CN112689642B

CN112689642B - T cell antigen conjugates with various construct optimizations

Info

Publication number: CN112689642B
Application number: CN201980047935.6A
Authority: CN
Inventors: 乔纳森·罗恩·布拉姆森; 克里斯托弗·W·赫尔森; 乔安妮·艾丽西亚·汉米尔; 肯尼斯·安东尼·牧瓦瓦司
Original assignee: McMaster University; Tramvera Immunization Usa Ltd
Current assignee: McMaster University; Tramvera Immunization Usa Ltd
Priority date: 2018-07-17
Filing date: 2019-07-17
Publication date: 2025-01-17
Anticipated expiration: 2039-07-17
Also published as: JP7404279B2; PH12021550114A1; AU2019307905B2; MX2021000541A; KR20210021593A; IL280049A; BR112021000735A2; WO2020018727A1; EP3823984A4; SG11202100307WA; CA3104887A1; CN112689642A; EP3823984A1; AU2019307905A1; JP2021530971A

Abstract

A trifunctional molecule is provided comprising (i) a target specific ligand, (ii) a ligand that binds to a protein associated with a TCR complex, and (iii) a T cell receptor signaling domain polypeptide. Variants of the molecules are provided, including variants that exhibit optimized surface expression, transduction efficiency, and effector function. Variations include, for example, different ligands that bind CD3 epsilon (e.g., OKT3, L2K, F6A, UCHT1, and humanized UCHT 1), different signaling domains, and different linkers between the domains.

Description

T cell antigen conjugates with various construct optimizations

Cross reference

The present application claims the benefits of U.S. provisional application number 62/699,173 submitted at 7.17.2018, U.S. provisional application number 62/703,037 submitted at 7.25.2018, U.S. provisional application number 62/773,120 submitted at 11.29.2019, U.S. provisional application number 62/826,853 submitted at 3.3.2019, U.S. provisional application number 62/828,879 submitted at 4.3.2019, U.S. provisional application number 62/839,235 submitted at 4.26.2019, U.S. non-provisional application number 16/442,274 submitted at 6.14.2019 and U.S. provisional application number 62/874,426 submitted at 7.15.2019, each of which is incorporated herein by reference in its entirety.

Sequence listing

The present application comprises a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy produced on day 7, month 17 of 2019 was named 55247704601_SL.txt, size 131,072 bytes.

Disclosure of Invention

In certain embodiments, disclosed herein are nucleic acid sequences encoding CD19 trifunctional T cell antigen conjugates (CD 19-TAC). In certain embodiments, the nucleic acid sequence encoding a CD19 trifunctional T cell antigen conjugate (CD 19-TAC) comprises (a) a first polynucleotide encoding a ligand that selectively binds to the CD19 antigen. In certain embodiments, the nucleic acid sequence encoding a CD19 trifunctional T cell antigen conjugate (CD 19-TAC) comprises (b) a second polynucleotide encoding a UCHT1 ligand that binds CD3. In certain embodiments, the nucleic acid sequence encoding a CD19 trifunctional T cell antigen conjugate (CD 19-TAC) comprises (c) a third polynucleotide encoding a TCR signaling domain polypeptide comprising a cytoplasmic domain and a transmembrane domain. In certain embodiments, the components encoded by the first, second, and/or third polynucleotides are linked in any suitable manner, e.g., in any suitable order and/or comprising any suitable linker. In certain embodiments, the component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused to each other directly or are linked by at least one linker. In certain embodiments, the ligand that selectively binds CD19 antigen is a single chain variable fragment (scFv). In certain embodiments, the ligand that selectively binds to the CD19 antigen comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 36. In certain embodiments, the UCHT1 ligand is a single chain antibody. In certain embodiments, the UCHT1 ligand comprises a Y182T mutation (SEQ ID NO: 72). In certain embodiments, the UCHT1 ligand is a humanized variant of the UCHT1 ligand (huUCHT 1) (SEQ ID NO: 44). In certain embodiments, the UCHT1 ligand is a humanized variant of UCHT1 comprising a Y177T mutation (huUCHT (Y177T)) (SEQ ID NO: 46). In certain embodiments, the UCHT1 ligand comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO. 14, SEQ ID NO. 72, SEQ ID NO. 44, or SEQ ID NO. 46. In certain embodiments, the cytoplasmic domain is a CD4 cytoplasmic domain and the transmembrane domain is a CD4 transmembrane domain. In certain embodiments, the polypeptide encoded by the third polynucleotide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 18. In certain embodiments, the component encoded by (a) and the component encoded by (c) are fused to the component encoded by (b). In certain embodiments, the component encoded by (b) and the component encoded by (c) are fused to the component encoded by (a). In certain embodiments, at least one linker connects the component encoded by (a) to the component encoded by (b). In certain embodiments, the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helix or a short helix. In certain embodiments, the at least one linker comprises a sequence having at least 80% identity to SEQ ID NO. 12 (G ₄ S flexible linker ("G ₄ S" is disclosed as SEQ ID NO: 73)), SEQ ID NO. 32 (large protein domain), SEQ ID NO. 30 (long helix) or SEQ ID NO. 28 (short helix), at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity. In certain embodiments, the CD3 is a CD3 of a TCR complex on a cell expressing the second polynucleotide. In certain embodiments, the binding of CD3 induces activation of a cell expressing the second polynucleotide. In certain embodiments, the CD19-TAC comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 64. In certain embodiments, the nucleic acid sequence does not encode a costimulatory domain. In certain embodiments, the nucleic acid sequence does not encode an activation domain.

In certain embodiments, disclosed herein are vector constructs comprising (a) a nucleic acid sequence disclosed herein (e.g., a nucleic acid sequence encoding CD 19-TAC), and (b) a promoter functional in mammalian cells.

In certain embodiments, disclosed herein are T cells comprising a nucleic acid sequence disclosed herein (e.g., a nucleic acid sequence encoding CD 19-TAC).

In certain embodiments, disclosed herein are pharmaceutical compositions comprising T cells disclosed herein and a pharmaceutically acceptable excipient.

In certain embodiments, disclosed herein are methods of treating a CD19 expressing cancer in an individual in need thereof, comprising administering to the individual a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising T cells comprising a nucleic acid sequence described herein, e.g., any one or more nucleic acid sequences described herein as encoding a CD19 trifunctional T cell antigen conjugate (CD 19-TAC). In certain embodiments, the cancer is a B cell malignancy. In certain embodiments, the cancer is a B-cell lymphoma, acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), or non-hodgkin's lymphoma. In certain embodiments, the pharmaceutical composition is administered arterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously or intraperitoneally.

In certain embodiments, disclosed herein are nucleic acid sequences encoding a trifunctional T cell antigen conjugate (Tri-TAC) comprising (a) a first polynucleotide encoding a target-specific ligand, (b) a second polynucleotide encoding a ligand that binds a protein associated with a TCR complex, and (c) a third polynucleotide encoding a T cell receptor signaling domain polypeptide, wherein the ligand that binds a protein associated with a TCR complex is selected from OKT3, F6A, or L2K. In certain embodiments, the component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused to each other directly or are linked by at least one linker. in certain embodiments, the component encoded by (a) and the component encoded by (b) are fused directly and linked to the component encoded by (c) by a linker. In certain embodiments, the component encoded by (b) and the component encoded by (c) are fused directly and linked to the component encoded by (a) by a linker. In certain embodiments, the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helix or a short helix. In certain embodiments, the at least one linker has a length of at least 80% with SEQ ID NO. 12 (G ₄ S flexible linker ("G ₄ S" is disclosed as SEQ ID NO: 73)), SEQ ID NO. 32 (large protein domain), SEQ ID NO. 30 (long helix) or SEQ ID NO. 28 (short helix), at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is OKT3. In certain embodiments, the ligand that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is F6A. In certain embodiments, the ligand that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is L2K. In certain embodiments, the ligand that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 26. In certain embodiments, the protein associated with the TCR complex is CD3. In certain embodiments, the target-specific ligand selectively binds a tumor antigen. In certain embodiments, the target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide or a single chain variable fragment (scFv). In certain embodiments, the target-specific ligand selectively binds CD19 antigen, HER2 antigen, or BCMA antigen. In certain embodiments, the target-specific ligand selectively binds HER-2 antigen and comprises an antigen binding domain of an antibody selected from trastuzumab, pertuzumab, lapatinib, lenatinib, trastuzumab-maytansinoid conjugate, rituximab, margetuximab, ti Mi Tuozhu mab, and Ertumaxomab. In certain embodiments, the target specific ligand selectively binds BCMA antigen comprising an antigen binding domain of an antibody selected from Belantamab mafodotin and GSK 2857916. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:36, SEQ ID NO:8 or SEQ ID NO: 34. In certain embodiments, the T cell receptor signaling domain polypeptide comprises a cytoplasmic domain and a transmembrane domain. In certain embodiments, the cytoplasmic domain is a CD4 cytoplasmic domain and the transmembrane domain is a CD4 transmembrane domain, or wherein the cytoplasmic domain is a CD8 cytoplasmic domain and the transmembrane domain is a CD8 transmembrane domain. In certain embodiments, the nucleic acid sequence further comprises a leader sequence. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 6, SEQ ID NO. 48 or SEQ ID NO. 50. In certain embodiments, the CD3 is a CD3 of a TCR complex on a cell expressing the second polynucleotide. In certain embodiments, the binding of CD3 induces activation of a cell expressing the second polynucleotide. In certain embodiments, the Tri-TAC comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 75, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59 or SEQ ID NO. 61. In certain embodiments, the Tri-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60 or SEQ ID NO: 62. In certain embodiments, the nucleic acid sequence does not encode a costimulatory domain. In certain embodiments, the nucleic acid sequence does not encode an activation domain.

In certain embodiments, disclosed herein are nucleic acid sequences encoding a trifunctional T cell antigen conjugate (Tri-TAC) comprising (a) a first polynucleotide encoding a target-specific ligand, (b) a second polynucleotide encoding a ligand that binds to a protein associated with a TCR complex, and (c) a third polynucleotide encoding a T cell receptor signaling domain polypeptide, wherein the nucleic acid sequences further comprise a leader sequence, and wherein the component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused to each other directly or linked by at least one linker. In certain embodiments, the target-specific ligand selectively binds a tumor antigen. in certain embodiments, the target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide or a single chain variable fragment (scFv). In certain embodiments, the target-specific ligand selectively binds CD19 antigen, HER2 antigen, or BCMA antigen. In certain embodiments, the target-specific ligand selectively binds HER-2 antigen and comprises an antigen binding domain of an antibody selected from trastuzumab, pertuzumab, lapatinib, lenatinib, trastuzumab-maytansinoid conjugate, rituximab, margetuximab, ti Mi Tuozhu mab, and Ertumaxomab. In certain embodiments, the target-specific ligand selectively binds BCMA antigen and comprises an antigen binding domain of an antibody selected from Belantamab mafodotin and GSK 2857916. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:36, SEQ ID NO:8, SEQ ID NO:34, SEQ ID NO:52, or SEQ ID NO: 54. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is selected from UCHT1, UCHT1 (Y182T), huUCHT1, huUCHT1 (Y177T), OKT3, F6A, or L2K. In certain embodiments, the ligand that binds to a protein associated with a TCR complex has an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 14, SEQ ID NO. 72, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 22, SEQ ID NO. 24 or SEQ ID NO. 26. In certain embodiments, the protein associated with the TCR complex is CD3. In certain embodiments, the T cell receptor signaling domain polypeptide comprises a cytoplasmic domain and a transmembrane domain. In certain embodiments, the cytoplasmic domain is a CD4 cytoplasmic domain and the transmembrane domain is a CD4 transmembrane domain, or wherein the cytoplasmic domain is a CD8 cytoplasmic domain and the transmembrane domain is a CD8 transmembrane domain. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 6, SEQ ID NO. 48 or SEQ ID NO. 50. in certain embodiments, the component encoded by (a) and the component encoded by (b) are fused directly and linked to the component encoded by (c) by a linker. In certain embodiments, the component encoded by (b) and the component encoded by (c) are fused directly and linked to the component encoded by (a) by a linker. In certain embodiments, the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helix or a short helix. In certain embodiments, the at least one linker has a length of at least 80% with SEQ ID NO. 12 (G ₄ S flexible linker ("G ₄ S" is disclosed as SEQ ID NO: 73)), SEQ ID NO. 32 (large protein domain), SEQ ID NO. 30 (long helix) or SEQ ID NO. 28 (short helix), at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity. In certain embodiments, the CD3 is a CD3 of a TCR complex on a cell expressing the second polynucleotide. In certain embodiments, the binding of CD3 induces activation of a cell expressing the second polynucleotide. In certain embodiments, the Tri-TAC comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 75, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59 or SEQ ID NO. 61. In certain embodiments, the Tri-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60 or SEQ ID NO: 62. In certain embodiments, the nucleic acid sequence does not encode a costimulatory domain. in certain embodiments, the nucleic acid sequence does not encode an activation domain.

In certain embodiments, disclosed herein are polypeptides encoded by the nucleic acid sequences disclosed herein.

In certain embodiments, disclosed herein are vector constructs comprising (a) a nucleic acid sequence disclosed herein, and (b) a promoter functional in mammalian cells.

In certain embodiments, disclosed herein are T cells comprising a nucleic acid sequence disclosed herein.

In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof, comprising administering to the individual a pharmaceutical composition disclosed herein. In certain embodiments, the subject is a mammal. In certain embodiments, the cancer is a solid cancer or a liquid cancer. In certain embodiments, the cancer is lung cancer, breast cancer, multiple myeloma, glioblastoma, gastric cancer, ovarian cancer, gastric cancer, colorectal cancer, urothelial cancer, endometrial cancer, or colon cancer. In certain embodiments, the cancer comprises a CD19 expressing cancer cell. In certain embodiments, the cancer is a B cell malignancy. In certain embodiments, the cancer is a B-cell lymphoma, acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), or non-hodgkin's lymphoma. In certain embodiments, the cancer comprises HER-2 expressing cancer cells. In certain embodiments, the cancer is breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, or gastric cancer. In certain embodiments, the cancer comprises BCMA-expressing cancer cells. In certain embodiments, the cancer is leukemia, lymphoma, or multiple myeloma. In certain embodiments, the pharmaceutical composition is administered to the subject arterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously or intraperitoneally. In certain embodiments, the pharmaceutical composition is in unit dosage form. In certain embodiments, the pharmaceutical composition comprises about 0.5-2 x 10 ⁹ T cells. In certain embodiments, the pharmaceutical composition is administered daily, weekly, biweekly, monthly, bi-monthly, or yearly.

In certain embodiments, disclosed herein are nucleic acid sequences encoding a CD19 trifunctional T cell antigen conjugate (CD 19-TAC) comprising (a) a first polynucleotide encoding a ligand that selectively binds to the CD19 antigen, (b) a second polynucleotide encoding a humanized variant of a UCHT1 ligand comprising a Y177T mutation that binds to CD3 (huUCHT 1) (huUCHT (Y177T)), and (c) a third polynucleotide encoding a polypeptide comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain, wherein the ligand encoded by (a), the ligand encoded by (b), and the polypeptide encoded by (c) are fused to each other directly or linked by at least one linker. In certain embodiments, the nucleic acid sequence does not encode a co-stimulatory domain, an activating domain, or both a co-stimulatory domain and an activating domain. In certain embodiments, the ligand that selectively binds CD19 antigen is a single chain variable fragment (scFv). In certain embodiments, the ligand that selectively binds CD19 antigen comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 36. In certain embodiments, the ligand that selectively binds to the CD19 antigen comprises the amino acid sequence of SEQ ID NO. 36. In certain embodiments, the huUCHT (Y177T) ligand is a single chain antibody. In certain embodiments, the huUCHT1 (Y177T) ligand comprises an amino acid sequence that has at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 46. In certain embodiments, the huUCHT1 (Y177T) ligand comprises the amino acid sequence of SEQ ID NO. 46. In certain embodiments, the polypeptide encoded by the third polynucleotide comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 18.

In certain embodiments, the third polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 18. In certain embodiments, the at least one linker is a G4S flexible linker, a large protein domain, a long helix or a short helix. In certain embodiments, the at least one linker comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 12, SEQ ID NO. 32, SEQ ID NO. 30, or SEQ ID NO. 28. In certain embodiments, the at least one linker comprises the amino acid sequence of SEQ ID NO. 12, SEQ ID NO. 32, SEQ ID NO. 30 or SEQ ID NO. 28. In certain embodiments, the CD3 is expressed on a cell expressing the second polynucleotide. In certain embodiments, the CD19-TAC comprises a nucleic acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises the sequence of SEQ ID NO:63 or SEQ ID NO: 64. In certain embodiments, disclosed herein are vector constructs comprising (a) a nucleic acid sequence disclosed herein, and (b) a promoter functional in mammalian cells. In certain embodiments, disclosed herein are compositions comprising a carrier and an excipient disclosed herein. In certain embodiments, disclosed herein are polypeptides encoded by the nucleic acid sequences disclosed herein.

In certain embodiments, disclosed herein is a nucleic acid sequence encoding a HER2 trifunctional T cell antigen conjugate (HER 2-TAC) comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 65, SEQ ID No. 67 or SEQ ID No. 75. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:66, SEQ ID NO:68 or SEQ ID NO: 76. In certain embodiments, the nucleic acid sequence does not encode a costimulatory domain. In certain embodiments, the nucleic acid sequence does not encode an activation domain.

In certain embodiments, disclosed herein are nucleic acid sequences encoding BCMA trifunctional T cell antigen conjugates (BCMA-TAC) comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 55, SEQ ID No. 57, SEQ ID No. 59 or SEQ ID No. 61. In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60 or SEQ ID NO: 62. In certain embodiments, the nucleic acid sequence does not encode a costimulatory domain. In certain embodiments, the nucleic acid sequence does not encode an activation domain.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1A is a schematic representation of natural T-cell activation.

Fig. 1B is a schematic diagram of CAR-based T-cell activation.

FIG. 1C is a schematic representation of T cell activation based on a trifunctional-T cell antigen conjugate (Tri-TAC).

