellular ligand-binding domains specific to a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CAIX and the second antigen comprises CD70.

[0010] In the embodiment, the CAR further comprises a transmembrane polypeptide, an intracellular signaling domain, and / or a co-stimulatory domain.

[0011] In the embodiment, the extracellular ligand-binding domain includes an antibody or a fragment thereof. For example, the antibody includes VH and / or VL as shown in Table 2, or any combination thereof. For example, the antibody includes VH and / or VL as shown in Table 4, or any combination thereof. For example, the extracellular binding domain includes VH and / or VL as shown in Tables 2 and 4, or any combination thereof. For example, the antibody includes CDR1, CDR2, and / or CDR3 as shown in Table 1, or any combination thereof. For example, the antibody includes CDR1, CDR2, and / or CDR3 as shown in Table 3, or any combination thereof. For example, the extracellular binding domain includes CDR1, CDR2, and / or CDR3 as shown in Tables 1 and 3, or any combination thereof.

[0012] In this embodiment, the manipulated cells express and secrete recombinant polypeptides.

[0013] In the embodiment, the recombinant polypeptide comprises an antibody or a fragment thereof, or a cytokine. For example, the recombinant polypeptide comprises an antibody or fragment thereof specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4. For example, the recombinant polypeptide comprises a cytokine including IL-12, IL-15, or IL-18.

[0014] In this embodiment, the recombinant polypeptide modulates the target immune system. For example, the recombinant polypeptide is an immune checkpoint blocking antibody.

[0015] In the embodiment, the recombinant polypeptide modulates tumor vasculogenesis. For example, the recombinant polypeptide may be specific to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1.

[0016] In the embodiment, the manipulated cells are T cells, NK cells, or NKT cells. For example, T cells are CD4+, CD8+, CD3+ panT cells, or any combination thereof. For example, T cells are a mixed population of CD4+ T cells and CD8+ T cells.

[0017] Aspects of this disclosure are further directed to nucleic acid constructs encoding chimeric antigen receptors. In embodiments, the chimeric antigen receptor comprises extracellular ligand-binding domains specific to a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CAIX and the second antigen comprises CD70.

[0018] In the embodiment, the nucleic acid construct further encodes a transmembrane polypeptide, an intracellular signaling domain, and / or a co-stimulatory domain.

[0019] In this embodiment, the nucleic acid construct further encodes a recombinant polypeptide.

[0020] Aspects of the disclosure are further directed to vectors comprising the nucleic acid constructs described herein.

[0021] Furthermore, aspects of the disclosure are directed to cells comprising the vectors described herein.

[0022] Aspects of the disclosure are also directed to methods for treating a subject afflicted with cancer. In embodiments, the method comprises administering to the subject a therapeutically effective amount of the engineered cells described herein.

[0023] Also, aspects of the invention are directed to methods of reducing cancer progression or promoting cancer regression in a subject. In embodiments, the method comprises administering to the subject a therapeutically effective amount of the engineered cells described herein.

[0024] Furthermore, aspects of the invention are directed to methods of reducing cell proliferation of cancer cells in a subject. In embodiments, the method comprises administering to the subject a therapeutically effective amount of the engineered cells described herein.

[0025] In embodiments, the cancer includes renal cell carcinoma.

[0026] Aspects of the disclosure are directed to a chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, wherein the extracellular ligand-binding domain is specific for a first antigen and a second antigen on the surface of cancer cells, the first antigen comprises CAIX, and the second antigen comprises CD70.

[0027] In embodiments, the CAR further comprises a transmembrane polypeptide, an intracellular signaling domain, and / or a co-stimulatory domain.

[0028] In embodiments, the extracellular ligand-binding domain comprises an antibody or a fragment thereof.

[0029] Furthermore, aspects of the invention are directed to cells comprising the chimeric antigen receptor (CAR) described herein.

[0030] Furthermore, aspects of the present invention are directed to engineered cells comprising a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises an extracellular ligand-binding domain specific to CAIX, and the second chimeric antigen receptor comprises an extracellular ligand-binding domain specific to CD70.

[0031] In this embodiment, the manipulated cells express and secrete recombinant polypeptides.

[0032] In this embodiment, the first chimeric antigen receptor and the second chimeric antigen receptor are expressed from a single nucleic acid construct. [Invention 1001] Engineered cells comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises extracellular ligand-binding domains specific to a first antigen and a second antigen on the surface of a cancer cell, the first antigen comprising CAIX, and the second antigen comprising CD70. [Invention 1002] The manipulated cell of the present invention 1001, wherein the CAR further comprises a transmembrane polypeptide and an intracellular signaling domain. [Invention 1003] The manipulated cell of the present invention 1002, wherein the CAR further comprises a co-stimulatory domain. [Invention 1004] The manipulated cell of the present invention 1001, wherein the extracellular ligand-binding domain contains an antibody or a fragment thereof. [Invention 1005] The manipulated cells of the present invention 1004, wherein the antibody comprises VH and / or VL, or any combination thereof, as shown in Table 2. [Invention 1006] The manipulated cells of the present invention 1004, wherein the antibody comprises VH and / or VL, or any combination thereof, as shown in Table 4. [Invention 1007] The manipulated cells of the present invention 1004, wherein the extracellular binding domain comprises VH and / or VL as shown in Tables 2 and 4, or any combination thereof. [Invention 1008] The manipulated cells of the present invention 1004, wherein the antibody comprises CDR1, CDR2, and / or CDR3, or any combination thereof, as shown in Table 1. [Invention 1009] The manipulated cells of the present invention 1004, wherein the antibody comprises CDR1, CDR2, and / or CDR3, or any combination thereof, as shown in Table 3. [Invention 1010] The manipulated cells of the present invention 1004, wherein the extracellular binding domain comprises CDR1, CDR2, and / or CDR3, or any combination thereof, as shown in Tables 1 and 3. [Invention 1011] Manipulated cells of the present invention 1001 that express and secrete recombinant polypeptides. [Invention 1012] The recombinant polypeptide comprises an antibody or a fragment thereof, or a cytokine, in the manipulated cells of the present invention 1011. [Invention 1013] The recombinant polypeptide modulates the target immune system in the manipulated cells of the present invention 1011. [Invention 1014] The recombinant polypeptide is an immune checkpoint blocking antibody, wherein the cells are manipulated according to the invention 1011. [Invention 1015] The recombinant polypeptide modulates tumor vasculogenesis in the manipulated cells of the present invention 1011. [Invention 1016] The engineered cells of the present invention 1015, wherein the recombinant polypeptide is specific to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1. [Invention 1017] The engineered cells of the present invention 1011, wherein the recombinant polypeptide comprises an antibody or fragment thereof specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4. [Invention 1018] The manipulated cells of the present invention 1012, wherein the cytokine comprises IL-12, IL-15, or IL-18. [Invention 1019] The manipulated cells of the present invention 1001, wherein the cells include T cells, NK cells, or NKT cells. [Invention 1020] The manipulated cells of the present invention 1019, wherein the T cells are CD4+, CD8+, CD3+ panT cells, or any combination thereof. [Invention 1021] The manipulated cells of the present invention 1019, wherein the T cells are a mixed population of CD4+ T cells and CD8+ T cells. [Invention 1022] A nucleic acid construct encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises extracellular ligand-binding domains specific to a first antigen and a second antigen on the surface of a cancer cell, the first antigen comprising CAIX, and the second antigen comprising CD70. [Invention 1023] The nucleic acid construct of the present invention 1022 further comprises a transmembrane polypeptide and an intracellular signaling domain. [Invention 1024] The nucleic acid construct of the present invention 1023, wherein the chimeric antigen receptor further comprises a costimulatory domain. [Invention 1025] A nucleic acid construct of the present invention 1023, further encoding a recombinant polypeptide. [Invention 1026] The nucleic acid construct of the present invention 1025, wherein the recombinant polypeptide can be secreted from the manipulated cell. [Invention 1027] A nucleic acid construct of the present invention 1025, wherein the recombinant polypeptide comprises an antibody or a fragment thereof, or a cytokine. [Invention 1028] The recombinant polypeptide is a nucleic acid construct of the present invention 1025, which modulates the target immune system. [Invention 1029] The nucleic acid construct of the present invention 1025, wherein the recombinant polypeptide is an immune checkpoint blocking antibody. [Invention 1030] The nucleic acid construct of the present invention 1025 wherein the recombinant polypeptide regulates tumor vasculogenesis. [Invention 1031] A nucleic acid construct of the present invention 1025, wherein the recombinant polypeptide comprises an antibody or fragment thereof specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4. [Invention 1032] The nucleic acid of the present invention 1027, wherein the cytokine comprises IL12, IL15, or IL18. [Invention 1033] The nucleic acid of the present invention 1030, wherein the recombinant polypeptide is specific to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1. [Invention 1034] A vector comprising the nucleic acid construct of the present invention 1022. [Invention 1035] Cells containing the vector of the present invention 1034. [Invention 1036] A method for treating a subject suffering from cancer, comprising administering a therapeutically effective amount of the manipulated cells of the present invention 1001 to the subject. [Invention 1037] A method for reducing or promoting the regression of cancer in a subject, comprising administering a therapeutically effective amount of the manipulated cells of the present invention 1001 to the subject. [Invention 1038] A method for reducing the proliferation of cancer cells in a subject, comprising administering a therapeutically effective amount of the manipulated cells of the present invention 1001 to the subject. [Invention 1039] A method according to any of items 1036 to 1038 of the present invention, wherein the cancer includes renal cell carcinoma. [Invention 1040] A chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, wherein the extracellular ligand-binding domain is specific to a first antigen and a second antigen on the surface of a cancer cell, the first antigen comprising CAIX, and the second antigen comprising CD70. [Invention 1041] The CAR of the present invention 1040 further comprises a transmembrane polypeptide and an intracellular signaling domain. [Invention 1042] CAR of the present invention 1041, further comprising a co-stimulatory domain. [Invention 1043] The CAR of the present invention 1040, wherein the extracellular ligand-binding domain comprises an antibody or a fragment thereof. [Invention 1044] Cells containing the chimeric antigen receptor (CAR) of the present invention 1040. [Invention 1045] Engineered cells comprising a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises an extracellular ligand-binding domain specific to CAIX, and the second chimeric antigen receptor comprises an extracellular ligand-binding domain specific to CD70. [Invention 1046] Manipulated cells of the present invention 1045 that express and secrete recombinant polypeptides. [Invention 1047] The recombinant polypeptide comprises an antibody or a fragment thereof, or a cytokine, in the manipulated cells of the present invention 1046. [Invention 1048] The recombinant polypeptide modulates the target immune system in the manipulated cells of the present invention 1046. [Invention 1049] The recombinant polypeptide is an immune checkpoint blocking antibody, wherein the cells are manipulated according to the invention 1046. [Invention 1050] The recombinant polypeptide modulates tumor vasculogenesis in the manipulated cells of the present invention 1046. [Invention 1051] The manipulated cells of the present invention 1046, wherein the recombinant polypeptide comprises an antibody or fragment thereof specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4. [Invention 1052] The manipulated cells of the present invention 1047, wherein the cytokine comprises IL12, IL15, or IL18. [Invention 1053] The nucleic acid of the present invention 1050, wherein the recombinant polypeptide is specific to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1. [Invention 1054] The cells described above include T cells or NK cells, and are manipulated cells according to the invention 1045. [Invention 1055] The manipulated cells of the present invention 1054, wherein the T cells are CD4+, CD8+, CD3+ panT cells, or any combination thereof. [Invention 1056] The manipulated cells of the present invention 1054, wherein the T cells are a mixed population of CD4+ T cells and CD8+ T cells. [Invention 1057] The manipulated cells of the present invention 1045, wherein the first chimeric antigen receptor and the second chimeric antigen receptor are expressed from a single nucleic acid construct. [Brief explanation of the drawing]