FIG. 1D is a schematic representation of natural T-cell activation.

Figure 1E is a schematic representation of CAR-based T-cell activation.

FIG. 1F is a schematic representation of T cell activation based on Tri-TAC.

FIG. 2A is a schematic diagram of a Tri-TAC configuration in which the UCHT1 domain is centrally located between the transmembrane domain (TM) and the antigen binding domain.

FIG. 2B is a schematic representation of a Tri-TAC configuration in which the UCHT1 domain is at the N-terminus, followed by an antigen binding domain and a transmembrane domain.

FIG. 2C is a schematic representation of a Tri-TAC molecule having a general antigen binding domain and a UCHT1 domain.

FIG. 3A is a schematic representation of a Tri-TAC molecule having a generic antigen binding domain.

FIG. 3B is a schematic representation of Tri-TAC with an anti-HER-2 DARPin antigen binding domain.

FIG. 3C is a schematic representation of Tri-TAC with an anti-CD 19 scFv antigen binding domain.

FIG. 3D is a schematic representation of Tri-TAC with an anti-BCMA scFv antigen binding domain.

FIG. 3E is a schematic representation of a Tri-TAC molecule having an anti-HER-2 DARPin antigen binding domain.

FIG. 3F is a schematic representation of a Tri-TAC molecule having an anti-BCMA scFv antigen binding domain.

FIGS. 4A-4D illustrate T cells engineered with Tri-TAC for HER-2 or CD 28-based CAR using DARPin. FIG. 4A illustrates the surface expression of the Tri-TAC and CAR compared to T cells that do not express the chimeric receptor. FIG. 4B illustrates the growth of three cell populations. Figures 4C-4D illustrate the percentage of engineered cells positive for various T cell activation markers following stimulation with antigen.

FIG. 5 shows a model of the structure of the CD19-TAC protein.

FIGS. 6A-6J show receptor surface expression and activation of various anti-HER-2 DARPin Tri-TAC controls. T cells were engineered with a Tri-TAC variant lacking a targeting element (-DARPin), a Tri-TAC variant lacking UCHT1 (-UCHT 1), or full length Tri-TAC. FIGS. 6A, 6D, and 6G show T cell transduction and Her2 binding capacity (left), FIGS. 6B, 6E, and 6H show degranulation (middle), and FIGS. 6C, 6F, and 6I show cytokine production (right). FIG. 6J shows that only full-length anti-HER-2 DARPin Tri-TAC was able to trigger a cytotoxic response.

Figures 7A-7C show anti-tumor activity, toxicity and cytokine production of T cells engineered with anti-HER-2 DARPin Tri-TAC or anti-HER-2 DARPin CD28 based CARs. Mice bearing established OVCAR-3 tumors were treated with T cells engineered with anti-HER-2 DARPin Tri-TAC or anti-HER-2 DARPin CAR. Fig. 7A illustrates the change in tumor growth relative to the day of T cell perfusion (day 35). Fig. 7B illustrates the change in body weight as a measure of toxicity in the same mice. Fig. 7C shows cytokine concentrations in mouse serum at day 7 after T-cell perfusion.

FIGS. 8A-8H show Tri-TACs designed with various alternatives to UCHT1 scFv-CD3 recruitment domains. FIG. 8A provides a schematic representation of TAC receptor constructs utilizing anti-HER-2 DARPin paired with UCHT1 or OKT3 anti-CD 3 scFv. FIG. 8B shows HER-2TAC surface expression of CD8+NGFR+ (left) or CD4+NGFR+T cells (right). FIGS. 8C, 8C1 show cytokine production by HER-2 specific TAC-T cells stimulated with antigen positive SK-OV-3 tumor cells. FIG. 8D shows the killing of SK-OV-3 tumor cells by HER-2TAC and carrier control (carrier carrying tNGFR only) T cells. Vector control T cells (circles) were compared against HER-2 specific TAC-T cells with UCHT1 (squares) or OKT3 (triangles). FIG. 8E provides a schematic representation of TAC receptor constructs utilizing anti-CD 19scFv paired with huUCHT, F6A, or L2K anti-CD 3 scFv. FIG. 8F shows CD19-TAC surface expression of CD8+NGFR+ (left) or CD4+NGFR+T cells (right). FIGS. 8G and 8G1 show cytokine production by CD 19-specific TAC-T cells stimulated with antigen-positive Raji tumor cells. Cytokine-producing cells were compared to TAC-T cells with huUCHT (squares), F6A (triangles) or L2K (diamonds). FIG. 8H shows the killing of NALM-6 tumor cells by CD19 TAC and carrier control (carrier carrying tNGFR only) T cells. Vector control T cells (circles) were compared against CD19 specific TAC-T cells with huUCHT a (squares), F6A (triangles) or L2K (diamonds).

FIGS. 9A-9H show the effect of various anti-CD 3 scFv on TCR surface expression. FIGS. 9A, 9E show TCR surface expression of T cells engineered with control vector (tNGFR), UCHT1 or OKT3 TAC variants. FIGS. 9B and 9F show that T cells engineered with OKT3-TAC have significantly reduced TCR surface expression relative to UCHT 1-TAC. FIGS. 9C, 9G show TCR surface expression of T cells engineered with control vector (tNGFR), huUCHT, F6A or L2K TAC variants. Fig. 9D, 9H show that T cells engineered with L2K TAC have significantly reduced TCR surface expression relative to huUCHT-TAC.

FIGS. 10A-10B illustrate linker domain variants. The domain that links the antigen binding domain to the TCR recruitment domain is referred to as the linker domain. FIG. 10A provides a schematic representation of TAC variants with different linker domains, (i) flexible linker, (ii) large domain linker (constructed from domains 3 and 4 derived from extracellular CD4 domain), (iii) long helical linker, and (iv) short helical linker. FIG. 10B provides exemplary amino acid sequences (SEQ ID NOS 69, 28, 30 and 32, respectively, in the order of appearance) for the domains represented in FIG. 10A.

FIGS. 11A-11E show exemplary in vitro parameters of CD19 TAC engineered with different linker variants. Fig. 11A shows surface expression of TAC variants in both CD4 and CD8 cells. FIG. 11B shows the surface expression of TACs comprising flexible linkers relative to TACs comprising helical or large domain linkers. Fig. 11C shows the overall transduction of TAC containing alternative linkers relative to flexible linkers. Fig. 11D, 11E show the relative cellular reactivity to antigen-positive Raji cells.

FIG. 12A shows in vitro cytotoxicity of BCMA Tri-TAC variants engineered with different linkers. Fig. 12B shows in vivo tumor control of BCMA Tri-TAC variants engineered with flexible linkers compared to short helical linkers.

FIGS. 13A-13C show the properties of CD 8. Alpha. Tri-TAC scFv against HER-2 and CD 8. Alpha. Tri-TAC DARPIN against HER-2. Fig. 13A, 13C show surface expression. Fig. 13B shows cytokine production.

FIGS. 14A-14D provide schematic diagrams of CD8 Tri-TAC variants. anti-HER-2-DARPin was used as an exemplary antigen binding domain and UCHT1 CD3 summoning domain was used as an exemplary summoning domain. FIG. 14A shows the Tri-TAC comprising the CD4 transmembrane and cytoplasmic domains (left) and the comparable region of the CD8 a/CD 8 β heterodimer (right). The regions critical for the function of the accessory receptor (arginine-rich domain and CXCP motif) are highlighted. FIG. 14B is a schematic representation of CD 8. Alpha. Tri-TAC comprising a cysteine-to-serine mutation and a CD 8. Alpha. Cytoplasmic domain for ensuring monomeric receptor distribution. Fig. 14C is a schematic representation of a cd8α+rβtri-TAC comprising a cysteine-to-serine mutation for ensuring monomeric receptor distribution and a chimeric cd8α cytoplasmic domain in which the arginine-rich region of cd8α is replaced with the arginine-rich region of cd8β. FIG. 14D is a schematic representation of a CD8β+Lck Tri-TAC comprising a cysteine-to-serine mutation to ensure monomer receptor distribution and a chimeric CD8β cytoplasmic domain in which the CD8α CXCP domain containing the Lck binding motif is added to the C-terminus of the CD8β cytoplasmic domain.

FIGS. 15A-15E show in vitro characterization of CD8 Tri-TAC variants relative to prototype Tri-TAC containing the CD4 region. FIGS. 15A-15B show the surface expression of CD8-Tri TAC variants relative to the prototype Tri-TAC. FIG. 15C shows the cytotoxicity in vitro of CD8-Tri TAC variants co-cultured with LOX IMVI (HER-2 negative) or A549, SKOV3, SKBR3 or MBA MB 231 (HER-2 positive). FIG. 15D shows cell division of T cells engineered with CD8 Tri-TAC variants or prototype Tri-TAC. FIG. 15E shows TCR surface expression of engineered T cells containing CD8 Tri-TAC variants or prototype Tri-TAC.

FIG. 16 shows various Tri-TACs.

FIG. 17 shows the TAC-CD19 insert in pCCL lentiviral vectors. FIG. 17 shows various domains of TAC-CD19 (CD 8a leader, FMC63 scFv, myc tag, huUCHT Y177T mutant and truncated CD4 anchored co-receptor domain).

FIG. 18 shows in vivo efficacy of TAC-CD19 produced from different donors.

FIGS. 19A-19C show in vitro examples of cytotoxicity of TAC-CD19 against tumor cell lines. FIG. 19A NALM-6 (acute lymphoblastic leukemia), FIG. 19B Jeko-1 (mantle cell lymphoma), and FIG. 19C Raji (Burkitt lymphoma).

Figure 19D shows a schematic of 3 different in vivo tumor models in NRG mice.

FIGS. 19E-19G show in vivo efficacy of CD19-TAC in NALM-6 (acute lymphoblastic leukemia) FIG. 19E, jeko-1 (mantle cell lymphoma) FIG. 19F and Raji (Burkitt lymphoma) FIG. 19G.

FIG. 20A shows experimental setup of TAC-CD19 treated mice with NALM-6 tumor. Following successful treatment, the mice were subsequently re-challenged with NALM-6 (CD 19 positive) or KMS11 (CD 19 negative) tumor cells.

Fig. 20B shows in vivo efficacy of mice treated with TAC-CD 19.

Fig. 21A shows the experimental design to evaluate the effect of dose regimen and dosing on potency and cell expansion.

FIG. 21B shows the in vivo survival of NALM-6-bearing mice treated with single or divided doses of TAC-CD 19.

FIGS. 22A-22B show experimental setup and data for in vivo expansion of TAC-CD19 after administration of split doses. Figure 22A shows gating strategy for identifying T cells in mouse blood. Fig. 22B shows in vivo results of T cell expansion in blood.

FIGS. 23A-23C show long-term in vivo studies of TAC-CD19 in mice. FIG. 23A shows the experimental procedure for the treatment of NALM-6 bearing mice with various controls and TAC-CD19 at two dosage levels. Fig. 23B shows in vivo efficacy of the control group compared to the TAC-CD19 treated group at both dose levels. Figure 23C shows the long term survival of mice treated with low doses of TAC-CD 19.

FIG. 24 shows the results of clinical chemistry analysis from mice treated with TAC-CD19 or non-transduced T cells.

FIG. 25 shows human cytokines released from mouse blood after treatment with TAC-CD19 or non-transduced T cells.

Fig. 26A-26C illustrate the performance of BCMA-TACs in different configurations. Fig. 26A shows the experimental design. Fig. 26B shows various controls and trials. Fig. 26C shows in vivo potency of various TAC constructs. FIGS. 26A-26C disclose "G ₄ S" as SEQ ID NO: 73.

Figure 27 shows that TAC proliferates when it encounters an antigen on a cell, but not when it is present on an artificial bead, regardless of whether the antigen is present on the bead or cell, the CAR proliferates.

Fig. 28A-28B show expansion of TAC engineered T cells in vivo and provide long term protection, indicating persistence of cells in a myeloma model. FIGS. 28A-28B show that BCMA-TAC T cells reject multiple myeloma tumors in a KMS-11 xenograft model engineered with nanoLuc (KMS 11-nanoLuc) (BCMA ^pos). After tumor implantation, mice were treated with BCMA TAC-T cells (with firefly luciferase). TAC-T cells proliferate significantly after administration. This is associated with tumor regression. Treated mice were resistant to tumor re-challenges, indicating long-term persistence of TAC-T cells.

FIG. 29 shows human cytokines released from mouse blood after treatment with TAC-CD19 or non-transduced T cells.

Fig. 30 shows exemplary histograms of TAC receptor surface expression in CD4 and CD8 engineered T cells. Cells were engineered with muIgG HER2 TAC (EF 1 alpha promoter), huIgG HER2 TAC (MSCV promoter), and muIgG HER2 TAC (MSCV promoter). After engineering, T cells were stained with TAC specific reagents and measured using flow cytometry. All constructs showed comparable levels of surface expression, with higher EF 1a driven expression compared to the MSCV construct.

Figure 31 shows the relative percentages of T cells expressing tnfα, ifnγ or IL-2 after co-culture with OVCAR3 (HER 2 positive) or LOX IMVI (HER 2 negative) cells. T cells were engineered with muIgG HER2 TAC (EF 1 alpha promoter), TAC constructs lacking HER2 binding domain (delta binding TAC; EF1 alpha promoter), huIgG HER2 TAC (MSCV promoter), or muIgG HER2 TAC (MSCV promoter). Delta-binding TAC control and HER2 TAC cells did not show meaningful cytokine expression when co-cultured with LOX-IMVI (HER 2 ^neg). All HER2 TAC engineered constructs co-cultured with OVCAR3 (HER 2 ^pos) showed similar ability to produce cytokines, whereas control T cells engineered with delta-binding TAC did not show significant cytokine production.

Figure 32 shows the in vivo efficacy of TAC engineered T cells in OVCAR3 solid tumor model. T cells were engineered with delta-binding TAC (EF 1 alpha promoter), muIgG HER2TAC (EF 1 alpha promoter), huIgG HER2TAC (MSCV promoter), or muIgG HER2TAC (MSCV promoter). Mice have been vaccinated subcutaneously with OVCAR3 (HER-2 positive) tumors. These tumors were allowed to grow to a size of about 100mm ³. Mice were then treated with a total of 6 million divided doses of HER2TAC engineered or delta conjugated TAC control T cells separated by 48h by tail vein injection. Tumor progression was followed by measurements once every two weeks. Delta binding to TAC did not show tumor control or tumor regression. All HER2TAC engineered T cells showed significantly reduced tumor progression, including tumor regression, relative to control mice. All HER2TAC engineered T cells had similar anti-tumor activity.

Detailed Description

Cancer is a major health challenge, and it is expected that over 15 tens of thousands of cancer cases will be diagnosed in canada alone. While early stage disease patients can sometimes be effectively treated by conventional therapies (surgery, radiation, chemotherapy), few options are available to patients with advanced disease and those options are often palliative in nature.

Active immunotherapy seeks to utilize the patient's immune system to clear tumors and provides an option for patients who fail conventional therapy. Typically, such treatment involves the perfusion of a patient with a large number of tumor-specific T cells. This method has proven successful in early clinical trials of a variety of diseases including melanoma, myeloma, leukemia, lymphoma and synovial sarcoma. As a specific example, several clinical studies demonstrated that immunotherapy using T cells was curative in patients with advanced melanoma, demonstrating the utility of this approach. In addition, patients suffering from Chronic Lymphocytic Leukemia (CLL) and Acute Lymphoblastic Leukemia (ALL) have also been effectively treated and cured by T cell immunotherapy.

The key challenge faced by the clinical application of adoptive T cell therapy is the source of T cells. Typically, T cells isolated from tumor bearing patients are grown ex vivo to large quantities and administered back to the patient to induce a robust anti-tumor immune response. Tumor specificity is achieved by either (i) isolating naturally occurring tumor-specific T cells from the patient, or (ii) engineering host T cells from peripheral blood to express tumor-specific receptors. Naturally occurring tumor-specific T cells are rare, and isolating such cells from cancer patients in therapeutic amounts is a laborious and expensive process. In contrast, engineering readily available peripheral blood T cells using tumor-specific receptors by genetic manipulation becomes increasingly efficient. Clinically viable techniques for this engineering process have been developed, and several clinical studies have demonstrated the feasibility and efficacy of genetically engineered T cells for the treatment of cancer.

To this end, most engineered T cell therapies involving genetic modification of T cells produce either (i) forced expression of T Cell Receptors (TCRs), or (ii) Chimeric Antigen Receptors (CARs) specific for antigen targets on tumors. Chimeric antigen receptors for engineered T cells have heretofore consisted of (i) a targeting domain, typically a single chain variable fragment (scFv), (ii) a transmembrane domain, and (iii) a cytoplasmic domain containing signaling elements from T cell receptors and related proteins. Such chimeric antigen receptors are also known as "T-bodies" or "chimeric immune receptors" (CIRs), but currently most researchers use the term "CAR". One advantage of the CAR approach is that it allows targeting immune cells of any patient against any desired target in a manner that is independent of the Major Histocompatibility Complex (MHC). This is attractive because MHC presentation is often defective in tumor cells.

CARs in modular form are considered and scientists spend a lot of time studying the effect of different cytoplasmic signaling domains on CAR function. Conventional CARs typically share two major components, (i) a cd3ζ cytoplasmic domain containing an Immune Tyrosine Activation Motif (ITAM) that is critical for T cell activation, and (ii) components that assist in stimulating receptors that trigger important survival pathways, such as the Akt pathway.

First generation CARs utilized a single signaling domain from either cd3ζ or fcsriy. The second generation CAR combines the signaling domain of CD3 zeta with the cytoplasmic domain of a co-stimulatory receptor from the CD28 or TNFR receptor family. Most CAR engineered T cells currently tested in the clinic utilize a second generation CAR in which cd3ζ is coupled to the cytoplasmic domain of CD28 or CD 137. These second generation CARs have demonstrated anti-tumor activity in CD19 positive tumors. Third generation CARs combine multiple co-stimulatory domains, but there is a concern that third generation CARs may lose antigen specificity.

While CAR engineered T cells have shown considerable promise in clinical applications, they rely on synthetic methods to replace the endogenous activation signal provided by the T Cell Receptor (TCR). Since such synthetic receptors are unable to deliver all signaling components associated with TCRs (e.g., ITAM on cd3γ, cd3δ, cd3ε), it is unclear whether or not T cells are optimally activated by a CAR or how CAR activation affects T cell differentiation (e.g., progression to memory T cells). Furthermore, since it is the nature of the CAR structure that separates the CAR signaling domains from their natural regulatory partners, there is an inherent risk that CARs may lead to low levels of constitutive activation, which may lead to off-target toxicity. Thus, the nature of the synthesis of the prototype CAR may disrupt the canonical mechanisms that limit TCR activation, and may exacerbate the severe toxicity normally associated with therapeutic doses of conventional CAR T cells.

In view of these limitations, T cells are preferably redirected to attack the tumor through their native TCR. For this purpose, a class of recombinant proteins called "bispecific T-cell adaptors" (bites) was created. These proteins utilize bispecific antibody fragments to crosslink T-cell TCR receptors with target antigens. This results in efficient T-cell activation, triggering cytotoxicity. Similarly, bispecific antibodies have been generated to achieve this goal, and some scientists simply use chemical linkages to link anti-CD 3 antibodies to tumor-specific antibodies. While these bispecific proteins have shown some activity in vitro, GMP production, short biological half-life and limited bioavailability represent significant challenges for successful use of these molecules in cancer therapy. In addition, these molecules also fail to correctly recapitulate native TCR signaling because they do not bind TCR accessory receptors (CD 8 and CD 4).

In view of the above, there remains a need for chimeric receptors with enhanced activity and safety.

An alternative chimeric receptor, known as a trifunctional T cell antigen conjugate (Tri-TAC or TAC) receptor, has been developed that utilizes unique biology to direct T cell attack on tumors. CARs are fully synthesized receptors that splice components of the T Cell Receptor (TCR) signaling complex together, while TAC receptors redirect the TCR to tumor targets and reenact the native TCR signaling structure. For example, in certain embodiments, the TACs disclosed herein activate native Major Histocompatibility Complex (MHC) signaling through T-cell receptors (TCRs) while retaining targeting that is not MHC restricted. In addition, the TACs disclosed herein recruit T-cell receptors (TCRs) in combination with co-receptor stimulation. Furthermore, in certain embodiments, the Tri-TAC disclosed herein exhibits enhanced activity and safety.

Certain terms

As used herein, the term "T cell" refers to a lymphocyte type that plays a central role in cell-mediated immunity. T cells, also known as T lymphocytes, differ from other lymphocytes such as B cells and natural killer cells in the presence of T-cell receptors (TCRs) on the cell surface. There are several T cell subsets with different functions including, but not limited to, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells and natural killer T cells.

The term "T cell antigen conjugate" or TAC may be used interchangeably with "trifunctional T cell antigen conjugate" or Tri-TAC and refers to an engineered nucleic acid construct or polypeptide that, when expressed on T cells, helps facilitate targeting of the T cells to a particular antigen. In certain embodiments, the TAC comprises (a) a target-specific ligand, (b) a ligand that binds to a protein associated with a T Cell Receptor (TCR) complex, and (c) a T cell receptor signaling domain.

As used herein, the terms "polynucleotide" and/or "nucleic acid sequence" and/or "nucleic acid" refer to a sequence of a nucleoside or nucleotide monomer consisting of bases, sugars, and inter-sugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymine, and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymine and uracil, and xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed by laboratory methods of genetic recombination, or obtained by chemical synthesis or other known protocols for producing nucleic acids.

As used herein, the term "isolated polynucleotide" or "isolated nucleic acid sequence" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The isolated nucleic acid is also substantially free of sequences naturally flanking the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the nucleic acid from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA and RNA and is double-stranded or single-stranded, and means the sense or antisense strand. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences.

As used herein, the term "recombinant nucleic acid" or "engineered nucleic acid" refers to a nucleic acid or polynucleotide that is not present in a biological organism. For example, recombinant nucleic acids can be formed by laboratory methods of genetic recombination (e.g., molecular cloning) to produce sequences that are not otherwise present in nature. Recombinant nucleic acids may also be produced by chemical synthesis or other known protocols for producing nucleic acids.

As used herein, the term "polypeptide" or "protein" describes a chain of amino acids. The polypeptides or proteins of the present disclosure are peptides, which generally describe a chain of amino acids. The term protein, as used herein, also describes macromolecules comprising one or more amino acid chains, and in certain embodiments is a fragment or domain of a protein or a full-length protein. Furthermore, as used herein, the term protein refers to a linear chain of amino acids, or to a chain of amino acids that has been processed and folded into a functional protein. Protein structures are divided into 4 different levels (1) primary structure, which refers to the amino acid sequence in the polypeptide chain, (2) secondary structure, which refers to regular local substructures on the polypeptide backbone chain, such as alpha-helices and beta-sheets, (3) tertiary structure, which refers to three-dimensional structures for monomeric or multimeric protein molecules, and (4) quaternary structure, which refers to three-dimensional structures of aggregates comprising two or more individual polypeptide chains that function as a single functional unit. In certain embodiments, the proteins of the present disclosure are obtained by isolating and purifying the proteins from the cells in which they are naturally produced, by enzymatic (e.g., proteolytic) cleavage, and/or by recombinant expression of nucleic acids encoding the proteins or fragments of the present disclosure. In certain embodiments, the proteins and/or fragments of the present disclosure are obtained by chemical synthesis or other known protocols for the production of proteins and fragments.

The term "isolated polypeptide" refers to a polypeptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, single chain antibodies, chimeric antibodies, and antibody fusions. The antibodies may be derived from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include, but is not limited to, fab ', F (ab') 2, scFv, dsFv, ds-scFv, dimer, minibody, diabody, and multimers, multispecific antibody fragments, and domain antibodies thereof.

As used herein, the term "vector" refers to a polynucleotide for delivering a nucleic acid into the interior of a cell. In certain embodiments, the vector is an expression vector comprising an expression control sequence (e.g., a promoter) operably linked to a nucleic acid to be expressed in a cell. Vectors known in the art include, but are not limited to, plasmids, phagemids, cosmids, and viruses.

As used herein, the term "tumor antigen" or "tumor-associated antigen" refers to an antigenic substance produced in tumor cells that triggers an immune response in a host (e.g., presented by an MHC complex). In certain embodiments, the tumor antigen is on the surface of a tumor cell.

As used herein, the term "T cell receptor" or TCR refers to a complex of integral membrane proteins involved in the activation of T cells in response to binding of an antigen. TCRs are disulfide-linked membrane-anchored heterodimers, typically composed of highly variable alpha (α) and beta (β) chains, expressed as part of a complex with an invariable CD3 (cluster 3) chain molecule. T cells expressing this receptor are called α: β (or αβ) T cells, although a few T cells express alternative receptors formed by variable gamma (γ) and delta (δ) chains and are called γδ T cells. CD3 is a protein complex consisting of 4 distinct chains. In mammals, the complex contains a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, and two CD3 zeta chains.

As used herein, the term "transmembrane and cytoplasmic domain" refers to a polypeptide comprising a transmembrane domain and cytoplasmic domain of a protein associated with a T Cell Receptor (TCR) complex. In certain embodiments, such transmembrane and cytoplasmic domains may include, but are not limited to, (a) protein domains associated with lipid rafts and/or (b) that bind Lck.

As used herein, "TCR accessory receptor" refers to a molecule that facilitates communication of a T Cell Receptor (TCR) with an antigen presenting cell, and may be considered to be part of a first signal that results in activation of the TCR. Examples of TCR accessory receptors include, but are not limited to, CD4, LAG3, and CD8.

As used herein, "TCR accessory stimulus" refers to a molecule that enhances the response of T cells to an antigen, and may be considered as a second signal that results in activation of the TCR. Examples of TCR accessory stimulators include, but are not limited to ICOS, CD27, CD28, 4-1BB (CD 137), OX40 (CD 134), CD30, CD40, lymphocyte fiction-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, and ligands that specifically bind CD 83.

As used herein, "TCR accessory inhibitor" or "checkpoint receptor" refers to a molecule that inhibits a T cell's response to an antigen. Examples of TCR accessory inhibitors include, but are not limited to, PD-1, TIM3, LAG-3, TIGIT, BTLA, CD, 160, and CD37.

The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer in certain embodiments to any mammalian subject, particularly a human, for whom offer free medical treatment breaks, treats, or therapies are desired. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as laboratory, zoo, sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In certain embodiments, the mammal is a human. None of these terms require supervision by medical personnel.

As used herein, the term "treatment" or the like is in certain embodiments intended to refer to administration of an agent or execution of a procedure for the purpose of achieving an effect. The effect may be prophylactic in terms of completely or partially arresting the disease or symptoms thereof and/or may be therapeutic in terms of affecting a partial or complete cure of the disease and/or symptoms of the disease. As used herein, "treating" or "treatment" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly in a human, and includes (a) preventing the occurrence of the disease or disease symptoms in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease (e.g., including a disease that may be associated with or caused by a primary disease), (b) inhibiting the disease, i.e., stopping its progression, and (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any sign of success in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as diminution, alleviation, diminishment of symptoms, making the patient more tolerant to the disease condition, slowing the rate of degeneration or decline, or making the end point of degeneration less debilitating. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of the examination conducted by the physician. Thus, the term "treating" includes administration of a compound or agent of the invention to prevent, delay, alleviate, terminate or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the alleviation, elimination or prevention of the disease, a symptom of the disease, or a side effect of the disease in the subject.

As used herein, no particular number of a reference includes a plurality of references unless the context clearly indicates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "an antibody" includes a plurality of antibodies in certain embodiments, and so forth.

As used herein, all numbers or ranges of numbers include all integers within or encompassing such ranges as well as fractions of values or integers within or encompassing such ranges unless the context clearly indicates otherwise. Thus, for example, references to a range of 90-100% include 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, references to a range of 1-5,000 times include 1,2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, etc., 2.1, 2.2, 2.3, 2.4, 2.5 times, etc., and so forth.

As used herein, "about" a number is meant to include the number and ranges from 10% lower to 10% higher than the number. "about" a range means from 10% lower than the lower limit of the range to 10% higher than the upper limit of the range.

"Percent (%) identity" refers to the degree to which two sequences (nucleotides or amino acids) have identical residues at identical positions in an alignment. For example, "amino acid sequence is Y X% identical to SEQ ID NO: Y" refers to the identity of the amino acid sequence to SEQ ID NO: Y, and specifies that X% of the residues in the amino acid sequence are identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary procedures for comparing and aligning pairwise sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman,1988; pearson, 1990) and gapped BLAST (Altschul et al, 1997), BLASTP, BLASTN or GCG (Devereux et al, 1984).

As used herein, the term "selectively binds" refers to a molecule (e.g., a target binding ligand for a protein such as TAC) that binds to its target molecule (e.g., a target antigen such as HER-2, BCMA or CD 19) with higher affinity than other molecules.

T cell antigen conjugate (Tri-TAC or TAC)

In certain embodiments, disclosed herein are nucleic acids encoding a trifunctional T cell antigen conjugate (Tri-TAC). In certain embodiments, the nucleic acid encoding Tri-TAC comprises (a) a first polynucleotide encoding a target-specific ligand, (b) a second polynucleotide encoding a ligand that binds to a TCR complex, and (c) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain. In certain embodiments, the nucleic acid encoding Tri-TAC does not encode a co-stimulatory domain. In certain embodiments, the nucleic acid encoding Tri-TAC does not encode a secondary activation domain.

Target specific ligands

The target-specific ligand, also referred to as an antigen binding domain, refers to any substance or molecule that binds directly or indirectly to a target cell. In certain embodiments, the target-specific ligand binds to an antigen on the target cell. In certain embodiments, the target cell is a cell associated with a disease state including, but not limited to, cancer, hematological malignancy, large B-cell lymphoma, diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, high grade B-cell lymphoma, or large B-cell lymphoma caused by follicular lymphoma. In certain embodiments, the target cell is a tumor cell. In certain embodiments, the target-specific ligand binds to a tumor antigen or a tumor-associated antigen on a tumor cell. In certain embodiments, the target antigen is a tumor antigen. In certain embodiments, the tumor antigen, when a protein, is a sequence of 8 or more amino acids up to the complete protein. In certain embodiments, the tumor antigen has any number of amino acids between 8 amino acids to a full-length protein comprising at least one antigenic fragment of the full-length protein presented in the Major Histocompatibility Complex (MHC). Examples of tumor antigens include, but are not limited to, CD19, HER-2 (erbB-2), B-cell maturation antigen (BCMA), alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), prostate-specific antigen (PSA), glioma-associated antigen, beta-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), enterocarboxylesterase, mut hsp70-2, M-CSF, prostase, PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin growth factor () -I, IGF-II, insulin receptor and IGF receptor and mesothelin.

In certain embodiments, the target-specific ligand includes, but is not limited to, antibodies and fragments thereof that bind to a target cell and/or antigen, e.g., single chain antibodies such as single chain antibodies (scFv), single domain antibodies, peptides, peptidomimetics, proteins, glycoproteins, or proteoglycans. In certain embodiments, the target-specific ligand includes, but is not limited to, a naturally occurring ligand such as a growth factor, enzyme substrate, receptor, or binding protein of a designed ankyrin repeat protein (DARPin), lectin, knottin, CENTRYRIN, ANTICALIN, or tumor antigen. In certain embodiments, the target-specific ligand includes a non-proteinaceous compound that binds to the target cell and/or antigen, including, but not limited to, a carbohydrate, lipid, nucleic acid, or small molecule. In certain embodiments, the target-specific ligand is a designed ankyrin repeat (DARPin) that targets a particular cell and/or antigen. In certain embodiments, the target-specific ligand is a single chain variable fragment (ScFv) that targets a particular cell and/or antigen.

In certain embodiments, the tumor antigen is a HER-2 antigen. In certain embodiments, the HER-2 specific ligand comprises an antigen binding domain of an antibody selected from trastuzumab, pertuzumab, lapatinib, lenatinib, trastuzumab-maytansinoid conjugate, rituximab, margetuximab, ti Mi Tuozhu mab, and Ertumaxomab. In certain embodiments, the target-specific ligand is DARPin that selectively binds to the HER-2 (erbB-2) antigen. In certain embodiments, the target-specific ligand is DARPin that specifically binds to the HER-2 (erbB-2) antigen. In certain embodiments, the HER-2 (erb-2) -targeted DARPin comprises SEQ ID NO 7 or SEQ ID NO 8.

In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 7. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 7. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 7. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 7. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 7. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 7. In certain embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO. 7.

In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 8. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 8. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 8. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 8. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 8. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 8. In certain embodiments, the target-specific ligand comprises the amino acid sequence of SEQ ID NO. 8.

In certain embodiments, the tumor antigen is a BCMA antigen. In certain embodiments, the BCMA specific ligand comprises an antigen binding domain of an antibody selected from Belantamab mafodotin and GSK 2857916. In certain embodiments, the target-specific ligand is an scFv that selectively binds BCMA. In certain embodiments, the target-specific ligand is an scFv that specifically binds BCMA. In certain embodiments, the scFv that binds BCMA comprises SEQ ID NO. 33 or SEQ ID NO. 34.

In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 33. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 77% sequence identity to SEQ ID NO. 33. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 33. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 33. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 33. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 33. In certain embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO. 33.

In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 34. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 34. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 34. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 34. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 34. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 34. In certain embodiments, the target-specific ligand comprises the amino acid sequence of SEQ ID NO. 34.

In certain embodiments, the tumor antigen is a CD19 antigen. In certain embodiments, the target-specific ligand is an scFv that selectively binds CD 19. In certain embodiments, the target-specific ligand is an scFv that specifically binds CD 19. In certain embodiments, the scFv that binds CD19 comprises SEQ ID NO:35 or SEQ ID NO:36.

In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 35. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 35. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 35. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 35. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 35. In certain embodiments, the first polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 35. In certain embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO. 35.

In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 36. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 36. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 36. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 36. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 36. In certain embodiments, the target-specific ligand comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 36. In certain embodiments, the target-specific ligand comprises the amino acid sequence of SEQ ID NO. 36.

Ligands that bind to TCR complexes

In certain embodiments, the TAC comprises a ligand that binds to a protein associated with the TCR complex. In certain embodiments, the ligand that binds to a protein associated with a TCR complex comprises a substance that directly or indirectly binds to a protein of the TCR. In certain embodiments, the ligand that binds to a protein associated with a TCR complex comprises a substance that selectively binds to a protein of the TCR. In certain embodiments, the ligand that binds to a protein associated with a TCR complex comprises a substance that specifically binds to a protein of the TCR. Proteins associated with the TCR include, but are not limited to, TCR alpha (alpha) chains, TCR beta (beta) chains, TCR gamma (gamma) chains, TCR delta (delta) chains, CD3 gamma chains, CD3 delta chains, and CD3 epsilon chains. In certain embodiments, the ligand that binds to a protein associated with a TCR complex is an antibody directed against the TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3 γ chain, CD3 δ chain, and/or CD3 epsilon chain. In certain embodiments, the protein associated with the TCR complex is CD3. In certain embodiments, the protein associated with the TCR complex is CD3 epsilon. Examples of CD3 antibodies include, but are not limited to. In certain embodiments, the antibody that binds CD3 is a single chain antibody, e.g., a single chain variable fragment (scFv). In certain embodiments, the ligand that binds to the TCR is an anti-CD 3 antibody or fragment thereof, e.g., moromimumab, oxybutynin, teplizumab, visilizumab, CD-12, MEM-57, 4D10A6, CD3D, or TR66.

In certain embodiments, the CD3 is a CD3 of a TCR complex on a cell expressing the second polynucleotide. In certain embodiments, the binding of CD3 induces activation of a cell expressing the second polynucleotide.