[0033] [Figure 1]A schematic diagram of bispecific tandem CAR T anti-CD70 and anti-CAIX scFvs combined in different permutations is shown by changing the order of two targeted scFvs with various linkers and hinges connected to the co-stimulatory domain (CD28, 41BB, CD28-41BB, or 41BB-CD28, non-limiting examples) and activation domain (CD3, etc.). Second-generation CAR T cell factories can be designed by introducing a second cassette anti-CD70 scFv to evaluate efficacy (e.g., addressing heterogeneity) and safety (e.g., limiting on-target off-tumor effects). [Figure 2] This study demonstrates that a second-generation CAR T cell factory can be established by combining a series of CARs (different scFvs, linkers, and hinges, etc.) with an immune checkpoint blocker as a payload. Based on these data, eight constructs were established by using G36 as an anti-CAIX scFv and B7 as an anti-CD70 scFv. Those skilled in the art will recognize that any scFv according to the present invention can be utilized. Dual-CAR engineering is the most important part, and the payload was replaced with zsgreen to demonstrate conversion efficiency. [Figure 3] The images show CAIX and CD70 upregulated and co-expressed in ccRCC. IHC staining of primary ccRCC cell lines generated from patient samples shows that both CAIX and CD70 are highly expressed and co-expressed in ccRCC. While we do not wish to be bound by theory, CAIX and CD70 are two potential targets for ccRCC therapy. To validate the targets, IHC was performed for CAIX and CD70 staining in primary cell lines generated from ccRCC patients. These images show 100% positivity for CAIX and CD70 staining, demonstrating that CAIX and CD70 are highly and co-expressed in ccRCC. This is now being validated in studies measuring CAIX and CD70 expression in ccRCC patients in at least 150 samples. [Figure 4]This shows CAIX and CD70 upregulated and co-expressed in ccRCC. IHC staining of patient samples with ccRCC shows that both CAIX and CD70 are highly expressed and co-expressed on ccRCC. [Figure 5-1] This document demonstrates the establishment of CRISPR-operated skrc-59 cell lines. For further in vitro evaluation, four CRISPR-operated skrc-59 cell lines were established with four distinct phenotypes: (i) CAIX+CD70+, (ii) CAIX+CD70-, (iii) CAIX-CD70+, and (iv) CAIX-CD70-. Four CRISPR-operated skrc-59 cell lines have been established. The four distinct phenotypes (i) CAIX+CD70+, (ii) CAIX+CD70-, (iii) CAIX-CD70+, and (iv) CAIX-CD70- are used in the in vitro assays described herein. The corresponding table shows the expression levels of CAIX and CD70 in the four distinct cell lines quantified. [Figure 5-2] Please refer to the explanation in Figure 5-1. [Figure 6] A graph of anti-CD70 minibodies showing selective binding to CD70+SKRC59 cells is shown. Phage display (panned against CD70+skrc-59 cells and subtracted against CD70-skrc-59 cells) shows that a series of anti-CD70 minibodies bound to CD70-positive ccRCC skrc-59 cells. Anti-CD70 minibodies were expressed via Expi293 cells in a 6-well plate. Three days after transfection, the supernatant was collected and IgG quantitative ELISA (Bethyl) was performed to estimate the concentration of minibodies in the supernatant. This approximate concentration was used to normalize the supernatant for the FACS binding curve. Staining was performed via a standard FACS staining protocol using anti-hFc-APC secondary. As can be seen, one of the hits of killing activity (#9) is highly nonspecific. The other two mono-CARs exhibiting killing activity (#3, 7) show good specificity to CD70. [Figure 7]This study demonstrates that anti-CD70 CAR T cells exhibit killing activity in the Celigo killing assay. These anti-CD70 scFv candidates were cloned into vectors, packaged with lentiviruses, and transduced into primary T cells. The CAR T cells were evaluated for antiproliferative activity in the Celigo assay. Results showed that cells #3, #7, and #9 were more effective than CD70 ligand CD27 CAR T cells. These hits were also cloned into pHAGE vectors and subjected to the Celigo killing assay. These graphs show that anti-CD70 CAR T cells exhibited killing activity in the Celigo killing assay, with an effector:target ratio of 2:1. Cell numbers at different time points were compared and normalized to untreated cells at the corresponding time points. Therefore, the Y-axis represents the percentage of treated / untreated cells, and the X-axis represents the different treatments. After 24 hours of co-culture with T cells, the number of SKRC59 cells in the treated group was significantly reduced compared to the untreated and untransduced T cell groups, demonstrating the antiproliferative activity of these CAR T cells. After 48 hours of co-culture, three candidate cells were found that were comparable to CD27, a ligand for CD70. Therefore, cells 3, 7, and 9 were used in further chromium 51 killing assays. [Figure 8] This study demonstrates that anti-CD70 CAR T cells exhibit killing activity against CAIX+CD70+ cells in a chromium 51 killing assay. CAR T cells were also evaluated for killing activity in a chromium 51 release assay. The results showed that CAR T cells #7 exhibited enhanced efficacy compared to CD27 (CD70 ligand) CAR T cells. A 4-hour chromium 51 release assay was performed. After 4 hours of incubation with chromium 51-labeled target cells, B7 was validated for killing activity against SKRC59 CD70+ cells. [Figure 9]This shows second-generation CAR-T cells. CAR T cells can be generated using zsGreen instead of immune checkpoint blockers. Based on these data, eight constructs were established by using G36 as an anti-CAIX scFv and B7 as an anti-CD70 scFv. Dual CAR engineering was the most important part, and the payload was replaced with zsGreen to demonstrate conversion efficiency. [Figure 10] This shows transfected 293T cells that bind to CAIX-PE. 293T cells were transfected with different constructs of dual CARs and subjected to binding assays with PE-labeled CAIX protein. All dual CARs bound to CAIX, and the different orientations of the two scFvs affected the EC50 of anti-CAIX scFv binding. Anti-CAIX scFv G36 preferred the second cassette after the linker. For example, 293T cells were transfected with these eight dual-specific constructs and their corresponding mono-CARs and stained with CAIX-PE. After normalization by transfection efficiency, the different orientations of the two scFvs affected the EC50 of anti-CAIX scFv binding. Anti-CAIX scFv G36 preferred the second cassette after the linker. [Figure 11] This shows transfected 293T cells that bind to CD70-APC. 293T cells were transfected with different constructs of dual CARs, and binding assays were performed using PE-labeled CAIX protein. All dual CARs bound to CD70, and the different orientations of the two scFvs did not affect the EC50 of anti-CD70 scFv binding. Anti-CD70 scFv B7 did not exhibit significant preference. For example, 293T cells were transfected with these eight dual-specific constructs and their corresponding mono-CARs and stained with CD70-PE. After normalization by transfection efficiency, the different orientations of the two scFvs did not affect the EC50 of anti-CD70 scFv binding. Anti-CD70 scFv B7 did not exhibit significant preference. [Figure 12]A B7-GGGGS3-G36 killing assay using Celigo is presented. For example, a selective killing assay was performed using B7-GGGGS3-G36 CAR cells against CAIX+ or CD70+ single-positive cells or CAIX+CD70+ double-positive cells mixed with CAIX-CD70- cells. B7-GGGGS3-G36 CAR T cells exhibited greater killing activity against target cells (CAIX+CD70+) than against non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-). [Figure 13] This report describes a B7-GGGGS3-G36 killing assay by FACS. Skrc-59 cells engineered with four different CRISPR protocols were transduced with BFP fluorescence. The cells were mixed in a 1:1:1:1 ratio and treated with B7-GGGGS3-G36 CAR T cells or culture medium. After treatment, the cells were stained with PE-labeled anti-CD70 antibody and APC-labeled anti-CAIX antibody, and flow cytometry was performed. B7-GGGGS3-G36 exhibited selective killing of CAIX+CD70+ cells, with its population decreasing from 26.7% to 18.5%. [Figure 14] This is a schematic diagram of a finely tuned anti-CAIX CAR T cell. Anti-CAIX scFv cells with various KDs were generated as CAR cells, and their corresponding killing activity was evaluated. To limit on-target off-tumor effects, a second-generation CAR T cell factory was designed by introducing a second cassette of anti-CD70 scFv cells. [Figure 15] This is a rendering of the figure of finely tuned anti-CAIX CAR-T cells. In validation experiments, CAR T cells were generated using zsGreen instead of immune checkpoint blockers. Eight constructs were established by using G36 as the anti-CAIX scFv and B7 as the anti-CD70 scFv. Dual CAR engineering was a key part, and the payload was replaced with zsGreen to demonstrate conversion efficiency. A series of antibodies against CAIX were identified with different KD values ​​ranging from 1.49 nM to 99.58 nM (see also Figure 16). Binding experiments classified the 13 antibodies into four groups. [Figure 16-1] A table is depicted showing the 19 identified anti-CAIX ScFv and their corresponding bindings. [Figure 16-2] Please refer to the explanation in Figure 16-1. [Figure 17] The graph shows the killing of anti-CAIX mono-CAR T cells against CAIX+CD70+ cells. These anti-CAIX CAR T cells were evaluated for antiproliferative activity in the Celigo assay. The results showed that the 19 CAR cells could be divided into four groups based on their killing activity: G37, G39, G125(++++) > G10, G21, G36, G40, G45, G57, G62, G98, G106, G119(+++) > G6, G9, G17, G27, G28(++) > G104(++). [Figure 18] The graph shows the killing of anti-CAIX mono-CAR T cells against CAIX+CD70+. These anti-CAIX CAR T cells were evaluated for antiproliferative activity in the Celigo assay. The results showed that the 19 CARs could be divided into four groups based on their killing activity: G37, G39, G125(++++) > G10, G21, G36, G40, G45, G57, G62, G98, G106, G119(+++) > G6, G9, G17, G27, G28(++) > G104(++). [Figure 19] This study demonstrates the killing of anti-CAIX mono-CAR T cells against CAIX+CD70+. These anti-CAIX CAR T cells were evaluated for antiproliferative activity in the Celigo assay. The results showed that the 19 CAR cells could be divided into four groups based on their killing activity: G37, G39, G125(++++) > G10, G21, G36, G40, G45, G57, G62, G98, G106, G119(+++) > G6, G9, G17, G27, G28(++) > G104(++). [Figure 20]Schematic diagram of a selective killing assay (e.g., using either the Celigo assay or the FACS assay). Two different selective assays were performed to investigate the selectivity of bispecific CAR for four different cell lines. For example, CAIX+CD70+ cells were mixed with monopositive cells, and then CAR cells were added. After incubation for a set period, the number of bipositive and monopositive cells was measured by Celigo. Alternatively, four different cell lines were mixed in a 1:1:1:1 ratio, and CAR cells were added. After 24 hours of co-culture, all cells were collected and stained with CAIX-APC and CD70-PE by FACS. [Figure 21] Graph showing tandem CAR killing against skrc-59 mixed cells. Selective killing assays were performed on CAIX+ or CD70+ single-positive cells or CAIX+CD70+ bipositive cells mixed with CAIX-CD70- cells. Bispecific CAR T cells had greater killing activity against target cells (CAIX+CD70+) than against non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-) with an E:T ratio of 5:1. [Figure 22] Graph showing tandem CAR killing against skrc-59 mixed cells. Selective killing assays were performed on CAIX+ or CD70+ single-positive cells or CAIX+CD70+ bipositive cells mixed with CAIX-CD70- cells. Bispecific CAR T cells had greater killing activity against target cells (CAIX+CD70+) than against non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-) with an E:T ratio of 5:1. [Figure 23] Graph showing tandem CAR killing in skrc-59 mixed cells. Selective killing assays were performed on CAIX+ or CD70+ single-positive cells or CAIX+CD70+ bipositive cells mixed with CAIX-CD70- cells. Bispecific CAR T cells had greater killing activity against target cells (CAIX+CD70+) than non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-) with an E:T ratio of 10:1. [Figure 24] CAIX and CD70 are up-adjusted with ccRCC and co-expressed. [Figure 25] This paper presents the design of CARs to address undesirable side effects previously associated with CART cell therapy. Efficacy and safety profiling are evaluated for the application of CART cell therapy to solid tumors. First, to restore effective anti-cancer immunity, a CAR-T cell factory was developed that can modulate the tumor microenvironment by locally secreting human anti-immune checkpoint blocker monoclonal antibodies (mAbs) at the tumor site, reversing T cell depletion. To limit on-target off-tumor side effects, a finely tuned CAR was designed with a reduced-affinity scFv to expand the therapeutic concentration range by limiting the recognition of tumor-associated antigens on normal tissue. Additionally, a bispecific CAR was designed by introducing a second cassette, such as anti-CD70 scFv, increasing preferential killing in the bi-positive population and resulting in improved safety profiling. [Figure 26] We demonstrate that the CAR-T cell factory exhibits enhanced killing activity. We designed a bicistronic lentiviral vector expressing anti-CAIX scFv linked to the CD28 and CD3z signaling domains in the first cassette and anti-PDL1 mAb in the second expression cassette. Thus, the CAR-T cell factory can target CAIX and secrete checkpoint blocker inhibitors at the tumor site, thereby transforming the suppressive tumor microenvironment. In an orthotopic RCC mouse model, 1E7 CAR-T cells were injected intravenously on day 0, and 2.5E6 CAR-T cells were injected on day 17. In our orthotopic RCC mouse model, the CAR-T cell factory showed enhanced killing activity compared to CAR-T cells secreting unrelated antibodies. [Figure 27] A schematic diagram of CAR-T evaluation in a humanized orthotopic ccRCC mouse model is shown. [Figure 28] MRI images of the RCC mouse model are shown. [Figure 29]A schematic diagram of experiments designed to establish a stress model and compare second and third generation CARs and CD8 vs. CD4 / 8 in an orthotopic ccRCC mouse model is shown. [Figure 30] This demonstrates that 41BB demonstrated superior killing ability in vivo. Efficient tumor regression was observed in the G36-41BB and G36-CD28-41BB treatment groups with 1E7 doses. The mixed CD4 / CD8 G36-41BB CAR-T demonstrated superior killing ability even with 3E6 doses. When comparing two second-generation CAR constructs and one third-generation CAR construct, G36-41BB was more efficient than G36-CD28 and G36-CD28-41BB. [Figure 31] The G36-41BB stress model is shown. 3E6 medication can be used in the G36-41BB stress model. [Figure 32] This demonstrates that the 41BB demonstrated superior lethality in vivo. [Figure 33] 41BB CAR-T cells showed the best proliferation in vivo. [Figure 34] This shows the difference between CD8 and CD4 / CD8. [Figure 35] This shows the in vivo proliferation of CD4 and CD8 T cells. [Figure 36] A schematic diagram of a finely tuned anti-CAIX CAR-T cell, designed to limit on-target off-tumor effects, is shown. While we do not wish to be constrained by theory, reducing the affinity of anti-CAIX scFv leads to the CAR recognizing only high-density CAIX on ccRCCs. A series of anti-CAIX scFv cells with various KDs were cloned into lentiviral vectors to generate corresponding CAR-T cells, and their killing activity was evaluated. [Figure 37]This is a detailed diagram of the "therapeutic concentration range." The "therapeutic concentration range" is a term originally derived from pharmacotoxicology and can refer to the dose range between efficacy and toxicity, achieving the best therapeutic effect without causing unacceptable toxicity. It is the range between the minimum effective dose (MED) and the maximum tolerated dose (MTD). This concept has been applied to optimize CAR-T therapy. To broaden the therapeutic concentration range, the affinity of the CAR to the antigen can be fine-tuned by assembling scFv with different KDs within the CAR construct, such as 1 nm to 100 nm. Ideally, after optimization, the CAR recognizes only high-density antigens on tumor cells, not low-density antigens on normal cells. Targeting antigens expressed only on tumor cells or only on insignificant tissues broadens the therapeutic concentration range because direct toxicity does not occur on the major tissues. On the other hand, targeting antigens expressed in important normal tissues / cells narrows the concentration range by reducing the MTD. [Figure 38] This is a schematic diagram illustrating the expansion of the therapeutic concentration range to address on-target off-tumor side effects. [Figure 39] IHC double staining of patient samples for CAIX and CD70 is shown. CD70 and CAIX were found to be highly expressed and co-expressed on ccRCCs. Therefore, CD70 is selected as the second target. [Figure 40] This shows IHC double staining of patient samples with CAIX and CD70. [Figure 41] As shown in the table, a panel of scFv for CAIX with different KD values ​​ranging from 1.49 nM to 99.58 nM is presented. Corresponding CAR-T cells were generated and screened by the Celigo killing assay. [Figure 42]The study demonstrated a correlation between scFv affinity and CART cell killing. Nineteen CARs were divided into four groups based on cytotoxicity and tested against skrc-59 cells with different CAIX expression levels: G37, G39, G125 > G10, G21, G36, G40, G45, G57, G62, G98, G106, G119 > G6, G9, G17, G27, G28 > G104. [Figure 43] It exhibits "Or" gating, capturing the heterogeneity of tumor cells but not killing healthy cells at low target densities. [Figure 44] CD70 is highly expressed in renal cancer, particularly clear cell renal cell carcinoma, suggesting it could be an ideal target as a secondary target for bispecific CARs. [Figure 45] IHC double staining of CAIX and CD70 in ccRCC patient samples is shown. CD70 and CAIX are highly expressed and co-expressed on ccRCC. Therefore, CD70 is selected as the second target. [Figure 46] IHC double staining of CAIX and CD70 in patient samples of ccRCC is shown. CD70 and CAIX are highly expressed and co-expressed on ccRCC. Therefore, CD70 is selected as the second target. [Figure 47] This shows CAIX and CD70 co-expressed in ccRCC, which have been up-adjusted. [Figure 48]This study demonstrates that anti-CD70 minibodies exhibit selective binding to CD70+ SKRC59 cells. Phage display (panning against CD70+ skrc-59 cells and subtracting against CD70-skrc-59 cells) revealed a series of anti-CD70 minibodies showing promising binding to CD70-positive ccRCC skrc-59 cells. Anti-CD70 minibodies were expressed via Expi293 cells in 6-well plates. Three days after transfection, the supernatant was collected, and IgG quantitative ELISA (Bethyl) was performed to estimate the concentration of minibodies in the supernatant. Using this approximate concentration, the supernatant was normalized for the FACS binding curve. Staining was performed via a standard FACS staining protocol using anti-hFc-APC secondary. One of the hits of killing activity (#9) was highly nonspecific. The other two mono-CARs exhibiting killing activity (#3, 7) showed good specificity for CD70. [Figure 49] This study demonstrates promising killing of CD70+skrc-59 cells by anti-CD70 B7 CAR-T cells. Phage display (panning against CD70+skrc-59 cells and subtracting against CD70-skrc-59 cells) revealed a series of anti-CD70 minibodies showing promising binding to CD70-positive ccRCC skrc-59 cells. These hits were cloned into lentiviral vectors, and Celigo killing assays were performed on corresponding CART cells. From the screening, B7 was identified as a candidate. [Figure 50] The bispecific CAR constructs are shown. The series of constructs were established at different rotations (or directions) with different linkers by using G36 as the anti-CAIX scFv and B7 as the anti-CD70 scFv. [Figure 51]This shows that anti-scFv G36 preferentially resides in the second cassette after the linker. 293T cells were transfected with these eight bispecific constructs and corresponding monoCARs and stained with CAIX-PE. After normalization by transfection efficiency, it shows that the different orientations of the two scFvs affect the EC50 of anti-CAIX scFv binding. Anti-CAIX scFv G36 preferentially resides in the second cassette after the linker. [Figure 52] Anti-CD70 scFv B7 does not exhibit preference. 293T cells were transfected with different constructs of dual CAR, and binding assays were performed with APC-labeled CD70 protein. After normalization by transfection efficiency, it was shown that the different orientations of the two scFvs did not affect the EC50 of anti-CD70 scFv binding. Anti-CD70 scFv B7 does not exhibit significant preference. [Figure 53] This document demonstrates the establishment of four CRISPR-engineered skrc-59 cell lines. For further in vitro evaluation, four CRISPR-engineered skrc-59 cell lines were established with four distinct phenotypes: CAIX+CD70+, CAIX+CD70-, CAIX-CD70+, and CAIX-CD70-. [Figure 54] This demonstrates that B7-GGGGS3-G36 exhibits preferential killing. Using B7-GGGGS3-G36 CAR cells as an example, selective killing assays were performed against CAIX+ or CD70+ single-positive cells, or CAIX+CD70+ double-positive cells mixed with CAIX-CD70- cells. B7-GGGGS3-G36 CAR-T cells were shown to have preferential killing activity against target cells (CAIX+CD70+) rather than non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-). After incubation for a set period, the number of double-positive and single-positive cells was measured by Celigo. Furthermore, B7-GGGGS3 was found to have a limited selective index against CAIX+CD70+ while mixed with CAIX+ cells. [Figure 55]This study demonstrates that B7-GGGGS3-G36 exhibits selective killing. Four different CRISPR-engineered skrc-59 cells were transduced with BFP fluorescence. The cells were mixed in a 1:1:1:1 ratio and treated with B7-GGGGS3-G36 CAR-T cells or culture medium. After treatment, the cells were stained with PE-labeled anti-CD70 antibody and APC-labeled anti-CAIX antibody and subjected to flow cytometry. B7-GGGGS3-G36 exhibited selective killing of CAIX+CD70+ cells, with its population reduced from 26.7% to 18.5%. This selective killing data from FACS also showed that B7-GGGGS3-G36 exhibited selective killing of CAIX+CD70+ cells, with its population reduced from 26.7% to 18.5%. [Figure 56] A schematic diagram of bispecific split CAR T is shown. Anti-CD70 and anti-CAIX scFv were expressed on the cell surface in different co-stimulatory domains. See, for example, dual(split)CAR T, Nat Rev Cancer 16(9):566-81. [Figure 57] This study demonstrates fragmented CAR killing in primary ccRCC cancer cells. Fractionated CAR T cells were evaluated for their killing activity against primary ccRCC cancer cells using mono-CAR T and tandem CAR T cells. It showed that fragmented CAR achieved superior killing at low E:T ratios, such as 1:1. [Figure 58] This provides multiple arrangements of the amino acid sequence of the anti-carbonic anhydrase IX (G250) scFv clone. [Figure 59] Provides alignment of human and mouse CAIX amino acid sequences. [Figure 60] The structure of carbonic anhydrase IX (G250) is provided. [Figure 61] This shows a CAR T cell killing assay using Skrc-59 transdermal cells. Celigo image cytometry is also shown. [Figure 62]This shows homology studies of 10 different species. Tree: The distance of each branch is equal to the number of differences between sequences (e.g., 0.1 means the difference between the two sequences is 10%), and the distance between two species is equal to the sum of the lengths of all the branches connecting them. Homology > 60% → Potential cross-reactivity. [Figure 63] This study demonstrates the establishment of stable skrc-59 cell lines expressing CAIX or CD70 from different species. CRISPR was used to knock out CAIX- / CD70-skrc-59 cells, which were then transduced with 10 different constructs (CD70 or CAIX from 5 different species) and classified by FACS. Half-transduction was performed using BFP (for Celigo). Staining was performed with commercially available antibodies. [Figure 64] The binding assay for anti-CAIX(G36)scFv is shown. Binding data from commercially available antibodies were normalized. Analysis was performed using nonlinear regression and log-(agonist) pair-response models. [Figure 65] This shows the killing assay for anti-CAIX(G36)CAR T cells. 100% of monkeys (same killing effect as humans, cross-reactivity), 50% of mice and hamsters (significant killing effect). [Figure 66] This shows an assay for killing anti-CD70(B7)CAR T cells. [Figure 67] This paper presents 20 different anti-CAIX CAR T cell killing assays. [Figure 68] This shows the G36 E:T 10:1 lethal assay with varying magnification. [Figure 69] The G36 lethal assay shows an E:T ratio of 5:1. [Figure 70] This shows the killing assay G36, n=2. [Figure 71] The killing assay showed 20 anti-CAIX scFv cells. [Figure 72] Show all candidate monkeys. [Figure 73] All candidate mice are shown. [Figure 74] Showing all candidate hamsters. [Figure 75] The B7 killing assay with magnification changes is shown. [Figure 76-1] The amino acid sequence and germline alignment of the anti-cd70 antibody are shown. [Figure 76-2] Please refer to the explanation in Figure 76-1. [Figure 77] The nucleic acid constructs of the fragmented car are shown. [Figure 78] The amino acid sequences and germline alignment of anti-PDL1 and anti-PD1 sequences are shown. [Figure 79] The amino acid sequence and germline alignment of the anti-CAIX antibody are shown. [Figure 80] Tandem CAR B7-GGGGS5-G36 exhibits cytotoxicity. [Figure 81-1] The amino acid sequence and germline alignment of the anti-TIGIT antibody are shown. [Figure 81-2] Please refer to the explanation in Figure 81-1. [Figure 81-3] Please refer to the explanation in Figure 81-1. [Figure 82] This shows the anti-PD-L1 amino acid sequence. [Figure 83-1] The anti-PD1 nucleic acid and amino acid sequence are shown. [Figure 83-2] Please refer to the explanation in Figure 83-1. [Figure 83-3] Please refer to the explanation in Figure 83-1. [Figure 83-4] Please refer to the explanation in Figure 83-1. [Figure 83-5] Please refer to the explanation in Figure 83-1. [Modes for carrying out the invention]

[0034] Chimeric antigen receptor (CAR) T-cell therapy represents an exciting area of ​​discovery that has already revolutionized the treatment of several blood-derived cancers. For example, in acute lymphoblastic leukemia (ALL), it has demonstrated remission rates of 80-90% and has been approved by the FDA. This technology can fight cancer by utilizing a patient's own immune cells and designing them to better recognize specific proteins located on cancer cells. For example, after the modifications are made in the laboratory, the immune cells can grow extensively in the lab, doubling their potential to kill cancer cells, and are eventually injected into the patient, where their increased efficacy and numbers allow them to attack cancer wherever it may be in the body.

[0035] CAR T-cell therapy has been largely elusive in solid tumors such as renal cell carcinoma (RCC) because cancer cells create a tumor microenvironment that turns off immune penetration. We have succeeded in creating an advanced mouse model that can provide crucial information for this technique. Our approach relies on creating humanized RCC in mice that can be studied to better understand their immunological fingerprint. By investigating how human RCC behaves in mice, the lab was able to identify antigens that can be targeted using CAR T-cell therapy in humans and other mammals, such as carbonic anhydrase IX [CAIX] and CD70. For the most part, these two antigens, namely CAIX and CD70, are found together only on renal cancer tumor cells, allowing the immune system to attack locally with minimal impact on healthy tissue. There has also been progress in counteracting immunosuppressive effects within the tumor microenvironment by manipulating T cells to produce and / or secrete checkpoint-blocking antibodies. Combining these approaches, dramatically improved efficacy within the tumor microenvironment should be possible.

[0036] Described herein are, for example, CAR T cells targeting CAIX and CD70, which can be administered to animals while closely monitoring changes in the tumor microenvironment and subsequent antitumor effects. Those skilled in the art will recognize that these approaches can also be combined with currently available technologies that have already had a positive impact on the clinical landscape of RCC, such as the use of immune checkpoint blockers.

[0037] A detailed description of one or more embodiments is provided herein. However, it will be understood that the present invention can be embodied in a variety of forms. Accordingly, the specific details disclosed herein should not be construed as limitations, but rather as representative grounds for the claims and for teaching those skilled in the art to use the invention in any suitable way.

[0038] The singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise. The use of the words “a” or “an” in the claims and / or specification with the term “to comprise” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.”

[0039] Whenever the phrases "for example," "etc.," and "including" are used herein, it is understood that they are always accompanied by the phrase "without limitation" unless otherwise explicitly stated. Similarly, "for example," "exemplary," etc., are understood to be non-limiting.

[0040] The term "substantially" allows for deviations from descriptive terms that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term "substantially," even if the word "substantially" is not explicitly listed.

[0041] The terms “comprising” and “including,” as well as “having” and “involving” (and similarly “comprises,” “includes,” “has,” and “involves”) are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general U.S. patent law definition of “comprising,” and is therefore interpreted as having the open-term meaning of “at least the following,” and also as not excluding additional features, limitations, aspects, etc. Thus, for example, “a process comprising steps a, b, and c” means that the process comprises at least steps a, b, and c. Whenever the terms “a” and “an” are used, they are understood as “one or more,” unless such interpretation is meaningless in the context.

[0042] As used herein, the term “approximately” can mean roughly, broadly, approximately, or within a range thereof. When the term “approximately” is used with a numerical range, it modifies that range by extending the upper and lower boundaries of the stated numerical value. Generally, the term “approximately” is used herein to mean changing a numerical value by a 20 percent variation (up or down) above or below the stated value.