In certain embodiments, the ligand that binds to the TCR complex is UCHT1 or a variant thereof. In certain embodiments, the ligand that binds to the TCR complex is UCHT1 (SEQ ID NO:13, SEQ ID NO:14, or homologs thereof). In certain embodiments, the UCHT1 ligand binds CD3. In certain embodiments, the UCHT1 ligand selectively binds CD3. In certain embodiments, the UCHT1 ligand specifically binds CD3. In certain embodiments, the UCHT1 ligand binds CD3 epsilon. In certain embodiments, the UCHT1 ligand selectively binds CD3 epsilon. In certain embodiments, the UCHT1 ligand specifically binds CD3 epsilon. In certain embodiments, the UCHT1 ligand is encoded by SEQ ID NO 13. In certain embodiments, the UCHT1 ligand comprises SEQ ID NO14. In certain embodiments, the UCHT1 ligand is mutated. In certain embodiments, the UCHT1 ligand comprises a Y182T mutation (also known as UCHT1 (Y182T)) (SEQ ID NO:71 and SEQ ID NO: 72). In certain embodiments, the UCHT1 (Y182T) ligand binds CD3. In certain embodiments, the UCHT1 (Y182T) ligand selectively binds CD3. In certain embodiments, the UCHT1 (Y182T) ligand specifically binds CD3. In certain embodiments, the UCHT1 (Y182T) ligand binds CD3 epsilon. In certain embodiments, the UCHT1 (Y182T) ligand selectively binds CD3 epsilon. In certain embodiments, the UCHT1 (Y182T) ligand specifically binds CD3 epsilon. In certain embodiments, the UCHT1 (Y182T) ligand is encoded by SEQ ID NO 71. In certain embodiments, the UCHT1 (Y182T) ligand comprises SEQ ID NO 72. In certain embodiments, the ligand that binds to the TCR complex is humanized UCHT1 (huUCHT 1). In certain embodiments, the ligand that binds to the TCR complex is huUCHT1 (SEQ ID NO 43, SEQ ID NO 44, or a homolog thereof). In certain embodiments, the huUCHT ligand binds CD3. In certain embodiments, the huUCHT ligand selectively binds CD3. In certain embodiments, the huUCHT ligand specifically binds CD3. In certain embodiments, the huUCHT1 ligand binds CD3 epsilon. In certain embodiments, the huUCHT1 ligand selectively binds CD3 epsilon. In certain embodiments, the huUCHT1 ligand specifically binds CD3 epsilon. In certain embodiments, the huUCHT1 ligand is encoded by SEQ ID NO 43. In certain embodiments, the huUCHT a ligand comprises SEQ ID NO 44. In certain embodiments, the huUCHT1 has a Y177T mutation (also known as huUCHT1 (Y177T)) (SEQ ID NO:45 and SEQ ID NO: 46). In certain embodiments, the huUCHT1 (Y177T) ligand binds CD3. In certain embodiments, the huUCHT (Y177T) ligand selectively binds CD3. In certain embodiments, the huUCHT (Y177T) ligand specifically binds CD3. In certain embodiments, the huUCHT1 (Y177T) ligand binds CD3 epsilon. In certain embodiments, the huUCHT1 (Y177T) ligand selectively binds CD3 epsilon. In certain embodiments, the huUCHT1 (Y177T) ligand specifically binds CD3 epsilon. In certain embodiments, the huUCHT1 (Y177T) ligand is encoded by SEQ ID NO 45. In certain embodiments, the huUCHT1 ligand comprises SEQ ID NO 46.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 13. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 13. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 13. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 13. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 13. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 13. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 13.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 14. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 75% sequence identity to SEQ ID No. 14. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 14. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 14. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 14. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 14. In certain embodiments, the ligand that binds to a TCR complex comprises the amino acid sequence of SEQ ID NO. 14.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 71. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 71. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 71. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 71. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 71. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 71. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 71.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 72. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 75% sequence identity to SEQ ID No. 72. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 72. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 72. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 72. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 72. In certain embodiments, the ligand that binds to the TCR complex comprises the amino acid sequence of SEQ ID NO: 72.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 43. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 43. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 43. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 43. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 43. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 43. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 43.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 44. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 75% sequence identity to SEQ ID NO 44. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 44. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 44. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO. 44. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 44. In certain embodiments, the ligand that binds to a TCR complex comprises the amino acid sequence of SEQ ID NO. 44.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 45. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 45. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 45. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 45. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 45. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 45. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 45.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 70% sequence identity to SEQ ID NO. 46. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 75% sequence identity to SEQ ID NO. 46. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO. 46. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO. 46. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO. 46. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 46. In certain embodiments, the ligand that binds to a TCR complex comprises the amino acid sequence of SEQ ID NO. 46.

In certain embodiments, the ligand that binds to CD3 is OKT3. In certain embodiments, the OKT3 ligand binds CD3. In certain embodiments, the OKT3 ligand selectively binds CD3. In certain embodiments, the OKT3 ligand specifically binds CD3. In certain embodiments, the OKT3 ligand binds to CD3 epsilon. In certain embodiments, the OKT3 ligand selectively binds to CD3 epsilon. In certain embodiments, the OKT3 ligand specifically binds to CD3 epsilon. In certain embodiments, the murine OKT3 ligand is encoded by SEQ ID NO 21. In certain embodiments, the OKT3 ligand comprises SEQ ID NO 22.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 21. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 21. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 21. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 21. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 21. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 21. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 21.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 75% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 22. In certain embodiments, the ligand that binds to a TCR complex comprises the amino acid sequence of SEQ ID NO. 22.

In certain embodiments, the ligand that binds to CD3 is F6A. In certain embodiments, the F6A ligand binds CD3. In certain embodiments, the F6A ligand selectively binds CD3. In certain embodiments, the F6A ligand specifically binds CD3. In certain embodiments, the F6A ligand binds CD3 epsilon. In certain embodiments, the F6A ligand selectively binds CD3 epsilon. In certain embodiments, the F6A ligand specifically binds CD3 epsilon. In certain embodiments, the murine F6A ligand is encoded by SEQ ID NO 23. In certain embodiments, the F6A ligand comprises SEQ ID NO 24.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 23. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 23. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 23. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 23. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 23. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 23. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 23.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 75% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24. In certain embodiments, the ligand that binds to a TCR complex comprises the amino acid sequence of SEQ ID NO. 24.

In certain embodiments, the ligand that binds to CD3 is L2K. In certain embodiments, the L2K ligand binds CD3. In certain embodiments, the L2K ligand selectively binds CD3. In certain embodiments, the L2K ligand specifically binds CD3. In certain embodiments, the L2K ligand binds CD3 epsilon. In certain embodiments, the L2K ligand selectively binds CD3 epsilon. In certain embodiments, the L2K ligand specifically binds CD3 epsilon. In certain embodiments, the murine L2K ligand is encoded by SEQ ID NO 25. In certain embodiments, the L2K ligand comprises SEQ ID NO 26.

In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 25. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 25. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 25. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 25. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 25. In certain embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 25. In certain embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO. 25.

In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 70% sequence identity to SEQ ID NO. 26. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 75% sequence identity to SEQ ID NO. 26. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO. 26. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO. 26. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO. 26. In certain embodiments, the ligand that binds to a TCR complex comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 26. In certain embodiments, the ligand that binds to a TCR complex comprises the amino acid sequence of SEQ ID NO. 26.

Transmembrane domain and cytoplasmic domain

In certain embodiments, the T cell antigen conjugate comprises a T cell receptor signaling domain polypeptide. In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain. In certain embodiments, the TCR signaling domain polypeptide comprises a cytoplasmic domain. In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and a cytoplasmic domain. In certain embodiments, the cytoplasmic domain and the transmembrane domain are optionally linked by a linker. In certain embodiments, the T cell receptor signaling domain polypeptide comprises a TCR accessory receptor domain. In certain embodiments, the T cell receptor signaling domain polypeptide does not comprise a TCR accessory stimulator domain. In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytoplasmic domain of a TCR accessory receptor. In certain embodiments, the TCR accessory receptor is CD4, CD8, LAG3, or a chimeric variant thereof.

In certain embodiments, the TCR accessory receptor is CD4. In certain embodiments, the TCR signaling domain polypeptide comprises the transmembrane and cytoplasmic domain of the CD4 accessory receptor encoded by SEQ ID No. 17. In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane and cytoplasmic domain of a CD4 accessory receptor comprising SEQ ID No. 18.

In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 17. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 17. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 17. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 17. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 17. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 17. In certain embodiments, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO. 17.

In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 18. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 18. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 18. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 18. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 18. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise the amino acid sequence of SEQ ID NO. 18.

In certain embodiments, the TCR accessory receptor is CD8. In certain embodiments, the TCR accessory receptor is cd8α. In certain embodiments, the TCR signaling domain polypeptide comprises the transmembrane and cytoplasmic domain of the CD 8a accessory receptor encoded by SEQ ID NO 37. In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane and cytoplasmic domain of a CD 8a accessory receptor comprising SEQ ID No. 38.

In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 37. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 37. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 37. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 37. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 37. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 37. In certain embodiments, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO. 37.

In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 38. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 38. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 38. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 38. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 38. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 38. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise the amino acid sequence of SEQ ID NO. 38.

In certain embodiments, the TCR signaling domain polypeptide comprises a chimera of a sequence or domain from a co-receptor. In certain embodiments, the TCR signaling domain polypeptide comprises a chimera of CD8 a and CD8 β, wherein the arginine-rich region of CD8 a is replaced with the arginine-rich region of CD8 β. In certain embodiments, the T cell receptor signaling domain polypeptide comprises the transmembrane and cytoplasmic domains of a CD8α+R (β) co-receptor chimera encoded by SEQ ID NO 39. In certain embodiments, the T cell receptor signaling domain polypeptide comprises the transmembrane and cytoplasmic domains of a CD8α+R (β) co-receptor chimera provided by SEQ ID NO. 40. In certain embodiments, the TCR signaling domain polypeptide comprises a chimera of CD8 a and CD8 β, and a CD8 a CXCP domain comprising an Lck binding motif is attached to the C-terminus of the CD8 β cytoplasmic domain. In certain embodiments, the T cell receptor signaling domain polypeptide comprises the transmembrane and cytoplasmic domain of a CD8β+Lck accessory receptor chimera encoded by SEQ ID NO. 41. In certain embodiments, the T cell receptor signaling domain polypeptide comprises the transmembrane and cytoplasmic domain of a CD8β+Lck accessory receptor chimera provided by SEQ ID NO. 42.

In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 39. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 39. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 39. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 39. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 39. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 39. In certain embodiments, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO. 39.

In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 40. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 40. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 40. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 40. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 40. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 40. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise the amino acid sequence of SEQ ID NO. 40.

In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 41. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 41. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 41. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 41. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 41. In certain embodiments, the third polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 41. In certain embodiments, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO. 41.

In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 42. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 42. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 42. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 42. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 42. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 42. In certain embodiments, the transmembrane domain and cytoplasmic domain comprise the amino acid sequence of SEQ ID NO. 42.

In certain embodiments, the T cell receptor signaling domain polypeptide comprises a TCR accessory stimulator domain. In certain embodiments, the TCR accessory stimulus is ICOS. In certain embodiments, the TCR accessory stimulus is CD27. In certain embodiments, the TCR accessory stimulus is CD28. In certain embodiments, the TCR accessory stimulus is 4-1BB (CD 137). In certain embodiments, the TCR accessory stimulus is OX40 (CD 134). In certain embodiments, the TCR accessory stimulus is CD30. In certain embodiments, the TCR accessory stimulus is CD40. In certain embodiments, the TCR accessory stimulus is lymphocyte fiction-associated antigen 1 (LFA-1). In certain embodiments, the TCR accessory stimulus is CD2. In certain embodiments, the TCR accessory stimulus is CD7. In certain embodiments, the TCR accessory stimulus is LIGHT. In certain embodiments, the TCR accessory stimulus is NKG2C. In certain embodiments, the TCR accessory stimulus is B7-H3. In certain embodiments, the TCR accessory stimulus is a ligand that specifically binds CD 83.

In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytoplasmic domain of a TCR helper inhibitor. In certain embodiments, the TCR accessory inhibitor is PD-1. In certain embodiments, the TCR accessory inhibitor is TIM3. In certain embodiments, the TCR accessory inhibitor is LAG-3. In certain embodiments, the TCR accessory inhibitor is TIGIT. In certain embodiments, the TCR accessory inhibitor is BTLA. In certain embodiments, the TCR accessory inhibitor is CD160. In certain embodiments, the TCR accessory inhibitor is CD37.

In certain embodiments, the TCR signaling domain polypeptide comprises both a cytoplasmic domain and a transmembrane domain of a TCR accessory receptor or accessory stimulator protein. In certain embodiments, the cytoplasmic domain and the transmembrane domain are from the same co-receptor or co-stimulator or from different co-receptors or co-stimulators. In certain embodiments, the TAC further comprises other polypeptides that act directly or indirectly to target or activate the T cells.

Connector, joint and arrangement

In certain embodiments, the nucleic acids disclosed herein take the order of (1) a first polynucleotide encoding a target-specific ligand, (2) a second polynucleotide encoding a ligand that binds to the TCR complex, and (3) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain. In certain embodiments, the nucleic acids disclosed herein take the order of (1) a first polynucleotide encoding a target-specific ligand, (2) a second polynucleotide encoding a ligand that binds to a TCR complex, and (3) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain, wherein the order is 5 'to 3' terminal. In certain embodiments, the nucleic acids disclosed herein take the order of (1) a first polynucleotide encoding a target-specific ligand, (2) a second polynucleotide encoding a ligand that binds to a TCR complex, and (3) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain, wherein the order is 3 'to 5' end. In certain embodiments, the nucleic acids described herein take the order of (1) a first polynucleotide encoding a ligand that binds to a TCR complex, (2) a second polynucleotide encoding a target-specific ligand, and (3) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain. In certain embodiments, the nucleic acids described herein take the order of (1) a first polynucleotide encoding a ligand that binds to a TCR complex, (2) a second polynucleotide encoding a target-specific ligand, and (3) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain, wherein the order is 5 'to 3' terminal. In certain embodiments, the nucleic acids described herein take the order of (1) a first polynucleotide encoding a ligand that binds to a TCR complex, (2) a second polynucleotide encoding a target-specific ligand, and (3) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain, wherein the order is 3 'to 5' end.

In certain embodiments, the first nucleic acid encodes a first polypeptide, the second nucleic acid encodes a second polypeptide, and the third nucleic acid encodes a third polypeptide. In certain embodiments, the first polypeptide, the second polypeptide, and the third polypeptide are fused directly. For example, both the target-specific ligand and the T cell receptor signaling domain polypeptide are fused to the ligand that binds to the TCR complex. In certain embodiments, the first polypeptide, the second polypeptide, and the third polypeptide are linked by at least one linker. In certain embodiments, the first polypeptide and the second polypeptide are fused directly and linked to the third polypeptide by a linker. In certain embodiments, the second polypeptide and the third polypeptide are fused directly and linked to the first polypeptide by a linker.

In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises 1 to 40 amino acids. In certain embodiments, the peptide linker comprises 1 to 30 amino acids. In certain embodiments, the peptide linker comprises 1 to 15 amino acids. In certain embodiments, the peptide linker comprises 1 to 10 amino acids. In certain embodiments, the peptide linker comprises 1 to 6 amino acids. In certain embodiments, the peptide linker comprises 30 to 40 amino acids. In certain embodiments, the peptide linker comprises 32 to 36 amino acids. In certain embodiments, the peptide linker comprises 5 to 30 amino acids. In certain embodiments, the peptide linker comprises 5 amino acids. In certain embodiments, the peptide linker comprises 10 amino acids. In certain embodiments, the peptide linker comprises 15 amino acids. In certain embodiments, the peptide linker comprises 20 amino acids. In certain embodiments, the peptide linker comprises 25 amino acids. In certain embodiments, the peptide linker comprises 30 amino acids.

In certain embodiments, the peptide linker comprises a G ₄S₃ linker (SEQ ID NO: 74). In certain embodiments, the peptide linker comprises SEQ ID NOs 11, 12, 15, 16, 19, 20 or variants or fragments thereof.

In certain embodiments, the peptide linker that links the target-specific ligand to a ligand that binds to the TCR complex (e.g., UCHT 1) is referred to as a linker to distinguish the protein domain from other linkers in the Tri-TAC. The joint has any size. In certain embodiments, the linker between the ligand that binds to the TCR complex and the target-specific ligand is a short helix comprising SEQ ID No. 28. In certain embodiments, the linker between the ligand that binds to the TCR complex and the target-specific ligand is a short helix encoded by SEQ ID No. 27. In certain embodiments, the linker between the ligand that binds to the TCR complex and the target-specific ligand is a long helix comprising SEQ ID No. 30. In certain embodiments, the linker between the ligand that binds to the TCR complex and the target-specific ligand is a long helix encoded by SEQ ID No. 29. In certain embodiments, the linker between the ligand that binds to the TCR complex and the target-specific ligand is a large domain comprising SEQ ID No. 32. In certain embodiments, the linker between the ligand that binds to the TCR complex and the target-specific ligand is a large domain encoded by SEQ ID No. 31.

In certain embodiments, a nucleic acid disclosed herein comprises a leader sequence. In certain embodiments, the leader sequence comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 5, 47 or 49. In certain embodiments, the leader sequence comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 5, 47 or 49. In certain embodiments, the leader sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 5, 47 or 49. In certain embodiments, the leader sequence comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 5, 47 or 49. In certain embodiments, the leader sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 5, 47 or 49. In certain embodiments, the leader sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 5, 47 or 49. In certain embodiments, the leader sequence comprises the nucleotide sequence of SEQ ID NO. 5, 47 or 49.

In certain embodiments, a nucleic acid disclosed herein comprises a leader sequence. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 70% sequence identity to SEQ ID No. 6, 48 or 50. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 75% sequence identity to SEQ ID No. 6, 48 or 50. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 6, 48 or 50. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 6, 48 or 50. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 6, 48 or 50. In certain embodiments, the leader sequence comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 6, 48 or 50. In certain embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO. 6, 48 or 50.

As disclosed herein, it is contemplated that the Tri-TAC exists in a variety of different configurations and combinations of (a) target-specific ligands, (b) ligands that bind to TCR complexes, and (c) TCR signaling domains.

In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) a single chain antibody (scFv) that binds CD3 epsilon, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) huUCHT (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) huUCHT1, and (c) the transmembrane and cytoplasmic domains of the CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) huUCHT1 (Y177T), and (c) the transmembrane and cytoplasmic domains of the CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) scFv, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) huUCHT (Y177T), and (c) the transmembrane and cytoplasmic domain of the CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) UCHT1, and (c) the transmembrane and cytoplasmic domains of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) UCHT1 (Y182T), and (c) the transmembrane and cytoplasmic domains of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) huUCHT1, and (c) the transmembrane and cytoplasmic domains of the CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) huUCHT (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) OKT3, and (c) the transmembrane and cytoplasmic domains of the CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) F6A, and (c) the transmembrane and cytoplasmic domains of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) BCMA-specific ScFv, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) BCMA-specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1 (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) a single chain antibody (scFv) that binds CD3 epsilon, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) huUCHT (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD4 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) huUCHT1, and (c) the transmembrane and cytoplasmic domains of the CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) huUCHT1 (Y177T), and (c) the transmembrane and cytoplasmic domains of the CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) DARPin, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) scFv, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) huUCHT (Y177T), and (c) the transmembrane and cytoplasmic domain of the CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) scFv, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) UCHT1, and (c) the transmembrane and cytoplasmic domains of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) huUCHT1, and (c) the transmembrane and cytoplasmic domains of the CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a target-specific ligand, (b) huUCHT (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) OKT3, and (c) the transmembrane and cytoplasmic domains of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) F6A, and (c) the transmembrane and cytoplasmic domains of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) HER-2 specific DARPin, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) BCMA-specific ScFv, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) BCMA-specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor.

In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1 (Y177T), and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) OKT3, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) F6A, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor. In certain embodiments, the Tri-TAC comprises (a) a CD19 specific ScFv, (b) L2K, and (c) a transmembrane and cytoplasmic domain of a CD8 co-receptor.

In certain embodiments, the Tri-TAC pulls CD3 and TCR into the lipid raft region of the membrane and brings Lck near the TCR, similar to natural MHC binding.

In certain embodiments, the TAC disclosed herein is an anti-HER-2 DARPin Tri-TAC (also referred to as configuration 1; SEQ ID NOs: 1 and 2), and comprises, in order:

anti-HER-2 Tri-TAC leader sequence (secretion Signal) (SEQ ID NOs: 5 and 6)

DARPin (SEQ ID NOs: 7 and 8) specific for HER-2 antigen

Myc tag (SEQ ID NO:9 and 10)

Joint (SEQ ID NO:11 and 12)

UCHT1 (SEQ ID NO:13 and 14)

Linker (SEQ ID NOs: 15 and 16)

CD4 (SEQ ID NOS: 17 and 18).

In certain embodiments, the TAC disclosed herein is HER2-TAC. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 65. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 65. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 65. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 65. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 65. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 65. In certain embodiments, the HER2-TAC comprises the nucleotide sequence of SEQ ID NO. 65.

In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 66. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 66. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 66. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 66. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 66. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 66. In certain embodiments, the HER2-TAC comprises the amino acid sequence of SEQ ID NO: 66.

In certain embodiments, the TAC disclosed herein is HER2-TAC. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 67. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 67. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 67. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 67. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 67. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 67. In certain embodiments, the HER2-TAC comprises the nucleotide sequence of SEQ ID NO: 67.

In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 68. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 68. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 68. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 68. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 68. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 68. In certain embodiments, the HER2-TAC comprises the amino acid sequence of SEQ ID NO. 68.

In certain embodiments, the TAC disclosed herein is HER2-TAC. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 75. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 75. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 75. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 75. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 75. In certain embodiments, the HER2-TAC comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 75. In certain embodiments, the HER2-TAC comprises the nucleotide sequence of SEQ ID NO. 75.

In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 76. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 76. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 76. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 76. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 76. In certain embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 76. In certain embodiments, the HER2-TAC comprises the amino acid sequence of SEQ ID NO. 76.

In certain embodiments, the TAC disclosed herein is BCMA-TAC. In certain embodiments, the BCMA-TAC comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 55, 57, 59 or 61. In certain embodiments, the BCMA-TAC comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 55, 57, 59 or 61. In certain embodiments, the BCMA-TAC comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO. 55, 57, 59 or 61. In certain embodiments, the BCMA-TAC comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 55, 57, 59 or 61. In certain embodiments, the BCMA-TAC comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 55, 57, 59 or 61. In certain embodiments, the BCMA-TAC comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 55, 57, 59 or 61. In certain embodiments, the BCMA-TAC comprises the nucleotide sequence of SEQ ID NO:55, 57, 59 or 61.

In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:56, 58, 60 or 62. In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:56, 58, 60 or 62. In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:56, 58, 60 or 62. In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:56, 58, 60 or 62. In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:56, 58, 60 or 62. In certain embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:56, 58, 60 or 62. In certain embodiments, the BCMA-TAC comprises the amino acid sequence of SEQ ID NO:56, 58, 60 or 62.

In certain embodiments, the TAC disclosed herein is CD19-TAC. In certain embodiments, the CD19-TAC comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO. 63. In certain embodiments, the CD19-TAC comprises the nucleotide sequence of SEQ ID NO. 63.

In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 64. In certain embodiments, the CD19-TAC comprises the amino acid sequence of SEQ ID NO. 64.

Polypeptide and vector constructs

In certain embodiments, disclosed herein are polypeptides encoded by the nucleic acid sequences disclosed herein. Also disclosed herein are vectors comprising the nucleic acid sequences disclosed herein. In certain embodiments, the vector further comprises a promoter. In certain embodiments, the promoter is functional in mammalian cells. Promoters are regions of DNA that initiate transcription of a particular nucleic acid sequence, and are well known in the art. "promoter functional in mammalian cells" refers to a promoter that drives expression of a related nucleic acid sequence in mammalian cells. Promoters that drive expression of a nucleic acid sequence are referred to as being "operably linked" to the nucleic acid sequence.

A variety of different delivery vectors and expression vectors are used to introduce the nucleic acids described herein into cells.

In certain embodiments, polynucleotides contained in vectors are disclosed herein to provide vector constructs, also referred to herein as vectors. In certain embodiments, the present disclosure provides a vector comprising:

a. A first polynucleotide encoding a target-specific ligand;

b. A second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex;

c. A third polynucleotide encoding a T cell receptor signaling domain polypeptide, and

D. promoters functional in mammalian cells.

In certain embodiments, the target of the target-specific ligand binds HER-2, BCMA or CD 19. In certain embodiments, the target-specific ligand is DARPin that selectively binds to the HER-2 (erbB-2) antigen. In certain embodiments, the target-specific ligand is DARPin that specifically binds to the HER-2 (erbB-2) antigen. In certain embodiments, the HER-2 (erb-2) -targeted DARPin comprises SEQ ID NO 7 or SEQ ID NO 8. In certain embodiments, the target-specific ligand is an scFv that selectively binds BCMA. In certain embodiments, the target-specific ligand is an scFv that specifically binds BCMA. In certain embodiments, the scFv that binds BCMA comprises SEQ ID NO. 33 or SEQ ID NO. 34. In certain embodiments, the target-specific ligand is an scFv that selectively binds CD 19. In certain embodiments, the target-specific ligand is an scFv that specifically binds CD 19. In certain embodiments, the scFv that binds CD19 comprises SEQ ID NO:35 or SEQ ID NO:36.

In certain embodiments, the ligand that binds to a protein associated with the TCR complex is UCHT1, humanized UCHT1 (huUCHT 1), OKT3, F6A, or L2K. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is UCHT1 or a variant thereof. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is UCHT1 and is encoded by SEQ ID NO: 13. In certain embodiments, the ligand that binds to a protein associated with a TCR complex is UCHT1 and comprises SEQ ID NO 14. In certain embodiments, the UCHT1 ligand that binds to a protein associated with a TCR complex has a Y182T mutation (UCHT 1 (Y182T)) and is encoded by SEQ ID NO: 71. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is UCHT1 (Y182T) and comprises SEQ ID NO:72. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is humanized UCHT1 (huUCHT 1) or a variant thereof. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is humanized UCHT1 (huUCHT 1) and is encoded by SEQ ID NO. 43. In certain embodiments, the ligand that binds to a protein associated with a TCR complex is huUCHT and comprises SEQ ID NO 44. In certain embodiments, the huUCHT ligand that binds to a protein associated with a TCR complex has a Y177T mutation (huUCHT (Y177T)) and is encoded by SEQ ID No. 45. In certain embodiments, the ligand that binds to a protein associated with a TCR complex is huUCHT (Y177T) and comprises SEQ ID NO 46.

In certain embodiments, the ligand that binds to a protein associated with the TCR complex is OKT3 or a variant thereof. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is OKT3 and is encoded by SEQ ID NO. 21. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is OKT3 and comprises SEQ ID NO. 22.

In certain embodiments, the ligand that binds to a protein associated with the TCR complex is F6A or a variant thereof. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is F6A and is encoded by SEQ ID NO. 23. In certain embodiments, the ligand that binds to a protein associated with a TCR complex is F6A and comprises SEQ ID NO. 24.

In certain embodiments, the ligand that binds to a protein associated with the TCR complex is L2K or a variant thereof. In certain embodiments, the ligand that binds to a protein associated with the TCR complex is L2K and is encoded by SEQ ID NO. 25. In certain embodiments, the ligand that binds to a protein associated with a TCR complex is L2K and comprises SEQ ID NO 26.

In certain embodiments, the protein associated with the TCR complex is CD3. In certain embodiments, the protein associated with the TCR complex is CD3 epsilon.

In certain embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytoplasmic domain of a TCR accessory receptor. In certain embodiments, the TCR accessory receptor is CD4, CD8, LAG3, or a chimeric variant thereof.

In certain embodiments, the first polynucleotide and third polynucleotide are fused to the second polynucleotide, and the coding sequence is operably linked to a promoter. In certain embodiments, the second polynucleotide and third polynucleotide are fused to the first polynucleotide, and the coding sequence is operably linked to a promoter. In certain embodiments, the vector is designed for expression in mammalian cells, such as T cells. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a retroviral vector.

In certain embodiments, useful vectors include vectors derived from lentiviruses, murine Stem Cell Viruses (MSCV), poxviruses, oncogenic retroviruses, adenoviruses, and adeno-associated viruses. Other useful delivery vectors include vectors derived from herpes simplex virus, transposons, vaccinia virus, human papilloma virus, simian immunodeficiency virus, HTLV, human foamy virus, and variants thereof. Other useful vectors include vectors derived from foamy viruses, mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, mammalian type D retroviruses and HTLV/BLV retroviruses. One example of a lentiviral vector useful in the disclosed compositions and methods is the pCCL < 4 > vector.

In certain embodiments, the nucleic acid is a recombinant or engineered nucleic acid. In certain embodiments, the first, second, and/or third polynucleotides are recombinant or engineered polynucleotides. In certain embodiments, the polynucleotides described herein are modified or mutated to optimize the function of the encoded polypeptide and/or the function, activity, and/or expression of the T cell antigen conjugate. In certain embodiments, the nucleic acid encodes a polypeptide.

In certain embodiments, modifications are made to the polynucleotide sequences and polypeptide sequences disclosed herein, including vector sequences. Modifications include substitutions, insertions or deletions of nucleotides or amino acids, or changes in the relative positions or order of nucleotides or amino acids.

Expression in T cells

In certain embodiments, disclosed herein are engineered T cells comprising a nucleic acid sequence disclosed herein or a vector disclosed herein. In certain embodiments, disclosed herein are human T cells engineered to express the Tri-TACs disclosed herein. In certain embodiments, the T cells express Tri-TAC as disclosed herein. Also disclosed herein are T cells transduced or transfected with T cell antigen conjugates or vectors comprising Tri-TAC. In certain embodiments, the T cell is an isolated T cell.

In certain embodiments, the human T cells engineered to express Tri-TAC exhibit equivalent function to conventional CARs in vitro. In certain embodiments, T cells engineered with the Tri-TAC exhibit superior function in vitro over conventional CARs. In certain embodiments, disclosed herein are human T cells engineered with Tri-TAC that exhibit enhanced safety compared to traditional CARs. In certain embodiments, human T cells engineered to express Tri-TAC exhibit enhanced safety compared to conventional CARs.

In certain embodiments, the T cells are obtained from a number of sources, including, but not limited to, blood (e.g., peripheral blood mononuclear cells), bone marrow, thymus tissue, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, spleen tissue, or tumor. In certain embodiments, the T cell is an autologous T cell. In certain embodiments, the T cells are obtained from a cell line of T cells. In certain embodiments, the T cells are obtained from a donor (allogeneic T cells). In certain embodiments, the T cells are obtained by differentiation of embryonic or adult stem cells or from induced pluripotent stem cells. In certain embodiments, regardless of the source of the T cells, the T cells have been modified such that they lack expression of endogenous TCRs and/or permanently or transiently lack expression of MHC/HLA molecules (universal donor T cells). In certain embodiments, the T cells are autologous to the subject. In certain embodiments, the T cells are allogeneic, syngeneic, or xenogeneic to the subject.

In certain embodiments, once obtained, the T cells are optionally enriched in vitro. In certain embodiments, the cell population is enriched by positive or negative selection. In addition, the T cells are optionally frozen or cryopreserved and then thawed at a later date.

In certain embodiments, the T cells are activated and/or expanded before or after the Tri-TAC is introduced into the T cells. In certain embodiments, the T cells are expanded by contacting the surface with an agent attached thereto that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a helper stimulator molecule on the surface of the T cells. In certain embodiments, the T cells are expanded by contact with one or more soluble agents that stimulate CD3/TCR complex signaling and accessory stimulator molecule signaling.

In certain embodiments, the T cells are transduced or transfected with a nucleic acid sequence. The transduced or transfected T cells express a protein encoded by the transfected or transduced nucleic acid sequence. Nucleic acids may be introduced into cells by physical, chemical or biological means. Physical means include, but are not limited to, microinjection, electroporation, ion bombardment, lipofection, and calcium phosphate precipitation. Biological means include the use of DNA and RNA vectors.

Viral vectors, including retroviral vectors, are used to introduce and express nucleic acids in T cells. Viral vectors include vectors derived from lentiviruses, murine Stem Cell Viruses (MSCV), poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses. The vector optionally includes a promoter (e.g., CMV promoter, eF a promoter, or MSCV promoter) that drives expression of the transduced nucleic acid molecule in T cells.

Any suitable assay may be used to confirm the presence and/or expression of the transduced nucleic acid sequence and/or polypeptide encoded by the nucleic acid in the T cell. Assays include, but are not limited to, southern and Northern blot analysis, RT-PCR and PCR, ELISA, western blot analysis, and flow cytometry.

The T cells expressing TAC have increased T cell activation in the presence of antigen as compared to T cells not expressing TAC and/or as compared to T cells expressing a conventional CAR. Increased T cell activation is determined by a number of methods including, but not limited to, increased tumor cell line killing, increased cytokine production, increased cell lysis, increased degranulation and/or increased expression of activation markers such as CD107 a, ifnγ, IL2 or tnfa. In certain embodiments, the increase is measured in a cell or cell population of the individual.

As used herein, the term "elevated" or "elevated" refers to an increase of at least 1%, 2%, 5%, 10%, 25%, 50%, 100% or 200% in a T cell or population of T cells expressing TAC compared to a T cell or population of T cells not expressing TAC and/or compared to a T cell or population of T cells expressing a conventional CAR.

Pharmaceutical composition

In certain embodiments, disclosed herein are pharmaceutical compositions comprising the engineered T cells disclosed herein (transduced with and/or expressing TAC) and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, buffers such as neutral buffered saline, phosphate buffered saline, and the like, sugars such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), or preservatives. In certain embodiments, the engineered T cells are formulated for intravenous administration.

The pharmaceutical composition is administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration is determined by factors such as the condition of the patient and the type and severity of the patient's disease, although the appropriate dosage is determined by clinical trials. The exact amount of the composition of the present invention to be administered is determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis and condition of the patient (subject) when indicating an "immunologically effective amount", "antineoplastic effective amount", "tumor transplantation effective amount" or "therapeutic amount".

In certain embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dose of 10 ¹ to 10 ¹⁵ cells per kg body weight, 10 ⁴ to 10 ⁹ cells per kg body weight, optionally 10 ⁵ to 10 ⁸ cells per kg body weight, 10 ⁶ to 10 ⁷ cells per kg body weight, or 10 ⁵ to 10 ⁶ cells per kg body weight, including all whole values within those ranges. In certain embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dose of greater than 10 ¹ cells per kg body weight. In certain embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dose of less than 10 ¹⁵ cells per kg body weight.

In certain embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dose of 0.5x10 ⁶ cells, 2x10 ⁶ cells, 4x10 ⁶ cells, 5x10 ⁶ cells, 1.2x10 ⁷ cells, 2x10 ⁷ cells, 5x10 ⁷ cells, 2x10 ⁸ cells, 5x10 ⁸ cells, 2x10 ⁹ cells, 0.5-2000x10 ⁶ cells, 0.5-2x10 ⁶ cells, 0.5-2x10 ⁷ cells, 0.5-2x10 ⁸ cells, or 0.5-2x10 ⁹ cells, including all integer values in those ranges.

In certain embodiments, the T cell composition is administered in multiple doses. In certain embodiments, the dose is administered singly or multiply, e.g., daily, weekly, biweekly or monthly, hourly, or after recurrence, recurrence or progression of the cancer being treated. In certain embodiments, the cells are administered using perfusion techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng.J.of Med.319:1676,1988).

The pharmaceutical composition is substantially free, e.g., free, of detectable levels of contaminants, e.g., selected from endotoxin, mycoplasma, replicating lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD 3/anti-CD 28 antibody coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, media components, vector packaging cells or plasmid components, bacteria, fungi, mycoplasma, IL-2, and IL-7.