[0043] Chimeric antigen receptor (CAR) T cell therapy Chimeric antigen receptor (CAR) T-cell therapy uses exogenous expression of CARs to redirect a patient's T cells to kill tumor cells. Referring to Figure 1, for example, a CAR is a transmembrane fusion protein that ligates the antigen-recognition domain of an antibody or fragment to the intracellular signaling domain of a T cell receptor and co-receptor. For example, a chimeric antigen receptor fuses an antigen-specific antibody fragment to the T cell costimulatory domain and the CD3 zeta intracellular signaling domain, enabling the redirection of T cells to the target cell, such as a tumor cell, where the antigen has been presented.

[0044] In this specification, the term “antibody” is used in its broadest sense and can refer to a molecule containing an immunoglobulin molecule and an immunoglobulin (Ig) molecule, i.e., an antigen-binding site that specifically binds to (and immunely reacts with) an antigen. “Specifically binds” or “immunely reacts with” can refer to an antibody that reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides. Antibodies of the present invention include, but are not limited to, polyclonal, monoclonal, humanized, fully human, bispecific, multispecific, chimeric, dAb (domain antibodies), single-chain antibodies, Fab, Fab' and F(ab')2 fragments, scFv, diabodies, minibodies, scFv-Fc fusions, and Fab expression libraries. Unless otherwise expressly stated, references to “antibody (singular)” or “antibody (plural)” made herein encompass any (or all) of these molecules, for example, insofar as they exhibit the desired antigen-binding activity.

[0045] Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH::VL heterodimers that can be expressed from gene fusions containing VH-coding and VL-coding genes linked by peptide-coding linkers. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). Numerous methods have been described for identifying the chemical structures necessary to convert naturally aggregated but chemically separated light and heavy polypeptide chains derived from the antibody V region into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patents 5,091,513, 5,132,405, and 4,946,778.

[0046] Solid tumors present unique challenges to CAR-T therapy. Unlike hematological malignancies, tumor-related target proteins are overexpressed in both tumor and healthy tissue, resulting in on-target / off-tumor T cell killing of healthy tissue. Furthermore, immunosuppression in the tumor microenvironment (TME) limits the activation of CAR-T cells that kill tumors. The embodiments of the disclosure address these issues. For example, embodiments include T cells containing a bispecific CAR that targets two antigens on cancer cells and mitigates on-target / off-tumor T cell killing. Referring to Figure 12, for example, B7-GGGGS-G36 CAR T cells had greater killing activity against targeted renal cancer cells (CAIX+CD70+) than against non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-). In embodiments, the bispecific CAR can also (b) secrete checkpoint-blocking antibodies that eliminate suppression in the tumor microenvironment. Other embodiments include a finely tuned CAR that recognizes only high-density antigens on tumor cells, rather than low-density antigens on normal cells. While we do not wish to be constrained by theory, this could be achieved, for example, by reducing the affinity of one or more antibodies associated with the CAR.

[0047] For example, Figure 1 provides a schematic diagram of a bispecific CAR targeting two antigens, such as CAIX and CD70. A bispecific CAR can refer to a CAR that has binding specificity to at least two different antigens. For example, a bispecific CAR can include a monoclonal antibody, such as a human or humanized antibody, or a fragment thereof. In this case, one of the binding specificities is CAIX and / or CD70. The second binding target is any other antigen, and advantageously, it may be a cell surface protein, receptor, or receptor subunit. For example, one of the binding specificities is for CAIX, and the second binding specificity is for CD70.

[0048] As reported (see, e.g., J Clin Oncol 24(2006)20-22; Molecular Therapy 21(2013)4), the first clinical trial of first-generation anti-CAIX G250 CAR-T cells in patients with renal cell carcinoma (RCC) failed to open the door to on-target off-tumor side effects. Two of the first three patients developed hepatitis due to CAIX expression in the bile ducts. The introduction of secondary antibodies such as secondary scFv may improve patient safety, as dual-target CAR T cells can reduce or eliminate on-target / off-tumor effects. For example, as shown in Figure 24, CAIX and CD70 expression are upregulated and co-expressed on ccRCC. On the other hand, CAIX is expressed in the bile ducts (mainly cytoplasmic), while CD70 is not expressed in the bile ducts. See, e.g., British Journal of Cancer 103(2010)676-684.

[0049] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Methods for preparing bispecific antibodies are known in the art. See, for example, U.S. Patent No. 8,329,178, which is incorporated herein by reference in its entirety.

[0050] Antibody molecules derived from humans belong to one of the IgG, IgM, IgA, IgE, and IgD classes, which differ from one another in the properties of the heavy chains present in the molecules. Certain classes also have subclasses such as IgG1, IgG2, and others. Furthermore, in humans, the light chain can be either a kappa chain or a lambda chain.

[0051] The term “antigen-binding site” or “binding region” can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly distinct stretches within the V regions of the heavy and light chains, called “hypervariable regions,” are inserted between more conserved adjacent stretches known as “framework regions” or “FR.” Thus, the term “FR” refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions of an immunoglobulin. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of the heavy and light chains are called “complementarity-determining regions” or “CDR.” See, for example, Tables 1 and 3, which provide CDRs for anti-CAIX and anti-CD70 antibodies.

[0052] A new mechanism associated with tumor progression is the immune checkpoint pathway, which involves cell interactions that prevent excessive activation of T cells under normal conditions, allowing T cell function in a self-limiting manner. As an evasion mechanism, many tumors can stimulate the expression of immune checkpoint molecules, resulting in an anergistic phenotype of T cells that cannot suppress tumor progression. For example, new clinical data highlight the importance of a single inhibitory ligand-receptor pair, programmed death ligand 1 (PD-L1, B7-H1 and CD274) and programmed death receptor 1 (PD-1, CD279), as an immune checkpoint that prevents the killing of cancer cells by cytotoxic T lymphocytes. The PD1 receptor is expressed in many cell types, including T cells, B cells, natural killer cells (NK), and host tissues. Tumor and antigen-presenting cells (APCs) expressing PD-L1 can block T cell receptor (TCR) signaling of cytotoxic T lymphocytes via binding to the receptor PD-1, reducing cytokine production and T cell proliferation. Overexpression of PD-L1 is found in many tumor types and mediates immunosuppressive function through interaction with other proteins, including CD80(B7.1), and blocks the ability to activate T cells via binding to CD28.

[0053] Genetic engineering of human lymphocytes expressing tumor-targeting chimeric antigen receptors (CARs) can generate anti-tumor effector cells that circumvent tumor immune evasion mechanisms due to abnormalities in the processing and presentation of protein antigens. Furthermore, these transgenic receptors can target tumor-associated antigens that are not protein-derived. In certain embodiments of this disclosure, there are lymphocytes modified to contain at least one CAR (CART), and in certain embodiments of the invention, a single CAR targets two or more antigens (e.g., bispecific CARs). In some embodiments, the cells contain fragmented CARs such as anti-CD70 and anti-CAIX scFv expressed on the cell surface having different co-stimulatory domains. Furthermore, some embodiments contain finely tuned CARs. In some embodiments, the CART is further modified to express and secrete one or more polypeptides, such as antibodies or cytokines such as IL-12, IL-15, or IL-18. Such CARTs are referred to herein as armed CARTs or CAR factories. Armed CART enables the simultaneous local secretion of polypeptides at the target site (i.e., the tumor site).

[0054] For example, referring to Figure 56, a split CAR contains two or more CARs on the surface of a cell such as a T cell or NK cell. The CARs may be specific to two or more antigens, such as CD70 and CAIX. Figure 77 provides an example of a nucleic acid construct encoding a split CAR. In this example, the first CAR is specific to CAIX and the second CAR is specific to CD70. As described herein, the CARs may be in any desired orientation. For example, the first CAR may be specific to CD70 and the second CAR may be specific to CAIX. As shown in the example, the first and second CARs can be expressed from a single nucleic acid construct. In such an example, the nucleic acid encoding a cleavable linker may be placed between the nucleic acid encoding the first CAR and the nucleic acid encoding the second CAR. In other embodiments, the two CARs may be from the same cell but from two separate nucleic acid constructs.

[0055] Modified TCRs called chimeric antigen receptors (CARs), such as CARs containing single-chain variable antibody fragments (scFvs) previously selected for their high affinity to specific tumor-associated antigens, represent a powerful new approach to cancer. The scFv presented in the CAR is linked to an intracellular signaling block containing CD3ζ, inducing T cell activation followed by antigen binding. This structure is characteristic of first-generation CARs and has been improved in second-generation CARs, which link CD28, 4-1BB, or OX40 signaling costimulatory endodomains to CD3, or in third-generation CARs, which tandem link the two elements to CD3ζ. These endodomains are necessary for complete T cell activation during TCR recognition by antigen-presenting cells (APCs), improving cytokine production and proliferation of CAR-T cells. Due to the difficulty in finding specific tumor-associated antigens, inefficient T cell homing to tumor sites, reduced T cell persistence in the body, and the immunosuppressive microenvironment of solid tumors, the effectiveness of CAR cells in treating solid tumors has been modest until now.

[0056] In certain cases, lymphocytes may contain chimeric, non-natural, and at least partially man-made receptors. In certain cases, the man-made chimeric antigen receptor (CAR) has one, two, three, four, or more components, and in some embodiments, one or more components facilitate the lymphocyte's targeting or binding to one or more tumor antigen-containing cancer cells.

[0057] The CAR according to the present invention comprises at least one transmembrane polypeptide comprising at least one extracellular ligand-binding domain and one transmembrane polypeptide comprising at least one intracellular signaling domain, wherein the polypeptides assemble together to form a chimeric antigen receptor. Exemplary CARS useful in embodiments of this disclosure include, for example, those disclosed in PCT / US2006 / 046350, PCT / US2015 / 067178, PCT / US2015 / 067225, and PCT / US2019 / 022272, each of which is incorporated herein by reference in whole.

[0058] As used herein, the term “extracellular ligand-binding domain” may refer to an oligonucleotide or polypeptide capable of binding to a ligand. The domain can interact with cell surface molecules. For example, an extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[0059] In particular, the extracellular ligand-binding domain may include an antigen-binding domain or antigen-recognition domain derived from an antibody against the target antigen. The antigen-binding domain or antigen-recognition domain may be an antibody fragment. An "antibody fragment" may be a molecule other than an intact antibody that includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. For example, referring to Figure 1, one embodiment includes a CAR with two scFv as the antigen-recognition domain. For example, referring to Figure 14, one embodiment includes a CAR with one scFv as the antigen-recognition domain.

[0060] The antigen recognition domain can target any desired antigen target. In embodiments, the desired antigen target is located on the surface of a cell, such as the surface of a cancer cell. Non-limiting examples of antigen targets include CAIX and / or CD70.

[0061] In some embodiments, CAR is specific to CAIX and / or CD70.

[0062] In embodiments, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) containing light chain (VL) and heavy chain (VH) variable fragments of a target antigen-specific monoclonal antibody conjugated by a flexible linker. Those skilled in the art will recognize that embodiments may include different linkers typically known in the art. See, for example, Chen, et al., “Fusion protein linkers: property, design and functionality,” Advanced drug delivery reviews 65.10(2013):1357-1369, which is incorporated herein by reference in its entirety. For example, different linkers can be used to fine-tune the dual-targeting CAR construct. The length of the linker may vary depending on the antibody of the dual-targeting CAR construct, the angle of their approach to the target epitope, and the topography of the target on the tumor cell membrane. For example, referring to Figure 2, flexible linkers may include GGGS1, GGGGS3, GGGGS5, or IgG1 hinges. In some embodiments, the number of Gs in the linker can be 2, 3, 4, 5, 6, or 7, in combination with any of S1, S2, S3, S4, S5, or S6. For example, the scFv antibody is specific to CAIX and / or CD70. For example, as shown in Figures 10 and 50, the orientation of the scFv relative to the linker can vary. In one nucleic acid construct shown in Figure 50, the anti-CAIX scFv may be in the first cassette (i.e., before the linker) and the anti-CD70 cassette may be in the second cassette (i.e., after the linker). Alternatively, the anti-CAIX scFv may be in the second cassette and the anti-CD70 scFv may be in the first cassette. For example, referring to Figure 51, the anti-CAIX scFv G36 may be in the first cassette and the anti-CD70 B7 may be in the second cassette. Alternatively, anti-CAIX scFv G36 may be in the second cassette, and anti-CD70 B7 may be in the first cassette. Linkers of various lengths and flexibility can be used as described herein. As shown, different orientations of the two scFvs may affect the coupling.For example, G36 exhibits higher binding affinity than the one operated as the second cassette. On the other hand, as shown in Figure 11, anti-CD70 scFv B7 does not have a significant preference.

[0063] Examples of antibodies useful for constructing CARs as disclosed herein include those detailed in Tables 1, 2, 3, or 4 herein. See also, for example, WO / 2007 / 065027 and WO / 2016 / 100985, whose contents are incorporated herein by reference in their entirety.

[0064] scFVs targeting antigen recognition domains useful for constructing CAR-Ts, such as CAIX and / or CD70, can be synthesized, manipulated, and / or produced using nucleic acids (e.g., DNA). DNA encoding antigen recognition domains can be cloned in frame into DNA encoding necessary CAR-T elements of immunologically interesting molecules, such as, but not limited to, CD28 and 41BB and CD3-zeta intracellular signaling domains, including, but not limited to, CD8 hinge regions, transmembrane domains, and costimulatory domains. See, for example, Figure 2.

[0065] As a non-limiting example, binding domains other than scFv, such as single-domain antibody fragments or receptor ligands from camelids, antibody-binding domains, antibody hypervariable loops, or CDRs, can also be used for predefined targeting of lymphocytes.

[0066] In one embodiment, the transmembrane domain further includes a stalk region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term “stalk region” may mean any oligo or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region is used to provide greater flexibility and accessibility to the extracellular ligand-binding domain. The stalk region may contain up to 300 amino acids, e.g., 10 to 100 amino acids, e.g., 25 to 50 amino acids. The stalk region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of the antibody constant region. Alternatively, the stalk region may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or it may be a completely synthetic stalk sequence. In one embodiment, the stalk region is part of a human CD8 alpha chain.

[0067] The signal-converting domain or intracellular signaling domain of the CAR of the present invention is involved in intracellular signaling after binding of the extracellular ligand-binding domain to its target, resulting in the activation of immune cells and an immune response. In other words, the signal-converting domain is involved in the activation of at least one of the normal effector functions of the immune cells on which the CAR expresses. For example, the effector function of a T cell may be cytolytic activity, including cytokine secretion, or helper activity. Therefore, the term “signal-converting domain” can refer to a portion of a protein that converts effector signaling function signals and instructs cells to perform specific functions.

[0068] The signal conversion domain can include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence can include a signaling motif known as an immune receptor-activating tyrosine motif, which is an ITAM. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention, in non-limiting examples, may include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In another embodiment, the signal conversion domain of the CAR can include a CD3 zeta signaling domain or an intraplasmic domain of an Fc epsilon RI beta or gamma chain. In another embodiment, signaling is provided by CD3 zeta along with co-stimulation provided by CD28 and tumor necrosis factor receptors (TNFr) (such as 4-1BB or OX40).

[0069] In one embodiment, the intracellular signaling domain of the CAR of the present invention includes a co-stimulatory signaling molecule. In some embodiments, the intracellular signaling domain includes two, three, four or more co-stimulatory molecules in tandem. The co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand necessary for an efficient immune response.

[0070] A "costimulatory ligand" can refer to a molecule on an antigen-presenting cell that specifically binds to a co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including but not limited to proliferative activation and differentiation, in addition to the primary signal provided by, for example, the binding of a peptide-carrying MHC molecule to the TCR / CD3 complex. Costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), cell adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor and ligand-binding agonists or antibodies, and Costimulatory ligands may include, but are not limited to, ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to ligands that specifically bind to, but are not limited to, costimulatory molecules present on T cells, such as, but are not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83.

[0071] A “costimulatory molecule” can refer to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, including but not limited to proliferation. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0072] In another specific embodiment, the signal conversion domain is a TNFR-related factor 2 (TRAF2) binding motif, which is the cytoplasmic tail of a co-stimulatory TNFR member family. The cytoplasmic tail of a co-stimulatory TNFR family member contains a TRAF2 binding motif consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD) (where X is any amino acid). TRAF proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.

[0073] Chimeric antigen receptors fuse an antigen-recognition domain with a signaling domain (also called a stimulating domain) that modulates (i.e., stimulates) cellular signaling. Non-exclusive examples of such stimulating domains include those of the CD28, 41BB, and / or CD3-zeta intracellular signaling domains. See, for example, Figure 2.

[0074] A distinguishing feature of a suitable transmembrane polypeptide is its ability to be expressed on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and to interact to induce a cellular response of immune cells against a given target cell. Different transmembrane polypeptides of the CAR of the present invention, including an extracellular ligand-binding domain and / or a signal-converting domain, interact together to participate in signal conversion after binding to a target ligand and induce an immune response. The transmembrane domain may be derived from either natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0075] As used herein, the term “part” may refer to any subset of a molecule, i.e., a shorter peptide. Alternatively, an amino acid sequence functional variant of a polypeptide may be prepared by mutations in the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions may be performed to arrive at the final construct, provided that the final construct has the desired activity and, in particular, exhibits specific anti-target cellular immune activity. The functionality of the CARs of the present invention in host cells is detectable by assays suitable for demonstrating the signaling ability of the CARs when a specific target is bound. Such assays are available to those skilled in the art. For example, such assays may enable the detection of signaling pathways triggered upon target binding, such as assays involving the measurement of increased calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or the production of interleukin (IL) 2, interferon gamma, GM-CSF, IL-3, and IL-4 resulting therefrom.

[0076] Carbonic anhydrase IX (CAIX) Several mAbs that react with surface antigens on RCCs have been identified. These include mAbs that recognize differentiated and overexpressed antigens, as well as mAbs that identify RCC-related antigens not expressed in normal kidneys (Michael, 2003; Yang, 2003). The CAIX gene, also known as G250 and MN, is located on chromosome 9p12-13, binds to zinc, and encodes a transmembrane protein with CA activity (Zavada, 1997; Grabmaier, 2000). In HeLa cells and RCC cell lines derived from human cervical cancer, CAIX / G250 / MN / is found in the plasma membrane and is observed as a nucleoprotein with apparent molecular weights of 58 and 54 kDa. It is N-glycosylated, and in the non-reduced state, it forms oligomers (Pastorekova, 1992). Sequence analysis of the predicted CAIX protein shows that it contains a signal peptide (aa1-37), an extracellular (EC) portion (aa38-414), a 20-amino acid hydrophobic transmembrane region (aa415-434), and a small 25-amino acid C-terminal extracellular portion (aa435-459). The amino acid sequences of human and mouse CAIX are shown in Figure 59. The extracellular portion consists of two distinct domains. The region between the signal peptide and the CA domain (aa53-111) shows significant homology (38% identity) to the keratan sulfate attachment domain of aggrecan, a large aggregated proteoglycan in humans (Doege, 1991). In the PG-like domain of CAIX, a hexapeptide motif with consensus EEDLPE (sequence number [ ]) is repeated seven times. The carbonic anhydrase domain is located near the plasma membrane (aa135-391). The CAIX antigen appears during malignant transformation and stains positively in approximately 95% of clear cell type RCC specimens, as well as in most renal cell metastases.

[0077] Epitopes expressed on the surface of tumor cells, unlike intracellular antigens, are excellent targets for humoral anticancer therapy because they are accessible to circulating antibodies in vivo. Human monoclonal antibodies (mAbs) are a well-tolerated treatment option for the increasing prevalence of cancer. The concept of selective tumor targeting by antibodies is based on the greedy interaction between antibodies and antigens expressed on malignant cells rather than normal tissues. Many mechanisms have been proposed for the ability of antibodies against tumors to mediate their effects in vivo. For example, the involvement of antibody Fc domains and effector cell FcγR leads to antibody-dependent cell-mediated cytotoxicity (ADCC). Some (antagonistic or inhibitory) antibodies can block signaling on tumor cells and thus synergistically act with the immune effector response by making tumor cells more sensitive to apoptosis or lytic cell death induced by immune effector cells (Baselga, 1998). Another method that utilizes antibodies involves the construction and functional expression of "T bodies" on T lymphocytes, also known as chimeric immunoreceptors or otherwise "designer T cells." The antigen-binding domain of a chimeric receptor may consist of an antigen-binding domain, e.g., a single-chain antibody (scFv), while the intracellular signaling domain derives from the cell membrane portion of a membrane-bound receptor that can induce cell activation (Maher, 2002; Pinthus, 2003). T lymphocytes transplanted with chimeric receptors have the combined advantages of MHC-independent and antibody-based antigen binding, with efficient T cell activation upon specific binding to the receptor ligand. This activation leads to the production and secretion of cytokines such as IL-2, interferon, GM-CSF, and TNF-α. Antigen-specific lysis of tumor cells has been reported both in vitro and in vivo. T lymphocytes can be permanently transplanted with antigen-specific chimeric receptors by retroviral transduction of a vector construct encoding a selected receptor molecule (Riviere review, 2004).

[0078] Embodiments of the present invention may include isolated human monoclonal antibodies or fragments thereof that immunospecifically bind to the carbonic anhydrase IX (G250) protein. Such antibodies can reduce the carbonic anhydrase activity of the protein. For example, such anti-CAIX antibodies may include those described in WO2007 / 065027 and US8,466,263, each of which is incorporated herein by reference in whole. See, for example, Figure 58, which provides a multiple arrangement of amino acid sequences of anti-carbonic anhydrase IX (G250) scFv clones. For example, embodiments of the present invention may include single-chain antibodies, e.g., scFv G6, G9, G10, G17, G21, G27, G28, G36, G37, G39, G40, G45, G57, G62, G98, G104, G119, or G125, as well as any other scFv identified according to the methods disclosed herein.

[0079] For example, Table 1 shows the amino acids of the CDR of an anti-CAIX antibody.

[0080] (Table 1) TIFF0007886110000001.tif211154TIFF0007886110000002.tif63154

[0081] For example, Table 2 shows the amino acid sequences of the VH and VL regions of the anti-CAIX antibody.

[0082] (Table 2) TIFF0007886110000003.tif139154TIFF0007886110000004.tif231154TIFF0007886110000005.tif231154TIFF0007886110000006.tif105154

[0083] Embodiments may also include consensus sequences of any of the amino acid sequences described herein. For example, if four or more clones have the same amino acid at a given position, that position in the consensus is designated by that amino acid.

[0084] CD70 CD70 is found on the surface of kidney tumor cells (e.g., clear cell carcinoma and papillary carcinoma), pancreas, larynx or pharynx, melanoma, ovarian, lung adenocarcinoma, colon, breast, and brain. See, for example, British Journal of Cancer 103(2010)676-684.

[0085] Embodiments of the present invention may include isolated human monoclonal antibodies or fragments thereof that bind immunospecifically to CD70. For example, embodiments of the present invention may include single-chain antibodies such as scFv A20, B2, B3, B5, B7, B8, or B9, as well as any other scFv identified according to the methods disclosed herein.

[0086] For example, Table 3 shows the amino acids of the CDR of the anti-CD70 antibody.

[0087] (Table 3) TIFF0007886110000007.tif71154

[0088] For example, Table 4 shows the amino acid sequences of the VH and VL regions of the anti-cd70 antibody.

[0089] (Table 4) TIFF0007886110000008.tif178154

[0090] Embodiments may also include consensus sequences of any of the amino acid sequences described herein. For example, if four or more clones have the same amino acid at a given position, that position in the consensus is designated by that amino acid.

[0091] cell Embodiments of this disclosure include cells expressing a CAR (i.e., CART). The cells may be of any kind, including immune cells capable of expressing a CAR for cancer treatment, or bacterial cells possessing an expression vector encoding a CAR. As used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable. All these terms also include their offspring, which are any subsequent generations. For example, due to planned or accidental mutations, not all offspring may be identical. In the context of expressing heterologous nucleic acid sequences, “host cell” may refer to a eukaryotic cell capable of replicating the vector and / or expressing the heterologous gene encoded by the vector. Host cells can and have been used as recipients of vectors. Host cells may be “transfected” or “transformed,” which refers to the process by which an exogenous nucleic acid is introduced into or into a host cell. Transformed cells include primary subject cells and their offspring. As used herein, the terms “engineered” and “recombinant” cells or host cells may refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Recombinant cells can therefore be distinguished from naturally occurring cells that do not contain the nucleic acid introduced by recombination. In embodiments of the present invention, host cells are T cells, including cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), and NK cells and NKT cells are also included in this disclosure.

[0092] Some vectors can utilize regulatory sequences that allow them to replicate and / or be expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above host cells can be incubated and maintained, and the conditions under which the vectors can be replicated. Also understood and known are the techniques and conditions that enable the large-scale production of vectors, as well as the production of nucleic acids encoded by the vectors and their homologous polypeptides, proteins, or peptides.

[0093] The cells may be autologous cells, syngeneic cells, allogeneic cells, or, in some cases, heterogeneic cells.

[0094] There are many situations where it may be desirable to be able to kill modified CTLs, such as when it is desirable to terminate treatment, when cells become neoplastic, in research where the absence of the cells after their presence is of interest, or for other reasons. For this purpose, it is possible to provide the expression of specific gene products that can kill modified cells under controlled conditions, such as inducible suicide genes.

[0095] Armed CART The present invention further includes CARTs modified to secrete one or more polypeptides. Such CARTs may be called CART factories, CAR T cell factories, or armed CARTs. The polypeptides may be, for example, antibodies or fragments thereof as described herein. For example, the polypeptides may be antibodies or cytokines. In embodiments, the antibodies are specific to TIGIT, CAIX, GITR, PD-L1, PD-L2, PD-1, CCR4, CTLA-4, VISTA, or CD70. For example, a CAR T cell factory may locally secrete a PD-L1 mAb at a tumor site to restore effective anti-cancer immunity and / or reverse T cell depletion. In embodiments, an armed CART secretes IL-12, IL-15, or IL-18.

[0096] For example, a second expression construct, which may be in the same DNA vector (e.g., antigen recognition domain) as the one encoding the CAR, or in a second, separate vector, can be used to encode a minibody (scFv-Fc) or an antibody or fragment thereof targeting one or more antigens of interest, and can be cloned after the internal ribosome entry site (IRES). Referring to the figure, the second expression cassette contains either a fluorescent molecule or an immunomodulatory minibody.

[0097] Armed CARTs have the advantage of simultaneously secreting polypeptides at target sites, such as tumor sites. For example, armed CARTs can secrete anti-TIGIT antibodies or fragments thereof. TIGIT is a T cell co-suppressive receptor that limits antitumor and other T cell-dependent chronic immune responses, such as CD8+ T cell-dependent immune responses. TIGIT is expressed in a subset of activated T cells and natural killer (NK) cells. For example, TIGIT is highly expressed in tumor-infiltrating T cells. In cancer models, antibody blockade of TIGIT contributed to improved effector function and tumor clearance of CD8+ T cells.

[0098] In this embodiment, the anti-TIGIT antibody of the armed CART comprises one or more anti-TIGIT antibody clones (or fragments thereof, e.g., FR1, FR2, FR3, FR4, CDR1, CDR2, CDR3, or any combination of the framework and / or CDR regions therein) as shown in Figure 81.

[0099] For example, an anti-TIGIT antibody may include a CDR1 of the VH region having the sequence GYTF....TSYG (sequence number [ ]), a CDR2 of the VH region having the sequence ISAY..NGNT (sequence number [ ]), a CDR3 of the VH region having the sequence ARDPGLWFGLTHDYYFDY (sequence number [ ]), a CDR1 of the VL region having the sequence SSNI....GSNT (sequence number [ ]), a CDR2 of the VL region having the sequence RN.......N (sequence number [ ]), and a CDR3 of the VL region having the sequence AAWDDSRSGPV (sequence number [ ]).

[0100] For example, an anti-TIGIT antibody may include a CDR1 in the VH region having the sequence GFTF....SDYS (sequence number [ ]), a CDR2 in the VH region having the sequence INSD..GSRT (sequence number [ ]), a CDR3 in the VH region having the sequence ARGPGFFGFDI (sequence number [ ]), a CDR1 in the VL region having the sequence RSNI....GRNS (sequence number [ ]), a CDR2 in the VL region having the sequence SN.......N (sequence number [ ]), and a CDR3 in the VL region having the sequence AAWDARLTGPL (sequence number [ ]).

[0101] For example, an anti-TIGIT antibody may include a CDR1 of the VH region having the sequence GYSF....TNYW (sequence number [ ]), a CDR2 of the VH region having the sequence INPV..NSRT (sequence number [ ]), a CDR3 of the VH region having the sequence ARYYYYAMEV (sequence number [ ]), a CDR1 of the VL region having the sequence SSNI....GSNT (sequence number [ ]), a CDR2 of the VL region having the sequence RN.......N (sequence number [ ]), and a CDR3 of the VL region having the sequence EAWDDSLNGPV (sequence number [ ]).

[0102] For example, an anti-TIGIT antibody may include a CDR1 of the VH region having the sequence GYTF....TNYG (sequence number [ ]), a CDR2 of the VH region having the sequence VDNN..NGNI (sequence number [ ]), a CDR3 of the VH region having the sequence ARGLFSSRWYLWFDP (sequence number [ ]), a CDR1 of the VL region having the sequence SSDVG...GYNY (sequence number [ ]), a CDR2 of the VL region having the sequence EV.......T (sequence number [ ]), and a CDR3 of the VL region having the sequence SSYTRSSTSYVV (sequence number [ ]).

[0103] For example, an anti-TIGIT antibody may include a CDR1 of the VH region having the sequence GGTF....SSYA (sequence number [ ]), a CDR2 of the VH region having the sequence ILPM..FGST (sequence number [ ]), a CDR3 of the VH region having the sequence ARGRDIVAPSNSGFDV (sequence number [ ]), a CDR1 of the VL region having the sequence SNNV....GNQG (sequence number [ ]), a CDR2 of the VL region having the sequence RN.......D (sequence number [ ]), and a CDR3 of the VL region having the sequence SAYDRSLNAWV (sequence number [ ]).

[0104] In other embodiments, the armed CART may secrete an anti-PDL1 antibody or a fragment thereof. For example, the armed CART may secrete an anti-PDL1 antibody or a fragment thereof disclosed in Provisional Patent Application No. 62 / 624,455, which is incorporated herein in its entirety by reference. Exemplary anti-PDL1 antibodies include antibodies having a VH amino acid sequence having SEQ ID NO: [ ] and / or a VL amino acid sequence having SEQ ID NO: [ ]. See, for example, Figures 78 and 81.

[0105] In the embodiment, the anti-PDL1 antibody of the armed CART includes one or more anti-PDL1 antibody clones (or fragments thereof, e.g., FR1, FR2, FR3, FR4, CDR1, CDR2, CDR3, or any combination of the framework and / or CDR regions therein) as shown in Figure 78 and / or Figure 81.

[0106] For example, the amino acid sequences of the complementarity-determining regions of the heavy and light chains of the PDL-1 antibody are as follows: TIFF0007886110000009.tif129134

[0107] For example, an anti-PDL1 antibody has a heavy chain having three CDRs containing the amino acid sequences of SEQ ID NOs: [ ], [ ], and / or [ ], and a light chain having three CDRs containing the amino acid sequences.

[0108] In other embodiments, the armed CART may secrete an anti-PD1 antibody or a fragment thereof. Exemplary anti-PD1 antibodies include antibodies having a VH amino acid sequence with SEQ ID NO: [ ] and a VL amino acid sequence with SEQ ID NO: [ ]. See, for example, Figures 78 and 83.

[0109] In embodiments, the anti-PD1 antibody of armed CART comprises one or more anti-PD1 antibody clones (or fragments thereof, e.g., FR1, FR2, FR3, FR4, CDR1, CDR2, CDR3, or any combination of frameworks and / or CDR regions therein) as described in Figures 78 and 83. For example, the anti-PD1 antibody comprises a heavy chain having three CDRs containing the amino acid sequences of SEQ ID NOs: [ ], [ ], and / or [ ], and a light chain having three CDRs containing the amino acid sequences of SEQ ID NOs: [ ], [ ], and / or [ ], respectively. See, for example, Figures 78 and 83.

[0110] In this embodiment, the amino acid sequences of the heavy chain and light chain complementarity-determining regions of the PD-1 antibody are shown below. PD-1 antibody heavy chain (V H Complementarity Determination Region (CDR) of ) TIFF0007886110000010.tif114134PD-1 antibody light chain (V L Complementarity Determination Region (CDR) of ) TIFF0007886110000011.tif114134

[0111] In other embodiments, armed CARTs may secrete anti-CCR4 antibodies or fragments thereof. For example, armed CARTs may secrete anti-CCR4 antibodies or fragments as described in WO2009 / 086514, WO2013 / 166500, PCT / US2015 / 054202, or PCT / US2016 / 026232.

[0112] For example, an anti-CCR4 protein antibody or fragment of armed CART may include an antibody having a VH CDR1 region with the amino acid sequence GYTFASYY (sequence number [ ]), a VH CDR2 region with the amino acid sequence WINPGNVNTKYNEKFKG (sequence number [ ]), a VH CDR3 region with the amino acid sequence STYYRPLDY (sequence number [ ]), and / or a VL CDR1 region with the amino acid sequence KSSQSILYSSNQKNYLA (sequence number [ ]), a VL CDR2 region with the amino acid sequence WASTRES (sequence number [ ]), and / or a VL CDR3 region with the amino acid sequence HQYLSSYT (sequence number [ ]).

[0113] For example, an anti-CCR4 protein antibody or fragment of armed CAR has a VH amino acid sequence. TIFF0007886110000012.tif22148 (Sequence ID [ ]) and / or VL amino acid sequence This can include an antibody having the sequence number TIFF0007886110000013.tif13148 (Sequence ID [ ]).

[0114] For example, an anti-CCR4 protein antibody or fragment of armed CART may include an antibody having the following properties:

[0115] VH chain of antibody 1-44 (SEQ ID NO: [ ]) TIFF0007886110000014.tif22149

[0116] VL chain of antibody 1-44 (Sequence ID [ ]) TIFF0007886110000015.tif13148

[0117] VH chain of antibodies 1-49 (SEQ ID NO: [ ]) TIFF0007886110000016.tif22150

[0118] VL chain of antibody 1-49 (Sequence ID [ ]) TIFF0007886110000017.tif13145

[0119] VH chain of antibody 2-1 (SEQ ID NO: [ ]) TIFF0007886110000018.tif22149

[0120] VL chain of antibody 2-1 (SEQ ID NO: [ ]) TIFF0007886110000019.tif13148

[0121] VH chain of antibody 2-2 (SEQ ID NO: [ ]) TIFF0007886110000020.tif22148

[0122] VL chain of antibody 2-2 (Sequence ID [ ]) TIFF0007886110000021.tif13148

[0123] VH chain of antibody 2-3 (SEQ ID NO: [ ]) TIFF0007886110000022.tif22149

[0124] VL chain of antibody 2-3 (SEQ ID NO: [ ]) TIFF0007886110000023.tif13148

[0125] The amino acid sequences of the complementarity-determining regions of the heavy and light chains of the anti-CCR4 antibody are shown in the table below. TIFF0007886110000024.tif135133

[0126] Armed CARTs can be constructed by including a nucleic acid encoding the target polypeptide after an intracellular signaling domain. In embodiments, there is an internal ribosome entry site (IRES) positioned between the intracellular signaling domain and the target polypeptide. Those skilled in the art will understand that by using multiple IRES sequences in tandem, two or more polypeptides can be expressed.

[0127] In one embodiment, the methods and compositions presented herein provide target-specific T cells, such as T cells having specificity for CAIX and / or CD70, which are capable of secreting polypeptides in the tumor microenvironment to combat T cell depletion. For example, myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early myeloid progenitor cells, immature granulocytes, macrophages, and dendritic cells at different stages of differentiation that constitute the tumor microenvironment. MDSCs are induced by pro-inflammatory cytokines and are found in large numbers in infectious and inflammatory conditions. While we do not wish to be bound by theory, their presence in the tumor microenvironment suggests a causal role in promoting tumor-associated immunosuppression. Human MDSCs express Siglec-3 / CD33 (GENBANK accession numbers NM_001772.4 (nucleotide sequence) and NP_001763.3 (amino acid sequence)) and exhibit heterogeneous expression of CD14 (GENBANK accession numbers NM_000591.4 (nucleotide sequence) and NP_000582.1 (amino acid sequence)) and CD15 (GENBANK accession numbers NM_002033.3 (nucleotide sequence) and NP_002024.1 (amino acid sequence)), indicating the existence of multiple subsets.Other MDSC markers useful for identifying MDSCs include B7-1 / CD8 (NM_001145873.1 (nucleotide sequence), NP_001139345.1 (amino acid sequence)), CCR2 (NM_001123041.2 (nucleotide sequence), NP_001116513.2 (amino acid sequence)), CD1d (NM_001766.3 (nucleotide sequence), NP_001757.1 (amino acid sequence)), CD2 (NM_001328609.2 (nucleotide sequence), NP_001315538.1 (amino acid sequence)), CD31 / PECAM-1 (NM_000442.5 (for nucleotide sequence), NP_000433 This includes, but is not limited to, CD43 (NM_001030288.3 (nucleotide sequence), NP_001025459.1 (amino acid sequence)), CD44 (NM_000610.4 (nucleotide sequence), NP_000601.3 (amino acid sequence)), gp130 (NM_002184.4 (nucleotide sequence), NP_002175.2 (amino acid sequence)), PD-L1 (NM_014143.4 (nucleotide sequence), NP_054862.1 (amino acid sequence)), and CD162 (NM_001206609.2 (nucleotide sequence), NP_001193538.1 (amino acid sequence)).

[0128] In one embodiment, the methods and compositions presented herein provide target-specific T cells, such as T cells having specificity for CAIX and / or CD70, which are capable of secreting polypeptides that target MDSCs in the tumor microenvironment. For example, the secreted polypeptides may target one or more MDSC markers (e.g., CD33, CD14, and / or CD15, or other MDSC markers listed herein).

[0129] Introducing the construct to CTL The expression vector encoding the CAR may be introduced as one or more DNA molecules or constructs, and may contain at least one marker that allows selection of host cells containing the construct(s).

[0130] The constructs can be prepared by conventional methods, and the genes and regulatory regions can be isolated, ligated, cloned into a suitable cloning host, and analyzed by restriction, sequencing, or other convenient means. In particular, PCR can be used to isolate individual fragments containing all or part of the functional units, and one or more mutations can be introduced using primer repair, ligation, in vitro mutagenesis, etc., as appropriate. Once completed and demonstrated to have the appropriate sequence, the construct(s) can then be introduced into CTLs by any convenient means. The constructs may be incorporated and packaged for infection or transduction into cells, such as non-replicating defective viral genomes, adenoviruses, adeno-associated viruses (AAVs), herpes simplex virus (HSV), or retroviral vectors or lentiviral vectors. The constructs may contain viral sequences for transfection. Alternatively, the constructs may be introduced by fusion, electroporation, bioristic methods, transfection, lipofection, etc. Host cells can be grown and enlarged in a culture before introducing the construct(s), and then subjected to appropriate treatment to incorporate the construct(s). Next, the cells are enlarged and screened for markers present in the construct(s). Various markers that can be successfully used include HPRT, neomycin resistance, thymidine kinase, and hygromycin resistance.

[0131] In some cases, if a construct is incorporated into a specific gene locus, it may have a target site for homologous recombination. For example, an endogenous gene can be knocked out and replaced (at the same locus or another location) with the gene encoded by the construct using materials and methods known in the art for homologous recombination. Either an OMEGA or O-vector can be used for homologous recombination. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338, 153-156.

[0132] The constructs can be introduced as a single DNA molecule encoding at least one CAR and, optionally, another gene, or as different DNA molecules having one or more genes. Other genes may include, for example, genes encoding therapeutic molecules or suicide genes. The constructs may be introduced simultaneously or sequentially, each having the same or different markers.

[0133] Vectors containing useful elements such as bacterial or yeast replication origins, selectable and / or amplified markers, and promoter / enhancer elements for expression in prokaryotes or eukaryotes, which can be used for preparing constructor DNA stocks and performing transfections, are well known in the art and many are commercially available.