In certain embodiments, the engineered T-cells disclosed herein are administered to a subject and subsequently re-bled (or apheresed), and the T-cells derived therefrom are activated and re-infused into the patient with the engineered T-cells. In certain embodiments, this process is performed multiple times every few weeks. T-cells from 10cc to 400cc of the blood sample were activated. T-cells from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc or 100cc evacuated blood samples are activated.

The modified/engineered T cells and/or pharmaceutical compositions are administered by methods including, but not limited to, aerosol inhalation, injection, perfusion, ingestion, infusion, implantation, or transplantation. The modified T cells and/or pharmaceutical compositions are administered to a subject arterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (i.v.) injection, by intravenous (i.v.) infusion or intraperitoneally. The modified/engineered T cells and/or pharmaceutical compositions thereof are administered to a patient by intradermal or subcutaneous injection. The modified/engineered T cells and/or pharmaceutical compositions thereof are administered by i.v. injection. The modified/engineered T cells and/or pharmaceutical compositions thereof are injected directly into a tumor, lymph node or infection site.

The modified/engineered T cells and/or pharmaceutical composition are administered in a volume of about 5mL、10mL、15mL、20mL、25mL、30mL、35mL、40mL、45mL、50mL、60mL、70mL、80mL、90mL、100mL、110mL、120mL、130mL、140mL、150mL、200mL、300mL、400mL or 500 mL.

The modified/engineered T cells and/or pharmaceutical composition are administered in a volume of up to about 5mL、10mL、15mL、20mL、25mL、30mL、35mL、40mL、45mL、50mL、60mL、70mL、80mL、90mL、100mL、110mL、120mL、130mL、140mL、150mL、200mL、300mL、400mL or 500 mL.

The modified/engineered T cells and/or pharmaceutical composition are administered in a volume of at least about 5mL、10mL、15mL、20mL、25mL、30mL、35mL、40mL、45mL、50mL、60mL、70mL、80mL、90mL、100mL、110mL、120mL、130mL、140mL、150mL、200mL、300mL、400mL or 500 mL.

The pharmaceutical composition is prepared by methods known per se for preparing a pharmaceutically acceptable composition for administration to a subject, such that an effective amount of said T cells is combined in a mixture with a pharmaceutically acceptable carrier. Suitable vectors are described, for example, in Remington pharmaceutical (Remington's Pharmaceutical Sciences, 20 th edition, mack Publishing Company, easton, pa., USA, 2000). On this basis, the composition comprises, but is not limited to, a solution of a substance associated with one or more pharmaceutically acceptable carriers or diluents, and is contained in a buffer solution having a suitable pH and being isotonic with physiological fluids.

Suitable pharmaceutically acceptable carriers include compositions that are chemically inert in nature and that are non-toxic, without interfering with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, glycerol solutions, N- (1 (2, 3-dioleyloxy) propyl) N, N, N-trimethylammonium chloride (DOTMA), dioleylphospholipid ethanolamine (DOPE), and liposomes. In certain embodiments, such compositions contain a therapeutically effective amount of the compound in combination with a suitable amount of carrier, so as to provide a form for direct administration to the patient.

Pharmaceutical compositions include, but are not limited to, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions which may also contain antioxidants, buffers, bacteriostats and solutes which render the composition substantially compatible with the tissue or blood of the intended recipient. Other components that may be present in such compositions include, for example, water, surfactants (e.g., tween), alcohols, polyols, glycerol, and vegetable oils. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.

The pharmaceutical compositions disclosed herein are formulated in a variety of different forms and administered by a variety of different means. The pharmaceutical dosage forms are administered orally, rectally or parenterally in dosage forms containing conventional acceptable carriers, adjuvants and vehicles as desired. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, or intrasternal injection and infusion techniques. Administration includes injection or infusion (including intra-arterial, intra-cardiac, intra-cerebral, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalation, transdermal, transmucosal, sublingual, buccal and topical (including epidermis, dermis, enema, eye drops, ear drops, intranasal, vaginal). In certain exemplary embodiments, the route of administration is by injection, e.g., intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

Liquid dosage forms include oral dosage forms, intravenous dosage forms, intranasal dosage forms, ophthalmic dosage forms, otic dosage forms, aerosols, and the like. In certain embodiments, a combination of the various dosage forms is administered. In certain embodiments, the composition is formulated for extended release.

Methods of treatment and use

In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof using the Tri-TAC disclosed herein. In certain embodiments, the target-specific ligands of TAC disclosed herein bind to a tumor antigen or a tumor-associated antigen on a tumor cell. In certain embodiments, the target-specific ligands of TAC disclosed herein bind selectively to a tumor antigen or a tumor-associated antigen on a tumor cell. In certain embodiments, the target-specific ligands of TAC disclosed herein specifically bind to a tumor antigen or a tumor-associated antigen on a tumor cell. In certain embodiments, the target antigen is a tumor antigen. Examples of tumor antigens include, but are not limited to, CD19, HER-2 (erbB-2), B-cell maturation antigen (BCMA), alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), prostate-specific antigen (PSA), glioma-associated antigen, beta-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), enterocarboxylesterase, mut hsp70-2, M-CSF, prostase, PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin growth factor () -I, IGF-II, insulin receptor and IGF receptor and mesothelin.

In certain embodiments, disclosed herein are methods of treating a cancer that expresses a target antigen in an individual in need thereof, the methods comprising administering to the individual the engineered T cells disclosed herein. In certain embodiments, the target antigen is CD19. In certain embodiments, the method of treating a CD19 expressing cancer in an individual in need thereof comprises administering to the individual engineered T cells comprising TAC containing a CD19 targeting ligand. In certain embodiments, examples of cancers treated with TAC comprising a CD19 targeting ligand include, but are not limited to, B cell malignancies. In certain embodiments, examples of cancers treated by TAC comprising a CD19 targeting ligand include, but are not limited to, B cell lymphomas, acute Lymphoblastic Leukemia (ALL), and Chronic Lymphocytic Leukemia (CLL). In certain embodiments, examples of cancers treated with TAC comprising a CD19 targeting ligand include, but are not limited to, non-hodgkin's lymphoma (NHL).

In certain embodiments, the target antigen is HER-2. In certain embodiments, the method of treating a cancer in which the cancer cells express HER-2 in an individual in need thereof comprises administering to the individual engineered T cells comprising TAC comprising a HER-2 targeting ligand. In certain embodiments, examples of cancers treated with TAC comprising HER-2 targeting ligands include, but are not limited to, breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and gastric cancer.

In certain embodiments, the target antigen is BCMA. In certain embodiments, the method of treating cancer in which cancer cells express BCMA in an individual in need thereof comprises administering to the individual engineered T cells comprising TAC comprising a BCMA targeting ligand. In certain embodiments, examples of cancers treated by TACs comprising BCMA targeting ligands include, but are not limited to, leukemia, lymphoma, and multiple myeloma.

Also disclosed herein is the use of the engineered T cells disclosed herein for the preparation of a medicament for treating cancer in an individual in need thereof. Also disclosed herein is the use of a T cell mixture comprising modified and unmodified cells, or a different population comprising modified cells with or without unmodified cells. Those of ordinary skill in the art will appreciate that therapeutic amounts of modified T cells are not necessarily homogeneous in nature.

In certain embodiments, the engineered T cells disclosed herein are part of a combination therapy. In certain embodiments, the effectiveness of the therapies disclosed herein is assessed multiple times. In certain embodiments, the patient is stratified based on the response to the treatment disclosed herein. In certain embodiments, the effectiveness of the treatment determines whether to enter the trial.

In certain embodiments, the cancer treated by engineered T cells comprising any of the TACs disclosed herein includes any form of neoplastic disease. In certain embodiments, examples of cancers treated include, but are not limited to, breast cancer, lung cancer, and leukemias such as Mixed Lineage Leukemia (MLL), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL). In certain embodiments, examples of cancers treated include, but are not limited to, large B-cell lymphomas, diffuse large B-cell lymphomas, primary mediastinal B-cell lymphomas, high grade B-cell lymphomas, or large B-cell lymphomas caused by follicular lymphomas. Other cancers include epithelial cancers, blastomas, melanomas, sarcomas, hematological cancers, lymphoid malignancies, benign and malignant tumors, and malignant tumors. In certain embodiments, the cancer comprises a non-solid tumor or a solid tumor. In certain embodiments, the cancer being treated includes non-vascularized or substantially non-vascularized tumors as well as vascularized tumors. In certain embodiments, the cancer is a solid cancer or comprises a solid tumor. In certain embodiments, the cancer is a liquid cancer or comprises a liquid tumor. In certain embodiments, the cancer is lung cancer, breast cancer, colon cancer, multiple myeloma, glioblastoma, gastric cancer, ovarian cancer, gastric cancer, colorectal cancer, urothelial cancer, endometrial cancer, or melanoma. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is glioblastoma. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is urothelial cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the cancer is melanoma.

TABLE 1 sequence listing

¹ Light chain, nucleotide 1-324, connector, nucleotide 3250387, heavy chain, nucleotide 388-750

² Light chain, amino acids 1-108, linker, amino acids 109-128, heavy chain, amino acids 129-250

³ Extracellular linker, nucleotides 1-66, transmembrane domain, nucleotides 67-132, cytoplasmic domain, nucleotides 133-254

⁴ Extracellular linker, amino acids 1-22, transmembrane domain, amino acids 23-44, cytoplasmic domain, amino acids 45-84

Examples

The following examples are provided for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any way. The present examples and methods described herein are representative of presently preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Those skilled in the art will recognize modifications and variations therefrom which are encompassed within the spirit of the invention as defined by the scope of the claims.

EXAMPLE 1 characterization of Tri-TAC technology

An overview of Tri-TAC technology is provided in fig. 1A-1C.

FIG. 1A shows an example of CD 8T-cell activation based on the co-assembly of different receptors and their associated protein partners. Initially, major histocompatibility complex I is presenting antigen (helix). This is recognized by the T Cell Receptor (TCR) complex which is capable of binding to the antigen. The TCR complex contains several individual subunits. The alpha/beta domain is capable of direct interaction with antigens presented on MHC-I. The α/β domain then interacts with several other domains (epsilon, gamma, delta, and zeta), all of which are involved in T-cell activation through a variety of different intracellular activation domains. The TCR complex and CD8 co-receptor interact simultaneously with MHC-I. The CD8 co-receptor binds to the MHC-I in an antigen-independent manner. CD8 interacts directly with Lck, a protein kinase important for activating the TCR receptor complex. The interaction of CD8 with Lck also ensures their association with lipid raft (membrane fraction) microdomains that are assumed to organize and encapsulate other relevant signaling components (dark spheres). Later stages of subsequent activation result in CD28 recruitment. If this cascade of interactions occurs several times in parallel, the T-cells become activated and are able to exert their cytotoxic effects.

Fig. 1B provides an overview of a Chimeric Antigen Receptor (CAR). CARs seek to reproduce the complex mechanism of T-cell activation by combining several key activation domains, such as cd3ζ and CD28, in a single synthetically engineered molecule. The CAR then interacts with the selected antigen using a specific binding domain. Depicted herein is ankyrin repeat protein (DARPin). Several such interactions, which are believed to occur in parallel, cause T-cell activation.

FIG. 1C is an overview of the Tri-TAC technology simulating the natural activation process. The Tri-TAC was developed to better reenact the natural signaling through the TCR while retaining targeting that is not MHC restricted. T-cell activation occurs after MHC is linked by TCR and T cell accessory receptors (CD 4 or CD 8) that bind simultaneously to conserved regions within the MHC molecule. The accessory receptors are specifically located within the membrane micro-domains of the "lipid raft" type that are particularly important for the formation of TCR signaling complexes. In addition to ensuring proper microdomain localization of the TCR-activating complex, these co-receptors also bind directly to Lck, a protein kinase critical for T-cell activation. Neither the traditional chimeric receptor nor the bifunctional protein is able to engage the accessory receptor molecule or Lck. A molecule was generated in which transmembrane and intracellular regions of the CD4 accessory receptor, which were localized to lipid rafts and to Lck binding, respectively, were fused to a CD3 binding single chain antibody (UCHT 1; SEQ ID NOS: 13, 14 and homologs thereof). This construct is designed to pull the CD3 molecule and TCR into the lipid raft region and bring Lck into proximity of the TCR, similar to natural MHC binding. To target this receptor, a designed ankyrin repeat (DARPin) was ligated to the CD4-UCHT1 chimera to generate a trifunctional T cell antigen conjugate (Tri-TAC). In this example, the DARPin is specific for the protooncogene HER-2 (erbB-2).

A variety of Tri-TAC configurations are possible (fig. 2A and 2B). In configuration 1 (fig. 2A), the antigen binding domain is located at the N-terminus, linked to the CD3 ligand binding domain, followed by the accessory receptor domain. In configuration 2 (fig. 2B), the CD3 ligand binding domain is located at the N-terminus, being linked to an antigen binding domain, which in turn is linked to a co-receptor domain.

Multiple classes of ligand binding domains can be incorporated into the Tri-TAC molecules (fig. 3A-3D). Examples herein show general schematic diagrams of configuration 1Tri-TAC (fig. 3A), tri-TAC with HER-2 specific DARPin (fig. 3B), tri-TAC with CD19 specific scFv (fig. 3C) and Tri-TAC with BCMA specific scFv (fig. 3D).

FIGS. 4A-4D show the function of Tri-TAC with HER-2 specific DARPin. Human T cells were engineered to express Tri-TAC as disclosed herein or a conventional CAR with the same DARPin. It was determined that in all cases the engineered T cells with Tri-TAC showed at least equivalent function to conventional CARs. Interestingly, for the 2 parameters (TNF- α production and CD107a mobilization), it was observed that in some cases the Tri-TAC was more active than the conventional CAR.

FIG. 4A shows surface expression of anti-HER-2 DARPin Tri-TAC compared to anti-HER-2 DARPin CAR and control T cells. Chimeric receptors were detected by incubation with recombinant HER-2. The anti-HER-2 DARPin Tri-TAC was well expressed on the surface of engineered T cells. FIG. 4B shows the growth of the engineered T cell culture. T cells were activated with anti-CD 3/anti-CD 28 antibody Dynabead and engineered with lentiviruses encoding Tri-TAC, CAR or non-encoding receptors (control). After 2 weeks, the CAR and control cultures grew to similar numbers, while the Tri-TAC cultures grew slightly slower. Fig. 4C and 4D show the functional properties of the engineered T cells. T cells engineered to express the Tri-TAC or CAR with HER-2DARPin were stimulated with plate-bound antigen. The T cells engineered to express Tri-TAC and CAR can perform all measured functions (TNF- α production, IFN- γ production and CD107a mobilization, fig. 3C and 3D). T cells engineered with Tri-TAC showed high frequency of CD107a positive cells after stimulation relative to T cells engineered with CAR (fig. 3D), indicating enhanced cytotoxicity on a per cell basis.

The data presented in FIGS. 6A-6J confirm the importance of both the ligand binding domain and UCHT1 CD3 binding domain to Tri-TAC function. T cells were engineered with full length Tri-TAC with HER-2DARPin (fig. 6G, 6H, 6I, bottom row), tri-TAC variants lacking DARPin (fig. 6A, 6B, 6C, top row), or Tri-TAC variants lacking UCHT1 (fig. 6D, 6E, 6F, middle row). All three engineered T cell populations were stimulated with HER-2 positive tumor cells. The T cells engineered with full length Tri-TAC can produce IFN-g, TNF-and IL-2 after stimulation, whereas the variants cannot produce any cytokines after stimulation. The three T cell populations were also co-cultured with D2F2/E2 cells (HER-2 expressing) or D2F2 cells (HER-2 negative) at a 4:1 effector to target ratio (FIG. 6J). T cells engineered with full length Tri-TAC showed robust killing of D2F2/E2 cells, but did not kill D2F2 cells. Other Tri-TAC variants lacking DARPin or UCHT1 did not exhibit killing.

Figures 7A-7C show the results of mice treated with vehicle control (NGFR), anti-HER-2 DARPin CAR, or anti-HER-2 DARPin Tri-TAC. Xenograft mouse models were used. OVCAR-3 tumor cells were subcutaneously administered to mice and allowed to grow until the tumor reached a size of 100-200mm ³. Fig. 7A shows the relative tumor progression normalized to the tumor size on the day of treatment. anti-HER-2 DARPin Tri-TAC engineered T-cells caused a rapid decrease in tumor volume, control was not effective, and CAR cells slowed tumor growth and showed a delayed decrease in tumor size. Fig. 7B shows the relative change in body weight after T-cell perfusion. Both control and anti-HER-2 DARPin Tri-TAC engineered cells showed no significant change in mouse body weight after treatment. In contrast, anti-HER-2 DARPin CAR treated mice showed significant weight loss, indicating severe toxicity. Fig. 7C shows cytokine concentrations in mouse serum at day 7 after T-cell perfusion. Cytokine levels were higher in CAR treated mice compared to Tri-TAC treated mice.

Example 2 substitution of UCHT1 affects Tri-TAC function

FIGS. 8A-8H illustrate the function of Tri-TAC with an optional CD3 binding domain. The domains are listed in fig. 8A and 8E. Tri-TAC containing UCHT1 (FIG. 8B), OKT3 (FIG. 8B) and huUCHT1 (FIG. 8F) showed high surface expression, while Tri-TAC containing F6A (FIG. 8F) and L2K (FIG. 8F) showed lower surface expression. Cells expressing Tri-TAC containing OKT3 showed low cytokine production (fig. 8C, fig. 8C 1) and moderate cytotoxicity (fig. 8D) after Tri-TAC ligation. Cells expressing Tri-TAC containing F6A showed strong cytokine production (fig. 8G, fig. 8G 1) and cytotoxicity (fig. 8H) after Tri-TAC ligation. Cells expressing Tri-TAC containing L2K showed low cytokine production (fig. 8G, fig. 8G 1) and moderate cytotoxicity (fig. 8H).