[0134] How to use The aspects of this disclosure relate to methods for treating subjects suffering from cancer.

[0135] For example, aspects of this disclosure relate to methods for killing cancer cells, such as renal cancer cells. Referring to Figure 12, for example, B7-GGGGS-G36 CAR T cells had greater killing activity against targeted renal cancer cells (CAIX+CD70+) than against non-target cells (CAIX+CD70-, CAIX-CD70+, CAIX-CD70-). Furthermore, referring to Figure 57, for example, bispecific fragmented CARs achieved superior killing compared to mono-CARs or bispecific CARs.

[0136] Aspects of this disclosure further relate to methods for stopping or reducing the progression of cancer in a subject, or for promoting the regression of cancer.

[0137] Furthermore, aspects of this disclosure relate to methods for reducing the proliferation of cancer cells in a subject. For example, see Figure 80.

[0138] "Cancer" and "cancerous" can refer to or describe, for example, a physiological condition in mammals characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancers. For example, cancer is renal cell carcinoma, such as ccRCC.

[0139] In cancer, normal intercellular interactions within tissues are disrupted, and the tumor microenvironment evolves to accommodate the growing tumor. The tumor microenvironment (TME) can refer to the cellular environment in which the tumor resides, including its components such as surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). The tumor microenvironment is complex and strongly influenced by the immune system.

[0140] The present invention provides, in particular (as described herein), CAR-T cell therapies for renal cell carcinoma. The secretion of monospecific, bispecific, or triplicate minibodies, antibodies, or minibodies / antibody fusion proteins by CAR-T cells at the tumor site may yield further benefits by altering (i.e., modulating) the immunosuppressive tumor microenvironment.

[0141] In embodiments, the method includes administering a therapeutically effective dose of the manipulated cells described herein to a subject suffering from cancer. The therapeutically effective dose may depend on the severity and course of the cancer, previous treatments, the subject's health status, weight, response to the drug, and the judgment of the attending physician.

[0142] The subjects may have cancer, such as humoral cancer (i.e., blood cancer) and / or solid cancer (i.e., tumors). Cancer can be benign or malignant and may be affected by the immune system.

[0143] Embodiments described herein can modulate the immune system to treat a subject suffering from cancer. "Modifying" can mean upcontrolling, inducing, stimulating, enhancing, and / or releasing inhibition, as well as inhibition, attenuation, and / or downcontrolling or suppressing. In embodiments, the activity of the immune system of the subject is modulated, or the microenvironment surrounding cancer cells and / or tumors is modulated, or both. For example, embodiments described herein can modulate (or enable) the immune system to kill tumor cells by altering the immunosuppressive tumor microenvironment and reducing microenvironment-dependent immunosuppression.

[0144] One embodiment relates to a method for treating subjects with renal cell carcinoma. Immunotherapy, such as that described herein, presents excellent treatment options for RCC. For example, the embodiment involves manipulating chimeric antigen receptor (CAR) T cells for RCC.

[0145] The “individual” or “subject” may be a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0146] The cells according to this disclosure can be used to treat cancer in patients who require cancer treatment. In another embodiment, the isolated cells according to the present invention can be used in the manufacture of pharmaceuticals for the treatment of cancer, autoimmune disorders, and viral infections in patients who require cancer treatment.

[0147] This disclosure relates to a method for treating a patient in need of cancer treatment, the method comprising at least one of the following steps: (a) providing chimeric antigen receptor cells according to the present invention, and (b) administering the cells to the patient.

[0148] The treatment may result in remission, cure, or prevention. It may be part of autoimmunotherapy or allogeneic immunotherapy. "Auto" means that the cells, cell lines, or cell populations used to treat the patient originate from the patient or from a human leukocyte antigen (HLA)-matched donor. "Allogeneic" means that the cells or cell populations used to treat the patient originate from a donor rather than from the patient.

[0149] The present invention is particularly suitable for allogeneic immunotherapy, insofar as it enables the transformation of T cells obtained from a donor into non-alloreactive cells. This can be carried out under a standard protocol and replicated as many times as needed. The resulting modified T cells can be pooled and administered to one or more patients, making them available as a "ready-made" therapeutic product.

[0150] The cells that can be used in the disclosed method are described in the preceding paragraph. The treatment can be used to treat patients diagnosed with cancer. Cancers that can be treated include non-angiogenic or substantially non-angiogenic tumors, as well as angiogenic tumors. Cancers may include non-solid tumors (e.g., hematological malignancies, e.g., leukemia and lymphoma) or solid tumors. Types of cancers that can be treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0151] This may be a treatment in combination with one or more therapies for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiotherapy.

[0152] According to embodiments of the present invention, the treatment can be administered to patients receiving immunosuppressive therapy. The present invention uses cells or cell populations that have been made resistant to at least one immunosuppressant by inactivating the genes encoding the receptors for the immunosuppressant. In this embodiment, the immunosuppressive therapy should help in the selection and expansion of T cells according to the present invention within the patient.

[0153] In further embodiments, the cell composition of the present invention is administered to a patient in combination with (e.g., before, simultaneously with, or after) a T-cell ablative therapy using any of the following: bone marrow transplantation, a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAM PATH. In another embodiment, the cell composition of the present invention is administered after a B-cell ablative therapy using a drug that reacts with CD20, such as rituximab. For example, in one embodiment, a subject may receive standard treatment with high-dose chemotherapy and then undergo peripheral blood stem cell transplantation. In a specific embodiment, after transplantation, the subject receives an infusion of the augmented immune cells of the present invention. In further embodiments, the augmented cells are administered before or after surgery. The modified cells obtained by any of the methods described herein can be used in specific embodiments of the present invention for treating patients in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Therefore, the scope of the present invention includes methods for treating patients in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising administering to the patient an effective amount of modified cells containing inactivated TCR alpha and / or TCR beta genes.

[0154] Cell administration This disclosure is particularly suitable for allogeneic immunotherapy, insofar as it allows for the transformation of donor T cells into non-alloreactive cells. This can be performed under standard protocols and replicated as many times as needed. The resulting modified T cells can be pooled and administered to one or more patients, making them available as a “ready-made” therapeutic product.

[0155] Depending on the properties of the cells, they can be introduced into host organisms, such as mammals, in a wide variety of ways. In certain embodiments, cells can be introduced into tumor sites, while in alternative embodiments, cells are "hone" or modified to become cancerous. The number of cells used depends on various circumstances, the purpose of introduction, the lifespan of the cells, the protocol used, such as the number of doses, the cell's ability to proliferate, and the stability of the recombinant construct. Cells can be applied, for example, as a dispersion injected into or near the site of interest. Cells may be in a physiologically acceptable medium.

[0156] In some embodiments, cells are encapsulated to inhibit immune recognition and then positioned at the tumor site.

[0157] Cells can be administered as desired. Various protocols can be used depending on the desired response, administration method, cell lifespan, and the number of cells present. The number of administrations depends, at least in part, on the factors mentioned above.

[0158] The administration of cells or cell populations according to the present invention can be carried out by any convenient method, including aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodulately, intramedullarily, intramuscularly, intravenously or intralymphatically, or intraperitoneally. In one embodiment, the cell composition of the present invention is administered by intravenous injection.

[0159] The administration of cells or cell populations may consist of 10⁴–10⁹ cells per kg of body weight, for example, 10⁵–10⁶ cells / kg of body weight, and includes all integer values ​​of the number of cells within this range. Cells or cell populations may be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the patient's clinical condition. Cells or cell populations can be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the optimal range of effective doses of a given cell type for a particular disease or condition is within the scope of this art. Effective dose means the amount that provides a therapeutic or preventive benefit. The dose administered will depend on the recipient's age, health condition and weight, the type of concurrent treatment if any, the frequency of treatment and the nature of the desired effect.

[0160] It should be understood that the system is affected by many variables that can change over time and in circumstances, such as the rate of loss of cellular activity as a result of cell loss or the expression activity of individual cells, including the cellular response to the ligand, expression efficiency, and, where appropriate, secretion levels, the activity of the expression product, and the specific needs of the patient. Therefore, with respect to individual patients, even if there are pluripotent stem cells that can be administered to the entire population, each patient is monitored for the appropriate dosage for the individual, and such practice of monitoring patients is commonplace in the art.

[0161] Nucleic acid-based expression systems The CARs of this disclosure can be expressed from an expression vector. Recombination techniques for producing such expression vectors are well known in the art.

[0162] DNA constructs, also known as “DNA vectors” as described herein, can be cloned into vectors used to transduce and produce chimeric antigen receptor T cells that secrete polypeptides and / or fragments thereof. For example, DNA constructs can be cloned into lentiviral vectors for the production of lentiviruses used to transduce and produce chimeric antigen receptor T cells that secrete monospecific, bispecific, or triplicate immunomodulatory antibodies / minibodies and / or antibody fusion proteins at tumor sites.

[0163] vector The term “vector” can refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell into which it can replicate. A nucleic acid sequence can be “exogenous,” meaning that it is either foreign to the cell into which the vector is introduced, or the sequence is homologous to an intracellular sequence but located in a position within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (such as YACs). Those skilled in the art will have sufficient equipment to construct vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0164] The term “expression vector” refers to any type of gene construct containing nucleic acids that encode transcriptionable RNA. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain various “regulatory sequences,” which refer to nucleic acid sequences necessary for the transcription and translation of the operably linked coding sequence in a particular host cell. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, as described below.

[0165] Promoter and enhancer A "promoter" is a regulatory sequence, a region of a nucleic acid sequence whose transcription initiation and rate are controlled. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, bind to initiate specific transcription of the nucleic acid sequence. The terms "operatably positioned," "operatably linked," "controlled," and "transcriptionally controlled" mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence to control the transcription initiation and / or expression of that sequence.

[0166] A promoter contains a sequence that functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters without a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyltransferase gene or the SV40 late gene, a separate element covering the start site itself helps to fix the start location. Additional promoter elements regulate the frequency of transcription initiation. These are located in a region 30-110 bp upstream of the start site, although some promoters have been shown to also contain functional elements downstream of the start site. To place the coding sequence under the "control" of the promoter, the 5' end of the transcription start site in the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of the DNA and promotes the expression of the encoded RNA.

[0167] In many cases, the spacing between promoter elements is flexible, so that the promoter's function is maintained even if the elements are reversed or moved relative to each other. In the tk promoter, the spacing between promoter elements can be widened to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in combination with "enhancers," which refer to cis-acting regulatory elements involved in the transcriptional activation of nucleic acid sequences.

[0168] A promoter may be naturally associated with a nucleic acid sequence, such as one that can be obtained by isolating a 5-prime 'non-coding sequence' located upstream of a coding segment and / or exon. Such a promoter may be called “endogenous.” Similarly, an enhancer may be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from other viruses, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are “not naturally occurring,” i.e., different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the most commonly used promoters in recombinant DNA construction include the lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthetically generating promoter and enhancer nucleic acid sequences, sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in relation to the compositions disclosed herein (see U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, regulatory sequences directing the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts can also be used.

[0169] Naturally, it will be important to use promoters and / or enhancers that effectively guide the expression of DNA segments in selected organelles, cell types, tissues, organs, or organisms for expression. Those skilled in the field of molecular biology are generally familiar with the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter used may be useful under appropriate conditions that direct high-level expression of the introduced DNA segment, such as constitutive, tissue-specific, inducible, and / or favorable for the large-scale production of recombinant proteins and / or peptides. Promoters may be heterogeneous or endogenous.

[0170] Furthermore, expression can also be promoted using any combination of promoters / enhancers. The use of T3, T7, or SP6 cytoplasmic expression systems is another embodiment. Eukaryotic cells can support cytoplasmic transcription from specific bacterial promoters if the appropriate bacterial polymerase is provided as part of a delivery complex or as an additional gene expression construct.

[0171] Assays for characterizing the identity of tissue-specific promoters or elements, as well as their activity, are well known to those skilled in the art.

[0172] Efficient translation of code sequences may also require specific start signals. These signals may include ATG start codons or adjacent sequences. It may be necessary to provide exogenous translation control signals, including ATG start codons. Those skilled in the art will readily determine this and provide the required signals.

[0173] In certain embodiments of this disclosure, internal ribosome entry site (IRES) elements are used to create multiple genes or polycistronic messages, which can be used in the present invention.

[0174] Vectors can contain multiple cloning sites (MCS), which are nucleic acid regions containing multiple restriction enzyme sites, any of which can be used to digest the vector in combination with standard recombination techniques. "Restriction enzyme digestion" refers to the catalytic cleavage of nucleic acid molecules by enzymes that function only at specific locations on the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, restriction enzymes that cleave within the MCS are used to linearize or fragment the vector so that foreign sequences can be ligated into the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombination techniques.

[0175] Splicing sites, termination signals, replication origins, and selectable markers can also be used.

[0176] Plasmid vectors In certain embodiments, plasmid vectors can be used to transform host cells. Plasmid vectors containing replicons and regulatory sequences derived from a species compatible with the host cell are used in association with these hosts. Vectors typically contain replication sites, as well as marking sequences that can provide phenotypic selection in transformed cells. In a non-limiting example, Escherichia coli (E. coli) is often transformed using derivatives of pBR322, a plasmid derived from the E. coli species. Since pBR322 contains genes for ampicillin and tetracycline resistance, transformed cells can be easily identified. The pBR plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, a promoter that the microorganism can use for the expression of its own proteins.

[0177] Furthermore, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, phage lambda GEM.TM.11 can be used in the creation of recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

[0178] Further useful plasmid vectors include the pIN vector (Inouye et al., 1985) and the pGEX vector, which are used to generate glutathione S-transferase (GST) soluble fusion proteins for later purification, isolation, or cleavage. Other suitable fusion proteins include those containing galactosidase, ubiquitin, etc.

[0179] Bacterial host cells containing an expression vector, such as E. coli, are grown in any many suitable media, e.g., LB. As those skilled in the art will understand, recombinant protein expression in a particular vector can be induced by contacting the host cells with a drug specific to a particular promoter, e.g., by adding IPTG to the medium, or by switching the incubation to a high temperature. After further culturing the bacteria, e.g., for 2-24 hours, the cells are harvested by centrifugation and washed to remove any residual medium.

[0180] Viral vector The ability of certain viruses to infect, invade, and integrate into host cell genomes via receptor-mediated endocytosis, and to stably and efficiently express viral genes, makes them attractive candidates for the introduction of foreign nucleic acids into cells (e.g., mammalian cells). Components of the present invention may be viral vectors encoding one or more CARs of the present invention. Non-limiting examples of viral vectors that may be used to deliver the nucleic acids of the present invention are described below.

[0181] Adenovirus vector Certain methods for nucleic acid delivery involve the use of adenoviral expression vectors. Adenoviral vectors are known to have a low ability to integrate into genomic DNA, but this feature is offset by the high gene transfer efficiency obtained with these vectors. An "adenoviral expression vector" means a construct that contains (a) adenoviral sequences sufficient to support packaging of the construct and (b) sufficient to ultimately express a tissue- or cell-specific construct cloned therein. Based on the knowledge that the genetic makeup or adenovirus is a 36 kb, linear, double-stranded DNA virus, large fragments of adenoviral DNA can be replaced with foreign sequences of up to 7 kb (Grunhaus and Horwitz, 1992).

[0182] AAV vector Nucleic acids can be introduced into cells using adenovirus-assisted transfection. Increased transfection efficiency has been reported in cell lines using an adenovirus coupled system (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use in the cells of the present invention because of its high frequency of integration and ability to infect non-dividing cells, and thus is useful for gene delivery to mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad infectious host range (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the production and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, which are incorporated herein by reference in their entirety.

[0183] Retroviral vector Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged into special cell lines (Miller, 1992).

[0184] To construct a retroviral vector, a nucleic acid (e.g., one encoding a sequence of interest) is inserted into the viral genome in place of certain viral sequences to produce a replication-defective virus. To generate virions, a packaging cell line that contains the gag, pol, and env genes but not the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into a special cell line (e.g., by calcium phosphate precipitation) along with the retroviral LTR and packaging sequences, the packaging sequences can package the RNA transcript of the recombinant plasmid into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). Next, the medium containing the recombinant retrovirus is collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect various types of cells. However, host cell division is required for integration and stable expression (Paskind et al., 1975).

[0185] Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997, U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are produced by multiple attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, resulting in biosafe vectors.

[0186] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. For example, a recombinant lentivirus can infect non-dividing cells that have been transfected with two or more vectors in which a suitable host cell possesses packaging functions, namely gag, pol, and env, as well as rev and tat as described in U.S. Patent No. 5,994,136, incorporated herein by reference. To target receptors of a specific cell type, recombinant viruses can be targeted by binding an antibody or a specific ligand to the envelope protein. For example, by inserting the sequence of interest (including regulatory regions) into a viral vector along with another gene encoding a ligand for a receptor on a specific target cell, the vector is now target-specific.

[0187] Other viral vectors In this invention, other viral vectors can be used as vaccine constructs. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus can be used. These offer several attractive properties to various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0188] Delivery using modified viruses The delivered nucleic acid may be contained within an infectious virus engineered to express a specific binding ligand. Thus, the viral particle specifically binds to a homologous receptor on the target cell, delivering its contents to the cell. A novel approach designed to enable the specific targeting of retroviral vectors was developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification enables specific infection of hepatocytes via the sialycoprotein receptor.

[0189] Another approach targeting recombinant retroviruses was designed, using biotinylated antibodies against retroviral envelope proteins and specific cell receptors. The antibodies were conjugated via the biotin component using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated in vitro infection of various human cells harboring these surface antigens by ecotropic viruses (Roux et al., 1989).

[0190] Vector delivery and cell transformation Suitable methods for nucleic acid delivery for cell transfection or transformation are known to those skilled in the art. Such methods include, but are not limited to, ex vivotransfection and direct delivery of DNA by injection. By applying techniques known in the art, cells can be transformed stably or transiently.

[0191] Ex vivo transformation Methods for transfecting eukaryotic cells and tissues extracted from living organisms in vitro are known to those skilled in the art. Therefore, cells or tissues can be extracted and transfected ex vivo using the nucleic acids of the present invention. In some embodiments, the transplanted cells or tissues can be placed in a living organism. In other embodiments, the nucleic acids are expressed in the transplanted cells.

[0192] The present invention kit Any of the compositions described herein may be included in the kit. In non-limiting examples, one or more cells for use in cell therapy containing a recombinant expression vector, and / or reagents for generating one or more cells for use in cell therapy, may be included in the kit. The components of the kit are provided in appropriate container means.

[0193] Some components of the kit may be packaged in an aqueous medium or in a lyophilized form. The kit's container means may include at least one vial, test tube, flask, bottle, syringe, or other container means in which the components are placed and preferably dispensed. If the kit has two or more components, the kit may also include a second, third, or other additional container in which additional components can be placed individually. However, various combinations of components may be contained in vials. The kit of the present invention may also include means for tightly sealing and containing the components for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0194] If the components of the kit are provided in the form of one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly useful. In some cases, the container means itself may be a syringe, pipette, and / or other similar device from which the formulation can be applied to the infected area of ​​the body, injected into an animal, and / or applied to and / or mixed with other components of the kit.

[0195] However, the components of the kit may be provided as dry powder(s). If the reagents and / or components are provided as dry powder, the powder may be reconstituted by the addition of a suitable solvent. For example, the solvent may also be provided in a separate container. The kit may also include a second container for sterile, pharmaceutically acceptable buffer and / or other diluents.

[0196] In certain embodiments of the present invention, the cells used in cell therapy are provided in a kit, and in some cases, the cells are essentially the sole component of the kit. The kit may include reagents and materials for producing the desired cells. In certain embodiments, the reagents and materials include primers, nucleotides, appropriate buffers or buffer reagents, salts, etc., for amplifying the desired sequence, and in some cases, the reagents include vectors and / or DNA encoding the CARs described herein, and / or modulating elements therefor.

[0197] In certain embodiments, a kit contains one or more devices suitable for extracting one or more samples from an organism. These devices may include syringes, scalpels, and the like.

[0198] In some cases of the present invention, the kit includes, in addition to embodiments of cell therapy, a second cancer therapy such as chemotherapy, hormone therapy, and / or immunotherapy. The kit(s) may be tailored to the specific cancer of an individual and may include each individual's second cancer therapy.

[0199] Combination therapy In certain embodiments of the present invention, the method of the present invention for clinical aspects is combined with other agents effective in treating hyperproliferative diseases, such as anticancer agents. “Anticancer” agents can adversely affect cancer in a subject by, for example, killing cancer cells, reducing the proliferation rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or extending the lifespan of a subject with cancer. These other compositions will be provided in combined amounts effective in killing or inhibiting cell proliferation. The process may involve contacting cancer cells with an expression construct and / or a drug(s) or a factor(s) simultaneously. This can be achieved by contacting cells with a single composition or a pharmacological formulation containing both drugs, or by contacting cells with two different compositions (one composition containing an expression construct and the other containing a second drug(s)) or formulations simultaneously.

[0200] Resistance of tumor cells to chemotherapy and radiotherapy agents is a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the effectiveness of chemotherapy and radiotherapy by combining them with other therapies. In one embodiment, cell therapy can be used in conjunction with chemotherapy, radiotherapy, or immunotherapy interventions, as well as with apoptosis-promoting or cell cycle-modulating agents.

[0201] Alternatively, the treatment of the present invention can precede or follow the treatment of other agents at intervals ranging from a few minutes to a few weeks. In embodiments where the other agent and the present invention are applied separately, generally, an effective period has not expired between each delivery time point to ensure that the agent and the treatment of the present invention can still exert an advantageous combined effect on the cells. In such cases, the cells can be contacted with both modalities within about 12 to 24 hours of each other (e.g., within about 6 to 12 hours of each other). In some situations, when several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration, it may be desirable to significantly extend the treatment period.

[0202] The treatment cycle will be repeated as necessary. For example, various standard therapies, as well as surgical interventions, can be applied in combination with the cell therapy of the present invention.

[0203] Chemotherapy Cancer treatment also includes various combination therapies using both chemical substances and radiation-based treatments. Combination chemotherapy includes, for example, Abraxane, Altretamine, Docetaxel, Herceptin, Methotrexate, Novantrone, Zoladex, Cisplatin (CDDP), Carboplatin, Procarbazine, Mechlorethamine, Cyclophosphamide, Camptothecin, Ifosfamide, Melphalan, Chlorambucil, Busulfan, Nitrosourea, Dactinomycin, Daunorubicin, Doxorubicin, Bleomycin, Plicomycin, Mitomycin, Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen receptor binders, Taxol, Gemcitabine, Navelbine, Farnesyl protein transferase inhibitors, Transplatinum, 5-Fluorouracil, Vincristine, Vinblastine, and Methotrexate, or variants of any of the aforementioned analogs or derivatives, and combinations thereof, but are not limited thereto.

[0204] In certain embodiments, chemotherapy for an individual may be employed in combination with the present invention, for example, before, during, and / or after administration of the present invention.

[0205] Radiation therapy Other factors that cause DNA damage and have been widely used include gamma rays, commonly known as X-rays, and / or the induced delivery of radioisotopes to tumor cells. Other forms of DNA damage factors, such as microwave and ultraviolet irradiation, are also useful. All of these factors are most likely to cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays is from 50–200 roentgens per day for long-term (3–4 weeks) doses to 2000–6000 roentgens per day for single doses. The dose range for radioisotopes varies considerably and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by newly formed cells.

[0206] In this specification, the terms “contact” and “exposure,” when applied to cells, are used to describe the process by which therapeutic constructs and chemotherapeutic or radiotherapeutic agents are delivered to or directly juxtaposed with target cells. To achieve cell toxicization or quiescence, both agents are delivered to the cells in a total amount effective in killing the cells or preventing them from dividing.

[0207] immunotherapy Immunotherapy relies on using immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies specific to certain markers on the surface of tumor cells. Antibodies can act as therapeutic effectors on their own or they can mobilize other cells to actually kill cells. Antibodies can also bind to drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and simply act as targeters. Alternatively, effectors may be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0208] Therefore, immunotherapies other than those described herein may be used in combination with this cell therapy as part of a combination therapy. Combination therapy approaches are described herein. For example, tumor cells must have several markers that are easily targeted, i.e., not present on most other cells. Many tumor markers exist, and any of these may be suitable for targeting under the circumstances of the present invention. Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate-specific antigen, urinary tract tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

[0209] gene In yet another embodiment, the second-line treatment is a gene therapy in which the therapeutic polynucleotide is administered before, after, or concurrently with the clinical embodiments of the present invention. The present invention includes a variety of expression products, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death regulators.

[0210] surgery Approximately 60% of cancer patients undergo some form of surgery, including prophylactic, diagnostic, staging, curative, and palliative surgeries. Curative surgery is a cancer treatment that can be used in combination with other therapies such as the treatments of the present invention, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0211] Curative surgery includes excision, in which all or part of the cancerous tissue is physically removed, cut out, and / or destroyed. Tumor excision refers to the physical removal of at least a portion of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure). For example, the present invention may be used in combination with the removal of superficial cancer, precancerous tissue, or an associated amount of normal tissue.

[0212] When all cancerous cells, tissue, or part of a tumor are removed, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or local application to the site with additional anticancer therapy. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also be in various dosages.

[0213] Other drugs In some embodiments, other agents can be used in combination with the present invention to improve the therapeutic effect of the treatment. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and GAP binding, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferating cells to apoptosis-inducing substances. Immunomodulators include tumor necrosis factor, interferon alpha, beta, gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. In some embodiments, upregulation of cell surface receptors or their ligands, e.g., Fas / Fas ligand, DR4, or DR5 / TRAIL, will enhance the apoptosis-inducing capacity of the present invention by establishing autocrine or paracrine effects on hyperproliferating cells. Increasing intercellular signaling by increasing the number of GAP bindings will increase the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell proliferation inhibitors or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors can also be used to improve the efficacy of the present invention. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. In some embodiments, other agents that enhance the sensitivity of hyperproliferating cells to apoptosis, such as the antibody c225, can be used in combination with the present invention to improve therapeutic efficacy.

[0214] Method for evaluating the activity of manipulated CAR T cells Aspects of this disclosure further relate to methods and kits for evaluating the killing capacity of engineered CAR T cells. Specifically, embodiments relate to immunocomplex analysis methods and kits for determining CAR T cell activity in co-culture with cancer cells. First, various target cancer cells (e.g., HEK293T, MDA-MB-231, MDA-MB-468, HCC38, and skrc59) are stained with a dye (e.g., ViaStain® Tracer Blue dye), seeded on a plate (e.g., a 96-well plate), and incubated for a period of time (e.g., 12 hours or overnight). Next, different T cell types (e.g., two different T cell types) are added to the wells (e.g., in an effector-to-target (E:T) ratio of 20:1) and co-cultured for a period of time (e.g., 24 hours). Finally, the plate is scanned and analyzed (e.g., using bright-field and blue fluorescence channels). Immune complexes were analyzed by confluence assays and compared to negative controls of untransduced T cells. As a result, the data plots displayed CAR T cell activity for all target and effector cell combinations tested. The use of an image cytometry platform allows for visual confirmation of the interaction between effector and target cells, resulting in highly accurate and robust results. [Examples]

[0215] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of constructing and carrying out the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and similar results can be obtained using alternative methods; therefore, these examples are for illustrative purposes only.

[0216] Example 1 Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and one of the 10 most common cancers in both men and women. Chimeric antigen receptor (CAR) T cells have proven to be a potent and clinically translatable immunotherapy against hematological malignancies. However, these results are not translatable to solid tumors due to inefficient homing of CAR T cells, a suppressive tumor microenvironment, and on-target off-tumor toxicity resulting from the sharing of CAR T-targeting epitopes on healthy tissue. To combat the suppressive microenvironment, immune checkpoint blockers have shown an enhanced effect on the antitumor response by restoring local antitumor immunity. The CAR T cell factory was designed to empower CAR T cells by secreting human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site. Our results show a dramatic improvement in CAR T-mediated killing of ccRCC in vivo and in vitro by reversing CAR T cell and tumor-infiltrating lymphocyte (TIL) depletion.

[0217] CAIX is an ideal target for ccRCC therapy and was used as the CAR target in the first clinical trials (see, e.g., Lamers, Sleijfer et al. 2006; Lamers, Willemsen et al. 2011). However, it caused serious side effects due to CAIX expression on the bile ducts. Therefore, it is important to develop a CAR with high efficacy and safety (e.g., limiting on-target off-tumor effects). To achieve this, second-generation CARs were developed by introducing a second targeted scFv (e.g., anti-CD70 scFv) together with the first targeted scFv (e.g., anti-CAIX scFv) into the CAR T cell factory, allowing the CAR to target two specific antigens simultaneously. See, for example, Figure 1. IHC staining of ccRCC patient samples revealed that targeted CD70 is highly expressed on ccRCC and co-expresses with CAIX, making it an ideal target to use as a second target.

[0218] Our 27 billion-member human scFv-phage display library was panned against antigen-expressing skrc-59 ccRCC cells and antigen-free skrc-59 ccRCC cells were subtracted to identify novel scFvs. Their binding kinetics (Kon / Koff) and affinity (Kd) were then measured via OctetRed 96 instrument to evaluate scFvs with desirable kinetics and their ability to bind to antigen-expressing cells. Candidates were cloned into vectors in which anti-CD70 and anti-CAIX scFvs were combined in different permutations by altering the order of two targeted scFvs with various linkers attached to the costimulatory domain (CD28, 41BB) and activating domain (CD3). Using a fourth-generation lentiviral packaging system, CAR lentiviruses were obtained, primary T cells isolated from PBMCs were transduced to express dual CARs, and these were tested in vitro against different cell lines. For further in vivo evaluation, a humanized orthotopic ccRCC mouse model was established by injecting luciferized ccRCC cells subcapsulate of the kidneys of NSG-SGM3 mice with a reconstituted human immune system.

[0219] In summary, a dual CAR T discovery platform was used to generate a series of CARs with different scFvs, linkers, and hinges. Those skilled in the art will recognize that embodiments may include different combinations of scFvs, linkers, and hinges. For example, different combinations of scFvs, linkers, and hinges can be used to treat different cancers.

[0220] Second-generation CAR T cells were discovered by combining the best dual CARs with payloads such as immune checkpoint inhibitor payloads. While we don't want to be bound by theory, second-generation CAR T cell factories could be used to treat cancers such as ccRCC, eliminating side effects on normal tissue.

[0221] References cited in this example Lamers, CH, S. Sleijfer, AGVulto, WHKruit, M. Kliffen, R. Debets, JW Gratama, G. Stoter and E. Oosterwijk (2006). “Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX:first clinical experience.” J Clin Oncol 24(13):e20-22. Lamers, CH, R. Willemsen, P. van Elzakker, S. van Steenbergen-Langeveld, M. Broertjes, J. Oosterwijk-Wakka, E. Oosterwijk, S. Sleijfer, R. Debets and JW Gratama (2011).”Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells.”Blood 117(1):72-82.

[0222] Example 2 Chimeric antigen receptor (CAR) T cells have proven to be a potent immunotherapy for hematological malignancies, but they have not been translated into solid tumors. Our CAR T cell factory empowers CAR T cells by locally secreting antibodies, such as human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs), at the tumor site, restoring the tumor microenvironment and achieving solid tumor remission. With the introduction of one or more additional scFvs, second-generation CARs offer improved efficacy and safety and are generating great promise in clinical practice.

[0223] The bispecific tandem CAR was designed to target tumor-associated antigens such as CAIX and CD70 (see, e.g., Figures 1 and 2), which are highly expressed and co-expressed on primary ccRCC cells by IHC (Figure 3). Successful panning of a 27 billion-member phage display library identified several anti-CD70 scFvs (see, e.g., Figure 6). We cloned the anti-CAIX scFv and anti-CD70 scFvs we previously discovered into pHAGE vectors with different linkers (see, e.g., Figure 9). The lentiviruses were then packaged and transduced into primary T cells. CAR T cells were obtained and evaluated in four different CRISPR-operated cell lines (see, e.g., Figure 5). Killing activity was assessed by Celigo and Cr51 release assays (see, e.g., Figures 12 and 13). Selectivity was demonstrated as described herein.

[0224] This CAR T cell factory can be used to treat cancers that overexpress CAIX and / or CD70, or in combination with other therapies.

[0225] Example 3 Quantification of CAIX and CD70 double IHC staining TIFF0007886110000025.tif149166

[0226] equivalent Those skilled in the art will recognize or be able to identify numerous equivalents to the specific substances and procedures described herein without using anything beyond routine experiments. Such equivalents are considered to fall within the scope of the present invention and are covered by the appended claims.