FIGS. 9A-9H show TCR surface expression on T cells engineered with the different Tri-TAC variants shown in FIGS. 8A and 8E. T cells engineered with the Tri-TAC variants comprising OKT3 (FIGS. 9A, 9E and 9B, 9F) or L2K (FIGS. 9C, 9G and 9D, 9H) exhibited lower TCR surface expression relative to T cells engineered with Tri-TAC comprising UCHT1 or huUCHT1, respectively. In contrast, T cells engineered with the Tri-TAC variant comprising F6A did not show TCR down-regulation relative to Tri-TAC with huUCHT a (fig. 9C, 9G and 9D, 9H). The F6A substitution reduces Tri-TAC receptor surface expression while preserving proper cytokine production and cytotoxicity. The L2K substitution suitably reduces surface expression and reduces cytokine production, but retains moderate cytotoxicity. The OKT3 substitution results in high surface expression of Tri-TAC, low cytokine production and moderate cytotoxicity. These data indicate that Tri-TAC surface expression and T cell effector function are not inherently proportional and that substitution of Tri-TAC domains alters effector function in some cases independent of surface expression levels. It is conceivable that TAC variants with reduced cytotoxicity and low surface expression may be valuable in certain clinical applications.

In many cases, the scFv replacement reduces the ability of the engineered T cells to make IFN- γ, TNF- α, and IL-2, but the engineered T cells still retain the ability to kill target cells. Excessive cytokine production has been associated with side effects in the clinical setting, limiting current CAR technology to life threatening diseases. The ability to modify TAC molecules to reduce their cytokine production while retaining appropriate cytotoxicity allows for the production of Tri-TAC receptors with the exact level of reactivity required to meet clinical efficacy and safety.

The ability of the OKT 3-containing Tri-TAC variants to inhibit TCR surface expression and cytokine production while retaining cytotoxicity may be of great value in allogeneic situations where inhibition of TCR may inhibit graft versus host disease.

These data confirm that scFv replacement of UCHT1 affects the function of Tri-TAC. Further modification will result in Tri-TAC that can be used in a variety of different applications (e.g. oncology, autoimmunity, allergy).

Example 3 introduction of various different linkers linking ligands that bind to TCR complexes to target binding ligand domains

FIGS. 10A-10B show several TAC variants with different linkers linking the ligand of the binding TCR complex to the target binding ligand domain. The flexible joint allows movement between the two domains. The large domain linker contains two folded domains and is very large and rigid. Small and long helical linkers also introduce rigidity, but are less restrictive when compared to the large domain linkers.

FIGS. 11A-11E show the effect of linker replacement on Tri-TAC surface expression, tri-TAC transduction efficiency and cytokine production after Tri-TAC ligation. Fig. 11A and 11B show that the helical connectors enhance surface expression and transduction efficiency when compared to the flexible connectors, while the large domain linker enhances transduction efficiency but does not enhance surface expression. FIGS. 11D and 11E show cytokine production by cells expressing Tri-TAC with short helices, long helices or large domain linkers.

FIG. 12A shows enhanced in vitro cytotoxicity of T cells expressing Tri-TAC with short helical junctions. FIG. 12B shows enhanced in vivo tumor control of T cells expressing Tri-TAC with short helical junctions. The short helical junctions are associated with high cytotoxicity in vitro and effective tumor control in vivo.

EXAMPLE 4 introduction of the CD8 alpha/beta cytoplasmic Domain

FIG. 13A shows surface expression of CD 8. Alpha. Tri-TAC paired with an anti-HER-2 scFv or in FIG. 13C with an anti-HER-2 DARIin. FIG. 13B shows cytokine production by T cells expressing CD 8. Alpha. Tri-TAC paired with anti-HER-2 scFv or anti-HER-2 DARIin.

FIG. 14A shows the CD4 Tri-TAC monomer and CD8 alpha/beta heterodimer. Both CD4 and CD8 TCR accessory receptors carry functional domains important for accessory receptor function. These regions include arginine-rich regions that are presumed to be important for lipid raft association and CXCP motifs required for Lck binding. Unlike CD4, which is a monomer, the CD8 co-receptor is a heterodimer consisting of alpha and beta subunits (fig. 14A). Both the α and β CD8 subunits contain arginine-rich regions, but only the α subunit contains the CXCP motif.

FIGS. 14B-14D provide schematic diagrams of variants of Tri-TAC incorporating elements from the CD8 co-receptor shown in FIG. 14A. The cysteines responsible for dimerization of CD 8a and CD8 β were replaced with alanine in all CD8 Tri-TAC variants. FIG. 14B is a schematic representation of CD 8. Alpha. Tri-TAC comprising a cysteine-to-serine mutation and a CD 8. Alpha. Cytoplasmic domain for ensuring monomeric receptor distribution. Fig. 14C is a schematic representation of a cd8α+rβtri-TAC comprising a cysteine-to-serine mutation for ensuring monomeric receptor distribution and a chimeric cd8α cytoplasmic domain in which the arginine-rich region of cd8α is replaced with the arginine-rich region of cd8β. FIG. 14D is a schematic representation of a CD8β+Lck Tri-TAC comprising a cysteine-to-serine mutation for ensuring monomeric receptor distribution and a chimeric CD8β cytoplasmic domain in which a CD8α CXCP domain comprising an Lck binding motif is added to the C-terminus of the CD8β cytoplasmic domain.

FIGS. 15A-15D show various phenotypic and functional properties of CD 8-based Tri-TAC variants relative to the prototype Tri-TAC. FIGS. 15A-15B show the surface expression of CD8-Tri TAC variants relative to the prototype Tri-TAC. Surface expression was comparable between different Tri-TACs. FIG. 15C shows the cytotoxicity in vitro of CD8-Tri TAC variants co-cultured with LOX IMVI (HER-negative) and A549, SKOV3, SKBR3 or MBA MB 231 (both HER-2 positive). All T cells engineered with Tri-TAC showed cytotoxicity. FIG. 15D shows cell division of T cells engineered with CD8Tri-TAC variants or prototype Tri-TAC (FIG. 15D). FIG. 15E shows TCR surface expression of engineered T cells containing CD8Tri-TAC variants or prototype Tri-TAC. All Tri-TAC variants have similar effects on TCR expression. Although CD4 co-receptors with both scFv and DARPin anti-HER-2 showed good surface expression and function, the CD 8a construct showed activity only in the case of the DARPin antigen binding domain. When testing different CD 8a cytoplasmic domains, all configurations contained the reported key sequence attributes associated with the function of the co-receptor (arginine-rich region and CXCP). All CD8 a/β constructs showed similar performance when compared to the CD4 prototype. This underscores that the retention of specific biochemical properties, such as lipid raft affinity and Lck binding, is more important for determining Tri TAC performance than for a specific cytoplasmic polypeptide sequence.

The growth of T cells engineered with cd8α+r (β) and cd8β+lck Tri-TAC was significantly impaired relative to the growth of T cells engineered with other variants. Despite having a significant impact on growth, these Tri-TACs all exhibit comparable ability to activate T cells. The reduced growth of the CD8α+R (β) and CD8β+Lck Tri-TAC may be advantageous for certain applications where maximum T cell expansion is not desired.

EXAMPLE 5 development of the CD19-TAC construct

FIG. 16 shows the stepwise development of CD19-TAC constructs. Several generations of lentiviral vectors were generated with various changes in the design elements to ensure CD 19-specific, appropriate TAC expression and GMP-grade lentiviral production. Each box represents a lentiviral vector and specifies 3 major design elements, (a) an antigen binding domain, (b) a TCR/CD3 binding domain, and (c) a co-receptor domain. The shaded area indicates the domain that was the subject of modification during vector development.

In a first step, the TAC comprises a HER-2 specifically designed ankyrin repeat protein (DARPin), a murine UCHT1 CD 3-specific scFv, and a flexible transmembrane and cytoplasmic CD4 polypeptide. The TAC was cloned into pCCL4 lentiviral vector.

To generate CD 19-specific Tri-TAC, HER-2-specific DARPin was replaced with a polypeptide comprising an N-terminal CD8a leader fused to an anti-CD 19 scFv. The heavy and light chains of the CD19 scFv are linked by a glycine-serine linker region.

The UCHT1 domain is replaced with a humanized version (huUCHT 1) to reduce immunogenicity. This TAC construct exhibits superior surface expression levels compared to its precursor.

To further increase receptor expression on the cell surface of T cells without compromising function, two independent modifications were evaluated in parallel. To increase single chain stability, the G ₄ S linker (SEQ ID NO: 73) used in the anti-CD 19 scFv was replaced with a more structured Whitlow linker. Separately, the Y177T mutation was introduced into the huUCHT domain. Both strategies enhance expression of TAC receptors and produce receptors with both Whitlow linker and Y177T mutation.

FIG. 17 shows the CD19-TAC insert in pCCL lentiviral vectors. The pCCL vector is characterized by a bi-directional promoter system in which ΔNGFR (hu) is under the control of the mCMV promoter and TAC expression is driven by the EF-1 alpha promoter. The Δngfr (hu) is a truncated human CD271 (tumor necrosis factor receptor superfamily member 16) with a transmembrane domain but lacking a cytoplasmic signaling domain. The expression product of Δngfr (hu) was used to quantify lentiviral transduction. The CD19-TAC#921 open reading frame is exaggerated to show the key elements of the TAC construct, CD8 alpha leader, FMC63 single chain (anti-CD 19 scFv), human c-Myc tag, huUCHT1 (Y177T) and ΔCD4 domain. The huUCHT (Y177T) mutation was identified by examining point mutations in residues that were subsequently introduced into the murine UCHT1 CD3 epsilon binding interface. The (Y177T) mutation was successfully identified in the screen. The (Y177T) mutation resulted in better Tri TAC surface expression while retaining T cell activation. Δcd4 lacks 4 CD4 extracellular immunoglobulin-like domains and retains extracellular linkers, transmembrane and cytoplasmic domains.

To generate GMP-grade lentiviral vectors, the CD19-Tri-TAC construct was cloned under the control of the MSCV promoter in the new lentiviral vector. The CD19-Tri-TAC construct is the same as shown in FIG. 17.

Example 6 ability to produce CD19-TAC expressing T cells from different donor materials

Figure 18 shows the potency of CD19TAC expressing T cells made from various donors. T cells expressing CD19-TAC were produced using T cells from three different donors and tested in NALM-6 tumor model. Mice bearing established NALM-6 tumors were treated with a single dose of 4X 10 ⁶ CD19TAC expressing T cells. Control mice showed rapid tumor growth and all mice reached the endpoint by the end of the study. T cell products from donors 1 and 2 resulted in complete control in all mice. T cell products from donor 3 lead to robust tumor control in all mice and long-term control in 2/4 treated mice. The study demonstrated that tumor rejection was achieved by CD19TAC expressing T cells derived from multiple healthy donors. The results of the NALM-6 tumor model in FIG. 18 indicate that effective CD19TAC was produced from materials from multiple donor sources.

Example 7 in vitro cytotoxicity and in vivo efficacy of CD19-TAC expressing T cells

To assess the ability of CD19-TAC to efficiently bind to a variety of different CD19 positive cells, tri-TAC engineered T cells were co-cultured with NALM-6 (acute lymphoblastic leukemia), raji (Burkitt's lymphoma) or Jeko-1 (mantle cell lymphoma). NALM-6, jeko-1 and Raji cells were engineered to have enhanced firefly luciferases to enable tracking of tumor burden in vitro and in living animals by bioluminescence imaging.

FIGS. 19A-19C show the killing of tumor cell lines by CD19-TAC expressing T cells. The effect is dose dependent and increases with increasing ratio of effector cells to target cells (E: T). As negative control, engineered cells or non-transduced T cells with Δtac (lacking antigen binding domain) were used. These results confirm that CD19-TAC expressing T cells kill CD19 positive tumor cells.

FIGS. 19D-19G show the design and results of in vivo studies assessing the efficacy of CD19-TAC in mice transplanted with NALM-6 (acute lymphoblastic leukemia), raji (Burkitt's lymphoma) or Jeko-1 (mantle cell lymphoma) liquid tumors. To elicit NALM-6, raji and Jeko-1 tumors, mice were vaccinated with NALM-6, raji or Jeko-1 cells, respectively, and kept for 4 or 7 days to allow implantation of tumors. On day 4 or 7, CD19-TAC expressing T cells were administered by tail vein intravenous injection. Tumor burden was measured at weekly intervals and the data plotted as average irradiance [ p/s/cm 2/sr ].

FIGS. 19E-19G illustrate that CD19-TAC engineered T cells were effective in inducing tumor regression and long-term tumor control in NALM-6 (acute lymphoblastic leukemia), raji (Burkitt's lymphoma) or Jeko-1 (mantle cell lymphoma) liquid tumors.

The results of the NAML-6, raji, or Jeko-1 tumor models in FIGS. 19A-19G indicate that CD19-TAC is effective in a variety of different CD 19-positive tumor models.

Example 8 persistent and sustained tumor immunity of CD19-TAC expressing T cells

FIGS. 20A-20B show the persistence of tumor immunity and resistance to re-challenge in mice receiving CD19-TAC expressing T cells. Mice bearing established NALM-6 tumors were treated with CD19-TAC expressing T cells.

FIG. 20A shows experimental setup for determining the persistence of CD19-TAC in mice. Mice were first vaccinated with NALM-6 cells, which were treated with CD19-TAC after a4 day implantation period. All mice showed tumor regression and complete tumor control. At 56 days post initial treatment, mice were re-challenged with NALM-6 (CD 19 positive) or KMS11 (CD 19 negative) liquid tumors. Naive mice were co-injected with tumor cells in all cases and used as negative controls. Tumor burden was tracked by luminescence signals.

FIG. 20B mice bearing established NALM-6 tumors are treated with CD19-TAC expressing T cells dosed as divided doses for a total of 4X 10 ⁶ engineered cells. As a control, a group of untreated animals was used. Following ACT, treated mice exhibited a durable anti-tumor response. In contrast, control mice showed an exponential increase in tumor mass and reached an endpoint associated with tumor burden. On day 56 post ACT, mice were re-challenged with NALM-6 tumor cells (CD 19 positive) or KMS11 tumor cells (CD 19 negative). Mice treated with CD19-TAC were still protected from NALM-6 (CD 19 positive) tumor cells, but from KMS11 (CD 19 negative) tumor cells.

The results of the re-challenge experiments in FIGS. 20A and 20B demonstrate that CD19-TAC, in some cases, differentiated into long-lived memory cells that retained anti-tumor properties.

Example 9 in vivo expansion and dose dependence of CD19-TAC expressing T cells

FIGS. 21 and 22 show the dose dependence, dose regimen (in fractions or single times) and expansion of T cells expressing CD19-TAC in the NALM-6 cancer model. Fig. 21A shows the experimental design. Mice received either a single dose of CD19-TAC expressing T cells on day 4 post tumor inoculation or divided doses delivered 7 days apart. Multiple doses of CD19-TAC expressing T cells were tested, 0.5X10 ⁶、1x10⁶ and 4X10 ⁶ cells. Figure 21B shows a control group of mice receiving 4x10 ⁶ untransduced cells or frozen medium (medium control).

FIG. 21B shows the survival of mice after NALM-6 injection and CD19-TAC injection. A dose-dependent increase in survival was observed in both the single dose and the split dose groups, with the highest single dose limiting tumor growth and increasing survival in mice.

FIG. 22A shows gating strategies for assessing T cell proliferation. The cells are first selected on the basis of forward and side scatter to select a lymphocyte population. Individual T cells are identified by the forward scattering region above the elevation gate. Living cells were identified by near IR gating. Human cells were identified by hCD45 gate. The resulting subset of cells was further divided into CD3 positive cells. These cells were then gated on CD4/CD8 and protein L. Staining strategies also contained mucd45_1 to identify murine blood cells. CD19 was included to stain NALM-6 cells.

Fig. 22B shows expansion of T cells in mice after divided dose adoptive T cell transfer (ACT). Blood samples were taken periodically after ACT and analyzed by flow cytometry. The values were normalized to the total T cell number present in the blood after ACT 1. Values were also normalized to the total number of CD45.1+ (murine) cells to account for differences in blood draw. T cells in mice treated with CD19-TAC engineered cells showed expansion in recipient mice within about 1-2 weeks after the first ACT (fig. 22B). The untransduced cells did not expand (fig. 22B).

The results of the various doses, dose regimens (FIG. 21B) and T cell counts (FIG. 22B) indicate that the potency of CD19-TAC is dose dependent, engineered T cells expand in vivo, and that such expansion is specific for CD19-TAC engineered cells in animals bearing CD19 positive tumors.

EXAMPLE 10 in vivo efficacy, long term efficacy and safety of CD19-TAC treatment

Figures 23-25 demonstrate long-term safety and efficacy (figure 23) and the absence of any treatment-related acute toxicity (figures 24-25).

Fig. 23A shows the experimental design. Mice were injected with 0.5 x 10 ⁶ rennet-engineered NALM-6 cells, which were allowed to implant for 4 days. Mice were then treated with two dose levels (4 and 12 x 10 ⁶ engineered cells) of CD 19-TAC-engineered T cells in a single dose. Tumor growth was then tracked by periodic luminescence measurements. Mouse health was assessed regularly by examining the behavior and physical characteristics (grooming behavior, mobility, fur integrity) of the mice.

Figure 23B shows tumor burden measured by luminescence after treatment with medium alone (frozen medium), non-engineered control cells (total T cell dose equal to total T cell dose of the highest engineered treatment group) and 4 or 12 x10 ⁶ engineered CD19-TAC engineered T cells. Both controls showed rapid tumor growth and no anti-tumor efficacy. The control agent caused a delay in tumor growth relative to the medium alone, presumably due to competition for graft habitat between high dose T cells and tumor cells. Engineered T cells showed tumor regression in all cases. The high dose treatment group showed complete tumor control in all cases. The 4 x10 ⁶ treatment group showed complete control of 3 mice, 1 mouse with delayed tumor growth, 1 mouse with controlled but high tumor burden.

Fig. 23C shows the overall survival for the different treatment groups. In both the vehicle and the non-engineered control mice, all mice died of the tumor within 23 to 35 days, respectively. In the case of high dose CD19-TAC treatment, all mice developed symptoms of GvHD and died of GvHD within 61 days. GvHD is the result of the mouse model itself rather than treatment with modified T cells. The low dose mice showed that 3 mice survived to the end of the study at 90 days, 1 mice died from high tumor burden, and 1 mice died from GvHD.