[0227] Sequence information SEQUENCE LISTING <110> DANA-FARBER CANCER INSTITUTE, INC. <120> CHIMERIC ANTIGEN RECEPTOR FACTORIES AND METHODS OF USE THEREOF <150> US 62 / 826,462 <151> 2019-03-29 <150> US 62 / 773,885 <151> 2018-11-30 <160> 352 <170> PatentIn version 3.5 <210> 1 <400> 1 000 <210> 2 <400> 2 000 <210> 3 <400> 3 000 <210> 4 <400> 4 000 <210> 5 <400> 5 000 <210> 6 <400> 6 000 <210> 7 <400> 7 000 <210> 8 <400> 8 000 <210> 9 <400> 9 000 <210> 10 <400> 10 000 <210> 11 <400> 11 000 <210> 12 <400> 12 000 <210> 13 <400> 13 000 <210> 14 <400> 14 000 <210> 15 <400> 15 000 <210> 16 <400> 16 000 <210> 17 <400> 17 000 <210> 18 <400> 18 000 <210> 19 <400> 19 000 <210> 20 <400> 20 000 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Gly Tyr Thr Phe Ala Ser Tyr Tyr 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Gly Tyr Thr Phe Ala Ser Gln Trp 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Gly Tyr Thr Phe Ala Ser Ser Trp 1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Gly Tyr Thr Phe Ala Ser Gln Tyr 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Gly Tyr Thr Phe Ala Ser Ala Trp 1 5 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Gln Ser Ile Leu Tyr Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Ile Asn Pro Gly Asn Val Asn Thr 1 5 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Trp Ala Ser Thr Arg Glu 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Ser Thr Tyr Tyr Arg Pro Leu Asp Tyr 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Ser Thr Trp Tyr Arg Pro Leu Asp Tyr 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Ser Thr Trp Tyr Arg Pro Asn Asp Tyr 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Thr Thr Arg Tyr Arg Pro Leu Asp Tyr 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Leu Thr Tyr Tyr Arg Pro Pro Asp Tyr 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 His Gln Tyr Leu Ser Ser Tyr Thr 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 His Gln Tyr Ile Ser Ser Tyr Thr 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 His Gln Tyr Lys Ser Ser Tyr Thr 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 His Gln Tyr Arg Ser Ser Tyr Thr 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 His Gln Tyr Tyr Ser Ser Tyr Thr 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 His Gln Tyr Met Ser Ser Tyr Thr 1 5 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Gly Gly Gly Ser 1 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Gly Gly Gly Gly Ser Ser Ser 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Gly Gly Gly Gly Ser Ser Ser Ser Ser 1 5 <210> 43 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Glu Glu Asp Leu Pro Glu 1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Thr Tyr Ala Met Thr 1 5 <210> 45 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Thr Gly Ser Arg Ser Asn Ile Gly Ala Asp Tyr Asp Val His 1 5 10 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Ser Tyr Ala Met Ser 1 5 <210> 47 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly Tyr Asn Val His 1 5 10 <210> 48 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 49 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 51 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly Tyr Asn Val His 1 5 10 <210> 52 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Asn Tyr Ala Met Thr 1 5 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Gly Gly Asn Asn Ile Gly Ser Lys Ser Val Glu 1 5 10 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Asn Ile Gly Ser Lys Ser 1 5 <210> 56 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Phe Asp Val His 1 5 10 <210> 57 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Thr Gly Thr Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His 1 5 10 <210> 58 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Ser Tyr Gly Met His 1 5 <210> 59 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 60 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Ile Tyr Ala Met Ser 1 5 <210> 61 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Lys Tyr Ala Met Ser 1 5 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Gln Gly Asn Ser Leu Arg Tyr Tyr Tyr Pro Ser 1 5 10 <210> 63 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Gly Gly Asp Asn Ile Gly Arg Lys Ser Val His 1 5 10 <210> 64 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Ala Val Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Ala Asn Asn Asn Arg Pro Ser 1 5 <210> 66 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Gly Asn Thr Asn Arg Pro Ser 1 5 <210> 68 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 69 Gly Asn Ser Asn Arg Pro Ser 1 5 <210> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Asp Asp Ile Asn Arg Pro Ser 1 5 <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 72 Leu Ile Ser Tyr Asp Gly Ser Val Thr His Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 73 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 73 Tyr Asp Ser Asp Arg Pro Ser 1 5 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 74 Ile Ser Tyr Asp Gly Ser Val Thr 1 5 <210> 75 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 Asp Asn Thr Asn Arg Pro Ser 1 5 <210> 76 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 76 Gly Asn Asn Asn Arg Pro Ser 1 5 <210> 77 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 77 Ala Ile Ser Gly Ser Gly Val Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 78 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Arg Asn Asn Gln Arg Pro Ser 1 5 <210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr His Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 81 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 81 Gly Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 82 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 82 Gly Lys Asn Asn Arg Pro Ser 1 5 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Asp Asp Arg Asp Arg Pro Ser 1 5 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Gly Pro Val Leu Arg Tyr Gly Phe Asp Ile 1 5 10 <210> 85 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Gln Ser Tyr Asp Ser Ser Leu Arg Ala Trp Val 1 5 10 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Ser His Ser Ser Gly Gly Phe Asp Tyr 1 5 <210> 87 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 Gln Ser Tyr Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 88 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile 1 5 10 <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Gln Ser Tyr Asp Arg Ser Leu Ser Trp Val 1 5 10 <210> 90 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Ala Thr Tyr Gly Asp Tyr Gly Ser Leu Asp Tyr 1 5 10 <210> 91 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Ala Ala Ala Gly Phe Asp Tyr 1 5 <210> 92 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Gly Ser Gly Tyr Gln Glu 1 5 <210> 93 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Gln Val Trp Asp Ser Ser Ser Asp His His Val Val 1 5 10 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 94 Ala Arg Gly Ser Gly Tyr Gln Glu His 1 5 <210> 95 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 95 Gln Ser Tyr Asp Ser Arg Leu Ser Ala Trp Val 1 5 10 <210> 96 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 96 Ile Gly Arg Tyr Ser Ser Ser Leu Gly Tyr 1 5 10 <210> 97 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 97 Gln Ser Tyr Asp Ser Gly Leu Arg Trp Val 1 5 10 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 98 Tyr Gly Asp Tyr Gly Ser Leu Asp Tyr 1 5 <210> 99 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 99 Gln Ser Tyr Asp Lys Ser Leu Ser Trp Val 1 5 10 <210> 100 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 100 Tyr Cys Ser Ser Thr Ser Cys Tyr Arg Gly Met Asp Val 1 5 10 <210> 101 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 101 Gln Ser Tyr Asp Lys Ser Leu Thr Trp Val 1 5 10 <210> 102 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 102 Gly Arg Ala Ala Arg Pro Pro Phe Asp Tyr 1 5 10 <210> 103 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Ala Ala Trp Asp Asp Ser Leu Asn Gly Val Val 1 5 10 <210> 104 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 104 Glu Ala Pro Tyr Ser Ser Ser Leu Asp Ala Phe Asp Ile 1 5 10 <210> 105 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 105 His Ser Arg Asp Asn Asn Gly His His Ile 1 5 10 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 106 Phe Ser Ala Tyr Ser Gly Tyr Asp Leu 1 5 <210> 107 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 107 Gln Ser Tyr Asp Ser Thr Leu Arg Val Trp Met 1 5 10 <210> 108 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Ser Ser Arg Ser Gly Tyr Phe Leu Pro Leu Asp Tyr 1 5 10 <210> 109 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 109 Ser Ser Arg Asp Asn Thr Asp Asn Arg Val Val 1 5 10 <210> 110 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 110 Ala Ala Val Thr Gly Gly Phe Asp Pro 1 5 <210> 111 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 111 Gln Val Trp Asp Ser Ser Ser Lys His Tyr Val 1 5 10 <210> 112 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 112 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Gly Thr Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Val Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Pro Val Leu Arg Tyr Gly Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Ile Val Ser Ser 115 <210> 113 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 113 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Ile Thr Ile Ser Cys Thr Gly Ser Arg Ser Asn Ile Gly Ala Asp 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ala Asn Asn Asn Arg Pro Ser Gly Val Pro Gly Arg Phe 50 55 60 Ser Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu Gly 100 105 110 <210> 114 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 114 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Ser Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 115 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 115 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 116 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 116 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 117 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 117 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Ser Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala His Tyr Tyr Cys Gln Ser Tyr Asp Arg Ser 85 90 95 Leu Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 118 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 118 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Gly Asp Tyr Gly Ser Leu Asp Tyr 100 105 <210> 119 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 119 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ala Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Arg Ala Trp Val 100 <210> 120 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 120 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ala Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 121 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 121 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asp Ile Asn Arg Pro Ser Gly Val Pro His Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu Gly 100 105 110 <210> 122 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (83)..(83) <223> Any amino acid <400> 122 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Xaa Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 123 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 123 Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Val Val Gln Pro Gly Thr 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Ser Tyr Asp Gly Ser Val Thr His Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Gly Tyr Gln Glu His Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 124 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 124 Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 Glu Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 125 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 125 Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Val Val Gln Pro Gly Thr 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Ser Tyr Asp Gly Ser Val Thr His Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Gly Tyr Gln Glu His 100 105 <210> 126 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 126 Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 His Val Val <210> 127 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 127 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 128 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 128 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Phe Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Thr Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Arg 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 129 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 129 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 130 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 130 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Ile Thr Ile Ser Cys Thr Gly Ser Arg Ser Asn Ile Gly Ala Asp 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ala Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Thr Asp Tyr Phe Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 131 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 131 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Gly Arg Tyr Ser Ser Ser Leu Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 132 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 132 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Gly 85 90 95 Leu Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Leu Leu Arg 100 105 110 <210> 133 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 133 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Gly Asp Tyr Gly Ser Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 134 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 134 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ala Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu Gly 100 105 110 <210> 135 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 135 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Arg Val 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Lys Ser 85 90 95 Leu Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg 100 105 110 <210> 136 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 136 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Val Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Cys Ser Ser Thr Ser Cys Tyr Arg Gly Met Asp Val Trp 100 105 110 Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 137 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 137 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 138 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 138 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Ile Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Val 35 40 45 Val Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Lys Ser 85 90 95 Leu Thr Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 139 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 139 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Ala Ala Arg Pro Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 140 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 140 Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Pro 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg 100 105 110 <210> 141 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 141 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Pro Tyr Ser Ser Ser Leu Asp Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 142 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 142 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Ser Arg Asp Asn Asn 85 90 95 Gly His His Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser 100 105 110 <210> 143 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 143 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 144 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 144 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Thr Asp Tyr Phe Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 145 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 145 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Ser Ala Tyr Ser Gly Tyr Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 146 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 146 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Lys Ser Ala Thr Ser Ala Ser Leu Thr Ile Thr Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Gly 85 90 95 Leu Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Leu Leu Gly 100 105 110 <210> 147 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 147 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 148 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 148 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Phe Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Thr Asp Tyr Phe Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Arg 100 105 110 <210> 149 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 149 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asn Gly Asn Tyr Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Ile Val Ser Ser 115 <210> 150 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 150 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Thr 85 90 95 Leu Arg Val Trp Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 151 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 151 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Pro Glu Phe Thr Phe Ser Lys Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ser Arg Ser Gly Tyr Phe Leu Pro Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 152 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 152 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asn Ser Leu Arg Tyr Tyr Tyr Pro 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Asn Thr Asp Asn Arg 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 <210> 153 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 153 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ala Val Thr Gly Gly Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 154 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 154 Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn Ile Gly Arg Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Ile Leu Val Ile Arg 35 40 45 Asp Asp Arg Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Val Asn Thr Ala Thr Leu Ile Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Lys His 85 90 95 Tyr Val Phe Gly Pro Gly Thr Lys Val Thr Ala Leu Gly 100 105 <210> 155 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 155 Ser Ser Asn Ile Gly Ser Asn Tyr 1 5 <210> 156 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 156 Gly Gly Thr Phe Ser Ser Gln Ala 1 5 <210> 157 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 157 Tyr Ser Val Phe His Ser Pro Asn Asn Lys Asn Tyr 1 5 10 <210> 158 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 158 Gly Phe Thr Val Ser Asn Tyr Ala 1 5 <210> 159 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 159 Ala Leu Pro Lys Lys Tyr 1 5 <210> 160 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 160 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 161 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 161 Gly Phe Thr Val Ser Thr Ser His 1 5 <210> 162 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 162 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 163 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 163 Gly Phe Ile Phe Ser Asp Tyr Tyr 1 5 <210> 164 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 164 Gln Asp Ile Gly Thr Asp 1 5 <210> 165 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 165 Ile Ser Gly Ser Gly Gly Ser Arg 1 5 <210> 166 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 166 Ile Ile Pro Phe Phe Gly Val Pro 1 5 <210> 167 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 167 Lys Ser Gly Ser Asp Gly Arg Thr 1 5 <210> 168 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 168 Lys Asp Ser Gly Gly Lys Thr 1 5 <210> 169 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 169 Ile Arg Ser Arg Arg Gly Glu Thr 1 5 <210> 170 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 170 Ala Arg Gly Arg Gly Gly His Gly Met Asp Val 1 5 10 <210> 171 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 171 Ala Ala Trp Asp Asp Ser Leu Asn Gly Leu Val 1 5 10 <210> 172 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 172 Ala Val Leu Lys Gly Arg Gly Asn Phe Asp Phe 1 5 10 <210> 173 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 173 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 174 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 174 Ala Lys Gly Ile Tyr Asp Val Thr Gly Ser Ser Phe Asp Ser 1 5 10 <210> 175 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 175 Tyr Ser Thr Asp Ser Ser Gly Asn His Lys Val 1 5 10 <210> 176 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 176 Gln Ser Tyr Asp Ser Gly Asn Arg Arg Val 1 5 10 <210> 177 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 177 Ala Arg Ala Arg Pro Ser Asp Pro Tyr Asp Gly Ser Gly Phe Asp Ala 1 5 10 15 Phe Asp Ile <210> 178 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 178 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 179 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 179 Ala Arg His Arg Lys Ser Phe Thr Asp Leu Asp Ala Phe Asp Leu 1 5 10 15 <210> 180 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 180 Gln His Phe Asn Asn Tyr Pro Ala Thr 1 5 <210> 181 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 181 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Ser Gly Gly Ser Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Gly Gly His Gly Met Asp Val 100 105 <210> 182 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 182 Gln Pro Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Leu Val 100 <210> 183 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 183 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Arg Ser Ser Gly Gly Thr Phe Ser Ser Gln 20 25 30 Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Phe Phe Gly Val Pro Thr Tyr Ala Gln Arg Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Pro Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Thr Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Leu Lys Gly Arg Gly Asn Phe Asp Phe 100 105 <210> 184 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 184 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Tyr Ser Val Phe His Ser 20 25 30 Pro Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gly Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Arg Ser Asn Trp Pro Leu Thr 100 <210> 185 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 185 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Arg Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Lys Ser Gly Ser Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Ile Tyr Asp Val Thr Gly Ser Ser Phe Asp Ser 100 105 110 <210> 186 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 186 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Lys Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val Leu Val Met Phe 35 40 45 Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Thr Asp Ser Ser Gly Asn His 85 90 95 Lys Val <210> 187 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 187 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Gly Asn Arg Arg Val 100 <210> 188 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 188 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Thr Ser 20 25 30 His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Gly Lys Asp Ser Gly Gly Lys Thr Tyr Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ala Arg Asp Asp Ser Leu Asn Thr Val Phe Leu 65 70 75 80 Gln Met Asn Asn Met Arg Asp Glu Asp Ser Gly Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Pro Ser Asp Pro Tyr Asp Gly Ser Gly Phe Asp Ala Phe 100 105 110 Asp Ile <210> 189 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 189 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln 20 25 30 Gly Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40 45 Ser Tyr Arg Asn Asn Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Ser Ser Leu 85 90 95 Ser Ala Trp Val 100 <210> 190 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 190 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Arg Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Ser Ile Arg Ser Arg Arg Gly Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Glu Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Arg Lys Ser Phe Thr Asp Leu Asp Ala Phe Asp Leu 100 105 110 <210> 191 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 191 Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Thr Asp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Asn Asn Tyr Pro Ala 85 90 95 Thr <210> 192 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 192 Gly Tyr Thr Phe Thr Ser Tyr Gly 1 5 <210> 193 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 193 Ile Ser Ala Tyr Asn Gly Asn Thr 1 5 <210> 194 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 194 Ala Arg Asp Pro Gly Leu Trp Phe Gly Leu Thr His Asp Tyr Tyr Phe 1 5 10 15 Asp Tyr <210> 195 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 195 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 196 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 196 Ala Ala Trp Asp Asp Ser Arg Ser Gly Pro Val 1 5 10 <210> 197 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 197 Gly Phe Thr Phe Ser Asp Tyr Ser 1 5 <210> 198 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 198 Ile Asn Ser Asp Gly Ser Arg Thr 1 5 <210> 199 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 199 Ala Arg Gly Pro Gly Phe Phe Gly Phe Asp Ile 1 5 10 <210> 200 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 200 Arg Ser Asn Ile Gly Arg Asn Ser 1 5 <210> 201 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 201 Ala Ala Trp Asp Ala Arg Leu Thr Gly Pro Leu 1 5 10 <210> 202 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 202 Gly Tyr Ser Phe Thr Asn Tyr Trp 1 5 <210> 203 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 203 Ile Asn Pro Val Asn Ser Arg Thr 1 5 <210> 204 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 204 Ala Arg Tyr Tyr Tyr Tyr Ala Met Glu Val 1 5 10 <210> 205 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 205 Glu Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 206 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 206 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 207 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 207 Val Asp Asn Asn Asn Gly Asn Ile 1 5 <210> 208 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 208 Ala Arg Gly Leu Phe Ser Ser Arg Trp Tyr Leu Trp Phe Asp Pro 1 5 10 15 <210> 209 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 209 Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 <210> 210 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 210 Ser Ser Tyr Thr Arg Ser Ser Thr Ser Tyr Val Val 1 5 10 <210> 211 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 211 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 212 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 212 Ile Leu Pro Met Phe Gly Ser Thr 1 5 <210> 213 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 213 Ala Arg Gly Arg Asp Ile Val Ala Pro Ser Asn Ser Gly Phe Asp Val 1 5 10 15 <210> 214 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 214 Ser Ala Tyr Asp Arg Ser Leu Asn Ala Trp Val 1 5 10 <210> 215 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 215 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 216 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 216 Ala Arg Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe 1 5 10 <210> 217 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 217 Gly Tyr Thr Leu Ser Ser His Gly 1 5 <210> 218 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 218 Ile Ser Ala His Asn Gly His Ala 1 5 <210> 219 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 219 Ala Arg Val His Ala Ala Leu Tyr Tyr Gly Met Asp Val 1 5 10 <210> 220 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 220 Ser Gly Ser Ile Asp Ser Asn Tyr 1 5 <210> 221 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 221 Gln Ser Tyr Asp Ser Asn Asn Arg His Val Ile 1 5 10 <210> 222 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 222 Asn Ile Gly Ser Lys Gly 1 5 <210> 223 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 223 Gln Val Trp Asp Ser Gly Ser Asp His Trp Val 1 5 10 <210> 224 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 224 Asn Ile Gly Asp Lys Gly 1 5 <210> 225 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 225 Gln Val Trp Asp Ser Ser Ser Asp His Trp Val 1 5 10 <210> 226 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 226 Asn Ile Gly Asn Lys Gly 1 5 <210> 227 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 227 Asn Ile Gly Gly Lys Gly 1 5 <210> 228 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 228 Gly Phe Thr Phe Asp Asp Tyr Ala 1 5 <210> 229 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 229 Ile Ser Trp Asn Ser Gly Ser Ile 1 5 <210> 230 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 230 Ala Ser Asp Tyr Gly Asp Lys Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 231 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 231 Gly Tyr Thr Phe Thr Thr Tyr Trp 1 5 <210> 232 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 232 Ile Tyr Pro Asp Asp Ser Asp Thr 1 5 <210> 233 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 233 Ala Phe Trp Gly Ala Ser Gly Ala Pro Val Asn Gly Phe Asp Ile 1 5 10 15 <210> 234 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 234 Gly Asp Ser Val Ser Ser Asp Asn Tyr Phe 1 5 10 <210> 235 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 235 Val Tyr Tyr Asn Gly Asn Thr 1 5 <210> 236 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 236 Ala Thr Glu Thr Pro Pro Thr Ser Tyr Phe Asn Ser Gly Pro Phe Asp 1 5 10 15 Ser <210> 237 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 237 Gly Tyr Thr Phe Asn Arg Phe Gly 1 5 <210> 238 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 238 Thr Asn Pro Tyr Asn Gly Asn Thr 1 5 <210> 239 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 239 Ala Arg Val Val Ala Val Asn Gly Met Asp Val 1 5 10 <210> 240 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 240 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 241 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 241 Ala Ser Gln Thr Val Ala Gly Ser Asp Tyr 1 5 10 <210> 242 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 242 Ala Ser Asp Tyr Gly Asp Lys Tyr Ser Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 243 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 243 Gly Phe Thr Phe Asp Asp Phe Ala 1 5 <210> 244 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 244 Ala Ala Trp Asp Gly Gly Leu Asn Gly Arg Gly Val 1 5 10 <210> 245 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 245 Ser Ser Asn Ile Gly Ala Gly Tyr Val 1 5 <210> 246 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 246 Ala Ala Trp Asp Asp Ser Leu Asn Ala Pro Val 1 5 10 <210> 247 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 247 Ser Asn Asn Val Gly Ala His Gly 1 5 <210> 248 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 248 Ser Ser Trp Asp Ser Ser Leu Ser Gly Tyr Val 1 5 10 <210> 249 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 249 Ser Gly Ser Ile Ala Ala Tyr Tyr 1 5 <210> 250 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 250 Gln Ser Tyr Asp Ser Ser Asn Leu Trp Val 1 5 10 <210> 251 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 251 Gln Val Trp His Ser Val Ser Asp Gln Gly Val 1 5 10 <210> 252 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 252 Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 253 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 253 Lys Ser Ser Gln Ser Ile Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 254 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 254 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 255 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 255 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30 Tyr Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Arg Pro Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 256 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 256 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 257 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 257 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Gln 20 25 30 Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Trp Tyr Arg Pro Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 258 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 258 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Ile Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 259 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 259 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Ser 20 25 30 Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Trp Tyr Arg Pro Asn Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 260 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 260 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Lys Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 261 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 261 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Ser 20 25 30 Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Thr Arg Tyr Arg Pro Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 262 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 262 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Arg Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 263 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 263 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Gln 20 25 30 Tyr Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Thr Tyr Tyr Arg Pro Pro Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 264 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 264 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Tyr Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 265 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 265 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Ala 20 25 30 Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Arg Pro Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 266 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 266 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Met Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 267 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 267 Gly Gly Gly Gly Ser 1 5 <210> 268 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 268 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Gly Asn Tyr Arg Gly Ser Leu Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 269 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 269 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Gly Thr Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Val Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Pro Val Leu Arg Tyr Gly Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 270 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 270 Gln Ser Val Leu Thr Leu Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly <210> 271 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 271 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg Gly 20 25 30 Tyr Asn Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asp Thr Asn Arg Pro Ser Gly Val Pro His Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu Gly 100 105 110 <210> 272 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 272 Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 273 <211> 459 <212> PRT <213> Homo sapiens <400> 273 Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu Ile Pro Ala 1 5 10 15 Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu Leu Leu Leu 20 25 30 Met Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro 35 40 45 Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp 50 55 60 Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu 65 70 75 80 Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro 85 90 95 Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp 100 105 110 Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn 115 120 125 Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly 130 135 140 Gly Asp Pro Pro Trp Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe 145 150 155 160 Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala 165 170 175 Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu 180 185 190 Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro 195 200 205 Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln 210 215 220 Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr 225 230 235 240 Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val Val His Leu Ser 245 250 255 Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu 260 265 270 Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala 275 280 285 Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser 290 295 300 Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp 305 310 315 320 Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys 325 330 335 Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser 340 345 350 Ala Lys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp 355 360 365 Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg 370 375 380 Val Ile Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala 385 390 395 400 Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu 405 410 415 Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu 420 425 430 Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser 435 440 445 Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala 450 455 <210> 274 <211> 437 <212> PRT <213> Mus sp. <400> 274 Met Ala Ser Leu Gly Pro Ser Pro Trp Ala Pro Leu Ser Thr Pro Ala 1 5 10 15 Pro Thr Ala Gln Leu Leu Leu Phe Leu Leu Leu Gln Val Ser Ala Gln 20 25 30 Pro Gln Gly Leu Ser Gly Met Gln Gly Glu Pro Ser Leu Gly Asp Ser 35 40 45 Ser Ser Gly Glu Asp Glu Leu Gly Val Asp Val Leu Pro Ser Glu Glu 50 55 60 Asp Ala Pro Glu Glu Ala Asp Pro Pro Asp Gly Glu Asp Pro Pro Glu 65 70 75 80 Val Asn Ser Glu Asp Arg Met Glu Glu Ser Leu Gly Leu Glu Asp Leu 85 90 95 Ser Thr Pro Glu Ala Pro Glu His Ser Gln Gly Ser His Gly Asp Glu 100 105 110 Lys Gly Gly Gly His Ser His Trp Ser Tyr Gly Gly Thr Leu Leu Trp 115 120 125 Pro Gln Val Ser Pro Ala Cys Ala Gly Arg Phe Gln Ser Pro Val Asp 130 135 140 Ile Arg Leu Glu Arg Thr Ala Phe Cys Arg Thr Leu Gln Pro Leu Glu 145 150 155 160 Leu Leu Gly Tyr Glu Leu Gln Pro Leu Pro Glu Leu Ser Leu Ser Asn 165 170 175 Asn Gly His Thr Val Gln Leu Thr Leu Pro Pro Gly Leu Lys Met Ala 180 185 190 Leu Gly Pro Gly Gln Glu Tyr Arg Ala Leu Gln Leu His Leu His Trp 195 200 205 Gly Thr Ser Asp His Pro Gly Ser Glu His Thr Val Asn Gly His Arg 210 215 220 Phe Pro Ala Glu Ile His Val Val His Leu Ser Thr Ala Phe Ser Glu 225 230 235 240 Leu His Glu Ala Leu Gly Arg Pro Gly Gly Leu Ala Val Leu Ala Ala 245 250 255 Phe Leu Gln Glu Ser Pro Glu Glu Asn Ser Ala Tyr Glu Gln Leu Leu 260 265 270 Ser His Leu Glu Glu Ile Ser Glu Glu Gly Ser Lys Ile Glu Ile Pro 275 280 285 Gly Leu Asp Val Ser Ala Leu Leu Pro Ser Asp Phe Ser Arg Tyr Tyr 290 295 300 Arg Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys Ser Gln Gly Val Ile 305 310 315 320 Trp Thr Val Phe Asn Glu Thr Val Lys Leu Ser Ala Lys Gln Leu His 325 330 335 Thr Leu Ser Val Ser Leu Trp Gly Pro Arg Asp Ser Arg Leu Gln Leu 340 345 350 Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg Thr Ile Glu Ala Ser 355 360 365 Phe Pro Ala Ala Glu Asp Ser Ser Pro Glu Pro Val His Val Asn Ser 370 375 380 Cys Phe Thr Ala Gly Asp Ile Leu Ala Leu Val Phe Gly Leu Leu Phe 385 390 395 400 Ala Val Thr Ser Ile Ala Phe Leu Leu Gln Leu Arg Arg Gln His Arg 405 410 415 His Arg Ser Gly Thr Lys Asp Arg Val Ser Tyr Ser Pro Ala Glu Met 420 425 430 Thr Glu Thr Gly Ala 435 <210> 275 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 275 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 276 <211> 95 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 276 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser 85 90 95 <210> 277 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 277 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys <210> 278 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 278 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Asn <210> 279 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 279 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 280 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 280 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro 100 <210> 281 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 281 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly <210> 282 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 282 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Lys Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Thr Asp Ser Ser Gly Asn His 85 90 95 <210> 283 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 283 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg <210> 284 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 284 Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln 20 25 30 Gly Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40 45 Ser Tyr Arg Asn Asn Asn Arg Pro Ser Gly Ile Ser Glu Arg Leu Ser 50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Ser Ser Leu 85 90 95 Ser Ala <210> 285 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 285 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe 100 105 <210> 286 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 286 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Asn <210> 287 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 287 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Asn Asn Arg His Val Ile 100 <210> 288 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 288 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp <210> 289 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 289 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asp Tyr Gly Asp Lys Tyr Ser Tyr Tyr Gly Met Asp Val 100 105 110 <210> 290 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 290 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly <210> 291 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 291 Gln Pro Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Phe Asp Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Gly Gly Leu 85 90 95 Asn Gly Arg Gly Val 100 <210> 292 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 292 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Gly <210> 293 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 293 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 294 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 294 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 <210> 295 <211> 98 <212> PRT <213> Homo sapiens <400> 295 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 296 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 296 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Gly Leu Trp Phe Gly Leu Thr His Asp Tyr Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 297 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 297 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Asn Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Arg 85 90 95 Ser Gly Pro Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu 100 105 110 <210> 298 <211> 98 <212> PRT <213> Homo sapiens <400> 298 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 299 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 299 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ser Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asn Ser Asp Gly Ser Arg Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Pro Gly Phe Phe Gly Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 300 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 300 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Arg Asn 20 25 30 Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Thr Asp Tyr Tyr Cys Ala Ala Trp Asp Ala Arg Leu 85 90 95 Thr Gly Pro Leu Phe Gly Gly Gly Thr Lys Leu Ser Val Leu 100 105 110 <210> 301 <211> 98 <212> PRT <213> Homo sapiens <400> 301 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg <210> 302 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 302 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Val Asn Ser Arg Thr Ile Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Tyr Tyr Ala Met Glu Val Trp Gly Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 303 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 303 Leu Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Thr Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Glu Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 304 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 304 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gly Gly Leu Glu Trp Met 35 40 45 Gly Trp Val Asp Asn Asn Asn Gly Asn Ile Asn Tyr Ala Gln Lys Phe 50 55 60 Leu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Phe Ser Ser Arg Trp Tyr Leu Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 305 <211> 99 <212> PRT <213> Homo sapiens <400> 305 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Leu <210> 306 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 306 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Thr Glu Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Ser Ser Tyr Thr Arg Ser 85 90 95 Ser Thr Ser Tyr Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 307 <211> 98 <212> PRT <213> Homo sapiens <400> 307 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 308 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 308 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Leu Pro Met Phe Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Leu Thr Leu Ile Ala Asp Glu Ser Thr Arg Thr Val Tyr 65 70 75 80 Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Asp Ile Val Ala Pro Ser Asn Ser Gly Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 309 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 309 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln 20 25 30 Gly Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40 45 Ser Tyr Arg Asn Asp Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln 65 70 75 80 Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Tyr Asp Arg Ser Leu 85 90 95 Asn Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 310 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 310 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 311 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 311 Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Ser His 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala His Asn Gly His Ala Ser Asn Ala Gln Lys Val 50 55 60 Glu Asp Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val His Ala Ala Leu Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 312 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 312 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Asn Asn Arg His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 313 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 313 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Leu Ala Pro Gly Gln 1 5 10 15 Ser Ala Arg Ile Ser Cys Gly Gly Asp Asn Ile Gly Ser Lys Gly Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Val Tyr 35 40 45 Asp Asp Arg Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Gly Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 314 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 314 Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Gly Gly Ser Asn Ile Gly Asp Lys Gly Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 315 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 315 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Asn Lys Gly Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 316 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 316 Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Arg Gly Asn Asn Ile Gly Gly Lys Gly Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Tyr Ser Arg Arg Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 His Ser Gly Ser Ala Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 317 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 317 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120 ccagggaagg gcctggagtg ggtctcaggt attagttgga atagtggtag cataggctat 180 gcggactctg tgaagggccg attcaccgtc tccagagaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagtgactac 300 ggtgacaaat actactacta cggtatggac gtctggggca aagggaccac ggtcaccgtc 360 tcctca 366 <210> 318 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 318 cagcctgggc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga agtaatactg tcaactggta tcagcaattc 120 cccggaaagg cccccaaact cctcatcttt aatgataatc agcggccctc aggggtccct 180 gaccgcttct ctgcttccaa gtctggcacc tcagcctccc tggccattag tggcctccag 240 tctgaggatg aggctgacta ttactgtgcg gcatgggatg gcggtctgaa tggtcgaggg 300 gtgttcggcg gagggaccaa actgaccgtc cta 333 <210> 319 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 319 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asp Tyr Gly Asp Lys Tyr Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 320 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 320 Gln Pro Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Phe Asn Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Gly Gly Leu 85 90 95 Asn Gly Arg Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 321 <211> 285 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 321 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 60 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 120 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 180 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 240 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaa 285 <210> 322 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 322 gcagagccca aatcttgtga caaaactcac acatgcccac cgtgccca 48 <210> 323 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 323 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 cccatcgaga aaaccatctc caaagccaaa 330 <210> 324 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 324 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 60 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 240 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 300 ctctccctgt ctccgggtaa atga 324 <210> 325 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 325 ggtcagccca aggctgcccc ctcggtcact ctgttcccgc cctcctctga ggagcttcaa 60 gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120 gcctggaagg cagatggcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180 caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 240 tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300 gcccctacag aatgttcatg a 321 <210> 326 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 326 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys <210> 327 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 327 Ala Glu P...

Claims

1. A modified cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular ligand-binding domain, the extracellular ligand-binding domain comprises a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain is specific to CAIX, the second antigen-binding domain is specific to CD70, and the extracellular ligand-binding domain comprises an antibody or its antigen-binding fragment. Antibodies or antigen-binding fragments specific to CAIX are as follows: VH CDR1, CDR2, and CDR3, respectively, are determined by the amino acid sequences SYAMS, AISANGGTTYYADSVKG, and NGNYRGAFDI, and VL CDR1, CDR2, and CDR3 are identified by their respective amino acid sequences TGSSSNIGAGFDVH, GNTNRPS, and QSYDSRLSAWV. Includes, Antibodies or antigen-binding fragments specific to CD70 are as follows: (a) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SGSIASNY, EDN, and QSYDSGNRRV; (b) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTFSSYA, ISGSGGSR, and ARGRGGHGMDV, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SSNIGSNY, RNN, and AAWDDSLNGLV; (c) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GGTFSSQA, IIPFFGVP, and AVLKGRGNFDF, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences YSVFHSPNNKNY, WAS, and QQRSNWPLT; (d) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences ALPKKY, EDS, and YSTDSSGNHKV; (e) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSTSH, KDSGGKT, and ARARPSDPYDGSGFDAFDI, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences SNNVGNQG, RNN, and SAWDSSLSAWV; or (f) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFIFSDYY, IRSRRGET, and ARHRKSFTDLDAFDL, VL CDR1, CDR2, and CDR3, respectively, based on their amino acid sequences QDIGTD, KAS, and QHFNNYPAT. including, Manipulated cells.

2. The manipulated cell according to claim 1, wherein the CAR further comprises a transmembrane polypeptide and an intracellular signaling domain.

3. The manipulated cell according to claim 2, wherein the CAR further comprises a stalk region located between the extracellular ligand-binding domain and the transmembrane domain.

4. The manipulated cell according to claim 2 or 3, wherein the CAR further comprises a co-stimulatory domain.

5. The manipulated cell according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment specific to CAIX comprises the following: Amino acid sequence EVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSS VH by, and VL is represented by the amino acid sequence QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGFDVHWYQQLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAITGLQAEDETDYYCQSYDSRLSAWVFGGGTKLTVLG.

6. The manipulated cell according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment specific to CD70 comprises the following: (a) VH and, based on the amino acid sequence QVQLVQSGGGLVQPRGSLRLSCAASGFTVSNYAMSWVRQAPGKGLEWVATKSGSDGRTYYADSVKGRFTIARDNSKNSLYLQMNSLRAADTAVYYCAKGIYDVTGSSFDS VL by amino acid sequence NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSGNRRV; (b) VH and, based on the amino acid sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSLISGSGGSRYYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCARGRGGHGMDV VL by amino acid sequence QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYYCAAWDDSLNGLV; (c) VH and, based on the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCRSSGGTFSSQAFSWVRQAPGQGLEWMGRIIPFFGVPTYAQRFQGRVTITADKSPTTAYMELTSLRSDDTAVYYCAVLKGRGNFDF VL based on the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSYSVFHSPNNKNYLAWYQQRPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLT; (d) VH and, based on the amino acid sequence QVQLVQSGGGLVQPRGSLRLSCAASGFTVSNYAMSWVRQAPGKGLEWVATKSGSDGRTYYADSVKGRFTIARDNSKNSLYLQMNSLRAADTAVYYCAKGIYDVTGSSFDS VL by amino acid sequence SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVMFEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDSSGNHKV; (e) VH and, based on the amino acid sequence EVQLVESGGGVVQPGRSLRLSCAASGFTVSTSHMSWVRQAPGKGLEWLSGKDSGGKTYYADSVRGRFTIARDDSLNTVFLQMNNMRDEDSGVYYCARARPSDPYDGSGFDAFDI VL by amino acid sequence SYELTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSYRNNNRPSGISERFSASRSGNTASLTITGLQPEDEADYYCSAWDSSLSAWV; or (f) VH and, based on the amino acid sequence QVQLVQSGGGLVKPRGSLRLSCAASGFIFSDYYMSWIRQAPGKGLQWVASIRSRRGETNYADSVKGRFTIARDNAEKSLYLQMNSLRAEDAAVYYCARHRKSFTDLDAFDL VL based on the amino acid sequence DIVMTQSPSTLSASVGDRVTITCRASQDIGTDLSWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQHFNNYPAT.

7. The manipulated cell according to any one of claims 1 to 4, wherein the extracellular ligand-binding domain comprises the VH and VL described in claim 5, and the VH and VL described in any one of claims 6(a) to (f).

8. A modified cell according to any one of claims 1 to 7, which expresses and secretes a recombinant polypeptide.

9. The engineered cell according to claim 8, wherein the recombinant polypeptide comprises an antibody or an antigen-binding fragment thereof, or a cytokine.

10. The engineered cell according to claim 8 or 9, wherein the recombinant polypeptide modulates the target immune system.

11. The engineered cell according to claim 8 or 9, wherein the recombinant polypeptide is an immune checkpoint blocking antibody.

12. The engineered cell according to claim 8 or 9, wherein the recombinant polypeptide regulates tumor vasculogenesis.

13. The engineered cell according to claim 12, wherein the recombinant polypeptide specifically binds to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1.

14. The manipulated cell according to claim 8, wherein the recombinant polypeptide comprises an antibody or antigen-binding fragment specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4.

15. The manipulated cell according to claim 9, wherein the cytokine comprises IL-12, IL-15, or IL-18.

16. The manipulated cells according to any one of claims 1 to 15, wherein the cells include T cells, NK cells, or NKT cells.

17. The manipulated cell according to claim 16, wherein the T cell is a CD4+, CD8+, CD3+ panT cell, or any combination thereof.

18. The manipulated cells according to claim 16, wherein the T cells are a mixed population of CD4+ T cells and CD8+ T cells.

19. A nucleic acid construct encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises extracellular ligand-binding domains specific to a first antigen and a second antigen on the surface of a cancer cell, the first antigen comprising CAIX, the second antigen comprising CD70, and the extracellular ligand-binding domain comprising an antibody or its antigen-binding fragment. Antibodies or antigen-binding fragments specific to CAIX are as follows: VH CDR1, CDR2, and CDR3, respectively, are determined by the amino acid sequences SYAMS, AISANGGTTYYADSVKG, and NGNYRGAFDI, and VL CDR1, CDR2, and CDR3 are identified by their respective amino acid sequences TGSSSNIGAGFDVH, GNTNRPS, and QSYDSRLSAWV. Includes, Antibodies or antigen-binding fragments specific to CD70 are as follows: (a) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SGSIASNY, EDN, and QSYDSGNRRV; (b) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTFSSYA, ISGSGGSR, and ARGRGGHGMDV, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SSNIGSNY, RNN, and AAWDDSLNGLV; (c) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GGTFSSQA, IIPFFGVP, and AVLKGRGNFDF, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences YSVFHSPNNKNY, WAS, and QQRSNWPLT; (d) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences ALPKKY, EDS, and YSTDSSGNHKV; (e) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSTSH, KDSGGKT, and ARARPSDPYDGSGFDAFDI, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences SNNVGNQG, RNN, and SAWDSSLSAWV; or (f) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFIFSDYY, IRSRRGET, and ARHRKSFTDLDAFDL, VL CDR1, CDR2, and CDR3, respectively, based on their amino acid sequences QDIGTD, KAS, and QHFNNYPAT. including, Nucleic acid constructs.

20. The nucleic acid construct according to claim 19, wherein the chimeric antigen receptor further comprises a transmembrane polypeptide and an intracellular signaling domain.

21. The nucleic acid construct according to claim 20, wherein the chimeric antigen receptor further comprises a costimulatory domain.

22. A nucleic acid construct according to any one of claims 19 to 21, further encoding a recombinant polypeptide.

23. The nucleic acid construct according to claim 22, wherein the recombinant polypeptide can be secreted from the manipulated cell.

24. The nucleic acid construct according to claim 22 or 23, wherein the recombinant polypeptide comprises an antibody or an antigen-binding fragment thereof, or a cytokine.

25. The nucleic acid construct according to any one of claims 22 to 24, wherein the recombinant polypeptide modulates the target immune system.

26. The nucleic acid construct according to any one of claims 22 to 24, wherein the recombinant polypeptide is an immune checkpoint blocking antibody.

27. The nucleic acid construct according to any one of claims 22 to 24, wherein the recombinant polypeptide regulates tumor vasculogenesis.

28. The nucleic acid construct according to claim 22, wherein the recombinant polypeptide comprises an antibody or antigen-binding fragment specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4.

29. The nucleic acid construct according to claim 24, wherein the cytokine comprises IL-12, IL-15, or IL-18.

30. The nucleic acid construct according to claim 27, wherein the recombinant polypeptide specifically binds to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1.

31. A vector comprising a nucleic acid construct according to any one of claims 19 to 30.

32. A cell containing the vector according to claim 31.

33. A pharmaceutical composition for treating a subject suffering from cancer, comprising a therapeutically effective amount of the manipulated cells described in any one of claims 1 to 18.

34. A pharmaceutical composition for reducing the progression or promoting the regression of cancer in a subject, comprising a therapeutically effective amount of the manipulated cells described in any one of claims 1 to 18.

35. A pharmaceutical composition for reducing the proliferation of cancer cells in a subject, comprising a therapeutically effective amount of the manipulated cells described in any one of claims 1 to 18.

36. The pharmaceutical composition according to any one of claims 33 to 35, wherein the cancer includes renal cell carcinoma.

37. A chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, wherein the extracellular ligand-binding domain is specific to a first antigen and a second antigen on the surface of a cancer cell, the first antigen comprising CAIX, the second antigen comprising CD70, and the extracellular ligand-binding domain comprising an antibody or an antigen-binding fragment thereof. Antibodies or antigen-binding fragments specific to CAIX are as follows: VH CDR1, CDR2, and CDR3, respectively, are determined by the amino acid sequences SYAMS, AISANGGTTYYADSVKG, and NGNYRGAFDI, and VL CDR1, CDR2, and CDR3 are identified by their respective amino acid sequences TGSSSNIGAGFDVH, GNTNRPS, and QSYDSRLSAWV. Includes, Antibodies or antigen-binding fragments specific to CD70 are as follows: (a) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SGSIASNY, EDN, and QSYDSGNRRV; (b) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTFSSYA, ISGSGGSR, and ARGRGGHGMDV, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SSNIGSNY, RNN, and AAWDDSLNGLV; (c) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GGTFSSQA, IIPFFGVP, and AVLKGRGNFDF, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences YSVFHSPNNKNY, WAS, and QQRSNWPLT; (d) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences ALPKKY, EDS, and YSTDSSGNHKV; (e) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSTSH, KDSGGKT, and ARARPSDPYDGSGFDAFDI, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences SNNVGNQG, RNN, and SAWDSSLSAWV; or (f) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFIFSDYY, IRSRRGET, and ARHRKSFTDLDAFDL, VL CDR1, CDR2, and CDR3, respectively, based on their amino acid sequences QDIGTD, KAS, and QHFNNYPAT. including, Chimeric antigen receptor.

38. The CAR according to claim 37, further comprising a transmembrane polypeptide and an intracellular signaling domain.

39. The CAR according to claim 38, further comprising a co-stimulatory domain.

40. A cell comprising a chimeric antigen receptor (CAR) according to any one of claims 37 to 39.

41. A modified cell comprising a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises an extracellular ligand-binding domain specific to CAIX, and the second chimeric antigen receptor comprises an extracellular ligand-binding domain specific to CD70. The extracellular ligand-binding domains specific to CAIX are as follows: VH CDR1, CDR2, and CDR3, respectively, are determined by the amino acid sequences SYAMS, AISANGGTTYYADSVKG, and NGNYRGAFDI, and VL CDR1, CDR2, and CDR3 are identified by their respective amino acid sequences TGSSSNIGAGFDVH, GNTNRPS, and QSYDSRLSAWV. The antibody contains or contains an antigen-binding fragment thereof, The extracellular ligand-binding domain specific to CD70 is as follows: (a) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SGSIASNY, EDN, and QSYDSGNRRV; (b) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTFSSYA, ISGSGGSR, and ARGRGGHGMDV, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences SSNIGSNY, RNN, and AAWDDSLNGLV; (c) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GGTFSSQA, IIPFFGVP, and AVLKGRGNFDF, VL CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences YSVFHSPNNKNY, WAS, and QQRSNWPLT; (d) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSNYA, KSGSDGRT, and AKGIYDVTGSSFDS, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences ALPKKY, EDS, and YSTDSSGNHKV; (e) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFTVSTSH, KDSGGKT, and ARARPSDPYDGSGFDAFDI, VL CDR1, CDR2, and CDR3, respectively, according to the amino acid sequences SNNVGNQG, RNN, and SAWDSSLSAWV; or (f) VH CDR1, CDR2, and CDR3, respectively, based on the amino acid sequences GFIFSDYY, IRSRRGET, and ARHRKSFTDLDAFDL, VL CDR1, CDR2, and CDR3, respectively, based on their amino acid sequences QDIGTD, KAS, and QHFNNYPAT. An antibody containing or an antigen-binding fragment thereof Manipulated cells.

42. The engineered cell according to claim 41, which expresses and secretes recombinant polypeptides.

43. The engineered cell according to claim 42, wherein the recombinant polypeptide comprises an antibody or an antigen-binding fragment thereof, or a cytokine.

44. The engineered cell according to claim 42 or 43, wherein the recombinant polypeptide modulates the target immune system.

45. The engineered cell according to claim 42 or 43, wherein the recombinant polypeptide is an immune checkpoint blocking antibody.

46. The engineered cell according to claim 42 or 43, wherein the recombinant polypeptide modulates tumor vasculogenesis.

47. The engineered cell according to claim 42, wherein the recombinant polypeptide comprises an antibody or antigen-binding fragment specific to TIGIT, GITR, PD-L1, PD-L2, PD-1, CTLA-4, VISTA, CD70, TIM-3, LAG-3, CD40L, or CCR4.

48. The manipulated cell according to claim 43, wherein the cytokine comprises IL-12, IL-15, or IL-18.

49. The engineered cell according to claim 46, wherein the recombinant polypeptide specifically binds to VEGF, VEGFR1, VEGFR2, PDGF, Ang-1, or AT1.

50. The manipulated cells according to any one of claims 41 to 49, wherein the cells include T cells or NK cells.

51. The manipulated cell according to claim 50, wherein the T cell is a CD4+, CD8+, CD3+ panT cell, or any combination thereof.

52. The manipulated cells according to claim 50, wherein the T cells are a mixed population of CD4+ T cells and CD8+ T cells.

53. The engineered cell according to any one of claims 41 to 52, wherein the first chimeric antigen receptor and the second chimeric antigen receptor are expressed from a single nucleic acid construct.

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