Figures 24 and 25 show clinical chemistry parameters and cytokine levels from medium control, non-engineered and CD19-TAC (4 and 12 x 10 ⁶ potent CD19-TAC engineered cells) treated mice. Mice were followed for 33 days after ACT and blood samples were taken on days 5, 12 and 33. Only CD19-TAC treated mice survived for 33 days. The vehicle control mice died from tumor burden before the third blood sample could be collected, and the non-engineered cell groups were sacrificed as early as day 26 before the mice would reach the tumor burden-related endpoint. All blood samples were analyzed for several clinical chemistry parameters and cytokine levels.

FIG. 24 shows that on days 5 and 12, CD19-TAC treated mice did not show significantly higher parameters than control mice. On day 33, all treated mice showed clinical chemistry parameters comparable to the early treatment time points, except for alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST), some of which experienced high levels, similar to mice treated with non-engineered cells sampled on day 26.

Figure 25 shows cytokine responses at days 5, 12 and 33. On day 5 post ACT, CD19-TAC but not control mice showed an increase in all cytokines tested. The cytokine increase is consistent with the inflammatory response of CD19-TAC engineered T cells to recognize and react with antigen-positive NALM-6 tumor cells. After their initial response, cytokine levels decreased by day 12, which correlates with subsequent induction of tumor regression and generally low tumor burden. On day 12, the cytokine levels of the CD19-TAC treated group were similar to or lower than those of the non-engineered T cell group, except for IL 10. At a later stage, all mice treated with non-transduced or CD19-TAC engineered T cells showed an increase in cytokines, presumably related to the onset of GvHD. See also fig. 29, which shows cytokine responses on days 5, 12, 26 and 33.

The results of long-term follow-up of mice treated with CD19-TAC and their clinical chemistry demonstrated that engineered T cells were safe for use and did not show any evidence of toxicity caused by the engineered specificity of CD 19-TAC. The results of the cytokine study demonstrated early inflammatory responses associated with anti-tumor efficacy, followed by a decrease in all cytokine levels, indicating a controlled inflammatory response.

EXAMPLE 11 in vivo efficacy of several BCMATri-TAC variants

FIG. 26 shows in vivo efficacy studies of various BCMA Tri-TAC constructs. Fig. 26A shows the overall experimental design. 1 million luciferase-engineered KMS11 (BCMA-positive) tumor cells were allowed to engraft for 12 days. Mice were then treated with a single effective dose of 4 million BCMA constructs and controls (fig. 26B). Tumor burden was assessed periodically by luminescence measurements. All mice exhibiting tumor regression and tumor control were then re-challenged with 1 million KMS11 cells on day 25 post ACT.

FIG. 26C control mice showed rapid growth of tumor cells after ACT reaching tumor-associated endpoints within 19 to 25 days. In contrast, all BCMA-TAC treated mice showed initial tumor regression. Tumor control was a function of the construct, with G ₄ S (SEQ ID NO: 73) 3625VH-VL exhibiting the lowest level of initial tumor control and short helix 3625VL-VH exhibiting the highest level of initial tumor control. After re-challenge, tumor control has been maintained until most of all constructs on day 25 remain protective against re-challenge.

The results of this in vivo study demonstrate that various BCMA Tri-TAC constructs are effective in controlling KMS11 (BCMA positive) liquid tumors. Certain preferred arrangements provide superior performance. In general, the helical linker region provides a relative benefit when compared to a flexible linker within the same scFv configuration.

Example 12 in vivo efficacy of TAC-Her2

Mice were vaccinated with OVCAR3 solid tumors in the posterior flank. Tumors were allowed to build and grow to a size of 100mm ³. Mice were then treated with tail vein injection of TAC-Her2 engineered T cells. Tumor volumes were measured periodically.

EXAMPLE 13 clinical trials

Clinical studies were performed in which subjects at least 18 years old, with CD19 positive diffuse large B-cell lymphoma, who failed or were not eligible for ASCT for at least two current first line therapies, including ASCT, were treated with CD19-TAC expressing T cells. The study was an open label, single arm, phase 1/2 two-phase trial characterized by an up-dosing phase for determining the Maximum Tolerated Dose (MTD) or recommended phase II dose (RPh 2D), followed by expansion of the cohort at the selected dose.

Following enrollment, subjects underwent leukocyte isolation to obtain T cells for the production of CD19-TAC expressing T cells. After successful manufacture, the subject enters the treatment phase. This stage involved chemotherapy with fludarabine and cyclophosphamide to clear lymphocytes, followed by Intravenous (IV) administration of CD19-TAC expressing T cells. Following treatment with CD19-TAC expressing T cells, subjects entered post-treatment follow-up and 2 years follow-up for safety, disease status and survival after their last dose of CD19-TAC expressing T cells. After completion of the study, subjects' survival, long-term toxicity and viral vector safety were followed in a separate long-term follow-up regimen until 15 years after their last dose of CD19-TAC expressing T cells.

In all groups, safety was assessed throughout the study period. T cell expansion was assessed from the time when the first dose of CD19-TAC expressing T cells was no longer detectable. Radiological disease assessment is performed by Positron Emission Tomography (PET) and/or Computed Tomography (CT) prior to treatment and about 3, 6, 9, 12, 18, and 24 months after the last dose of CD19-TAC expressing T cells or until the progressive disease or treatment with additional anticancer therapies.

EXAMPLE 14 production of CD 19-TAC-expressing T cell medicine

The process of manufacturing a CD19-TAC expressing T cell drug comprises selecting CD4/CD 8T cells from a leukocyte apheresis product, activating the CD4/CD8 positive cells, transducing the cells with a lentiviral vector comprising a CD19-TAC construct (as described in example 5), expanding the transduced cells to a level sufficient for the proposed dosing regimen, and harvesting and cryopreserving the final product.

The patient's leukocyte individual material with associated unique object identifier (UPN) is received at the manufacturing site and provided with a unique sample number (ISN). CD4/CD8 cells were selected and cryopreserved until the incubation process step began.

The cryopreserved CD4/CD8 positive selected T cells were thawed at 37 ℃, resuspended in a suitable medium and inoculated into a culture bag containing an activating reagent, and the culture incubated overnight at 37 ℃ per 5% co 2.

The cells were transduced with CD19-TAC lentiviral vectors at the appropriate multiplicity of infection (MOI) and incubated overnight at 37℃per 5% CO 2. On the next few days, the cultures were supplemented with complete medium to maintain the desired cell concentration and eventually pooled in transfer bags, centrifuged, resuspended, and inoculated into larger culture bags at the target cell density.

For pharmaceutical dosage forms, the harvested cell suspension is resuspended in vehicle and stored frozen in a controlled-speed refrigerator and then transferred to LN2 storage.

The product is transported in a frozen state to a clinical site, thawed on site and administered intravenously.

Prior to clinical trials, engineering manufacturing runs were performed using white blood cell apheresis material from healthy donors, including all in-process and release tests. In addition to in-process and release testing, research supporting regulatory documentation has also been conducted on the final drug from these engineering runs. These studies included post-thaw stability, initiation of long-term stability, residue testing to ensure clearance of growth-promoting cytokines, and early evaluation of assays that indicate potential function/potency.

EXAMPLE 15 preclinical development of BCMA-specific T-cell antigen conjugate (TAC) therapy for the treatment of BCMA-positive malignancies

Figure 27 shows that TAC proliferates when it encounters an antigen on a cell but does not proliferate when the antigen is present on an artificial bead, but CAR proliferates regardless of whether the antigen is present on a bead or cell.

Fig. 28A-28B show that TAC engineered T cells expand in vivo and provide long term protection in a myeloma model, indicating persistence of the cells. FIGS. 28A-28B show that BCMA-TAC T cells reject multiple myeloma tumors in a KMS-11 xenograft model (BCMA ^pos) engineered with nanoLuc (KMS 11-nanoLuc). Following tumor implantation, mice were treated with BCMA TAC-T cells (with firefly luciferase). TAC-T cells expand significantly after administration. This is associated with tumor regression. The treated mice were resistant to tumor re-challenges, indicating long-term persistence of TAC-T cells.

The data suggests that TAC-T cells may destroy tumor cells by a mechanism that mimics the natural process of T cell activation. The TAC technology shows 1) strong efficacy in liquid tumors, 2) proliferation in vivo, 3) T cell persistence, protecting mice against re-challenges, and 4) cell expansion after T cell administration.

EXAMPLE 16 in vivo and in vitro Activity of hu-or muIg HER-TAC with MSCV or EF1 alpha promoter

CD4 and CD 8T cells were engineered with viruses expressing a variety of different TACs. One group of cells was engineered with a lentivirus that utilized the MSCV promoter to express TAC specific for HER-2 using a murine IgG signal peptide [ mu IgG TAC (MSCV) ]. Another group of cells was engineered with another lentivirus that uses the MSCV promoter to express TAC specific for HER-2 using a human IgG signal peptide [ huIgG TAC (MSCV) ]. The third group of cells was engineered with another lentivirus that uses the EF1 alpha promoter to express TAC [ mu IgG TAC (EF 1 alpha) ] specific for HER-2 using a murine IgG signal peptide. As a negative control, TAC lacking HER2 binding domain (delta binding domain TAC) was used. The engineered cells were then characterized in vitro for their re-surface expression and specific activity, and in vivo for activity in OVCAR3 HER2 positive solid tumor models.

Figure 30 shows T cell surface expression of the human or murine IgG leader peptide HER2 TAC receptor under the control of MCSV or EF1 alpha promoter. Surface expression is a key requirement for biological activity and this suggests that HER2 TAC receptor expression is not affected by the species source of IgG signal peptide.

Figure 31 demonstrates that T cells engineered with human or murine IgG leader peptide HER2 TAC construct under the control of MCSV or EF1 alpha promoter induced cytokine production when co-cultured with HER2 positive target cells (OVCAR 3) but not with HER2 negative cells (LOX IMVI). This suggests that HER2 TAC receptor engineered T cells are able to specifically bind to HER2 expressing target cells but are not reactive with antigen negative cells.

Figure 32 demonstrates the in vivo efficacy of the human or murine IgG leader peptide HER2 TAC construct under the control of MCSV or EF1 alpha promoter. Following divided dosing of engineered T cells, HER2 TAC engineered cells showed significant effects on tumor growth, including tumor regression, relative to negative control delta-conjugated TAC. This in vivo experiment demonstrates that all HER2 TAC engineered cells show significant activity against solid tumor models in vivo.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and to cover methods and structures within the scope of these claims and their equivalents.

Sequence listing

<110> University of Marst, michausta (MCMASTER UNIVERSITY)

Tetramera immune America limited (TRIUMVIRA IMMUNOLOGICS USA, INC.)

<120> T cell antigen conjugates with various construct optimizations

<130> 55247-704.601

<140>

<141>

<150> 62/839,235

<151> 2019-04-26

<150> 62/828,879

<151> 2019-04-03

<150> 62/826,853

<151> 2019-03-29

<150> 62/773,120

<151> 2018-11-29

<150> 62/703,037

<151> 2018-07-25

<150> 62/699,173

<151> 2018-07-17

<160> 77

<170> PatentIn version 3.5

<210> 1

<211> 1521

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 1

atgtcacggg gctccgacct gggcaaaaag ctgctggagg ccgctagggc cgggcaggac 60

gatgaagtga gaatcctgat ggccaacggg gctgacgtga atgctaagga tgagtacggc 120

ctgacccccc tgtatctggc tacagcacac ggccatctgg agatcgtgga agtcctgctg 180

aaaaacggag ccgacgtgaa tgcagtcgat gccattgggt tcactcctct gcacctggca 240

gcctttatcg gacatctgga gattgcagaa gtgctgctga agcacggcgc tgacgtgaac 300

gcacaggata agttcggaaa aaccgctttt gacatcagca ttggcaacgg aaatgaagac 360

ctggctgaaa tcctgcagaa actgaatgaa cagaaactga ttagcgaaga agacctgaac 420

cccgggggag gaggagggag cgggggagga ggcagcggcg ggggaggctc tggaggagga 480

gggagcggat ccatggacat ccagatgact cagaccacaa gctccctgtc tgcaagtctg 540

ggcgaccggg tgacaatctc ctgcagagcc tctcaggata ttaggaacta cctgaattgg 600

tatcagcaga aacctgatgg cacagtcaag ctgctgatct actataccag ccggctgcac 660

tcaggcgtgc caagcaaatt ctcaggaagc ggctccggga ctgactactc cctgaccatc 720

tctaacctgg agcaggaaga tattgctacc tatttctgcc agcagggcaa tacactgccc 780

tggacttttg ccggaggcac caaactggag atcaaggggg gaggcgggag tggaggcggg 840

ggatcaggag gaggaggcag cggaggagga gggtccgagg tccagctgca gcagagcgga 900

ccagaactgg tgaagcccgg agcaagtatg aaaatctcct gtaaggcctc aggatacagc 960

ttcaccggct atacaatgaa ctgggtgaaa cagtcccatg gcaagaacct ggaatggatg 1020

gggctgatta atccttacaa aggcgtcagc acctataatc agaagtttaa agacaaggcc 1080

acactgactg tggataagtc tagttcaacc gcttacatgg agctgctgtc cctgacatct 1140

gaagacagtg ccgtgtacta ttgtgctcgg tctggctact atggggacag tgattggtac 1200

ttcgatgtct ggggacaggg cactaccctg accgtgtttt ctactagtgg cggaggagga 1260

tcactcgaga gcggacaggt gctgctggaa tccaatatca aagtcctgcc cacttggtct 1320

acccccgtgc agcctatggc tctgattgtg ctgggaggag tcgcaggact gctgctgttt 1380

atcgggctgg gaattttctt ttgcgtgcgc tgccggcacc ggagaaggca ggccgagcgc 1440

atgagccaga tcaagcgact gctgagcgag aagaaaacct gtcagtgtcc ccatagattc 1500

cagaagacct gttcacccat t 1521

<210> 2

<211> 525

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 2

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala

20 25 30

Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly

35 40 45

Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu

50 55 60

Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn

65 70 75 80

Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His

85 90 95

Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val Leu Leu Lys

100 105 110

His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe

115 120 125

Asp Ile Ser Ile Gly Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln

130 135 140

Lys Leu Asn Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Pro Gly

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Gly Ser Met Asp Ile Gln Met Thr Gln Thr Thr Ser

180 185 190

Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala

195 200 205

Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp

210 215 220

Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly

225 230 235 240

Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu

245 250 255

Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln

260 265 270

Gln Gly Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

275 280 285

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu

305 310 315 320

Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly

325 330 335

Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly

340 345 350

Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser

355 360 365

Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys

370 375 380

Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp

385 390 395 400

Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp

405 410 415

Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Leu Thr Val Phe Ser

420 425 430

Thr Ser Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu

435 440 445

Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met

450 455 460

Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly

465 470 475 480

Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala

485 490 495

Glu Arg Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys

500 505 510

Gln Cys Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

515 520 525

<210> 3

<211> 1647

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 3

atggactttc aggtgcagat tttctctttt ctgctgattt ccgcaagcgt catcgagctc 60

gggggggggg ggtcaggatc catggacatc cagatgactc agaccacaag ctccctgagc 120

gcatccctgg gcgaccgagt gacaatctca tgcagagcca gccaggatat taggaactac 180

ctgaattggt atcagcagaa acctgacggc acagtcaagc tgctgatcta ctatacttcc 240

cggctgcact ctggcgtgcc aagtaaattc tctgggagtg gatcaggcac tgactactca 300

ctgaccatca gcaacctgga gcaggaagat attgctacct atttctgcca gcagggcaat 360

acactgccct ggacttttgc aggcgggacc aaactggaga tcaagggcgg cggcggaagt 420

ggaggaggag gctcaggcgg aggagggagc ggcggaggag gcagcgaggt ccagctgcag 480

cagagcggac cagaactggt gaagcctggc gcatccatga aaatctcttg taaggcctct 540

gggtacagtt tcaccggata tacaatgaac tgggtgaaac agtctcatgg caagaacctg 600

gaatggatgg gcctgattaa tccttacaaa ggcgtcagca cctataatca gaagtttaaa 660

gacaaggcca cactgactgt ggataagtct agttcaaccg cttacatgga gctgctgtca 720

ctgacaagcg aagactccgc cgtgtactat tgcgctagga gcggatacta tggcgactcc 780

gattggtact tcgatgtctg ggggcaggga actaccctga ccgtgtttag cactagtgga 840

ggaggaggct ctggaggagg agggagtgga ggcgggggat caggaggagg aggcagcgat 900

atcatgtcac ggggctccga cctgggcaaa aagctgctgg aggccgctag ggccgggcag 960

gacgatgaag tgagaatcct gatggccaac ggggctgacg tgaatgctaa ggatgagtac 1020

ggcctgaccc ccctgtatct ggctacagca cacggccatc tggagatcgt ggaagtcctg 1080

ctgaaaaacg gagccgacgt gaatgcagtc gatgccattg ggttcactcc tctgcacctg 1140

gcagccttta tcggacatct ggagattgca gaagtgctgc tgaagcacgg cgctgacgtg 1200

aacgcacagg ataagttcgg aaaaaccgct tttgacatca gcattggcaa cggaaatgaa 1260

gacctggctg aaatcctgca gaaactgaat gaacagaaac tgattagcga agaagacctg 1320

aacgtcgacg gaggaggagg gtctggagga gggggaagtg gcgggggagg cagcggggga 1380

ggcgggtctc tcgagagtgg ccaggtgctg ctggaaagca atatcaaggt cctgccaact 1440

tggtccaccc cagtgcagcc tatggctctg attgtgctgg gaggagtcgc aggactgctg 1500

ctgtttatcg gcctggggat tttcttttgc gtgcgctgcc ggcaccggag aaggcaggct 1560

gagcgcatgt ctcagattaa gcgactgctg agcgagaaga agacctgtca gtgcccccat 1620

agattccaga aaacctgttc acccatt 1647

<210> 4

<211> 547

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 4

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Glu Leu Gly Gly Gly Gly Ser Gly Ser Met Asp Ile Gln Met

20 25 30

Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr

35 40 45

Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

50 55 60

Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser

65 70 75 80

Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala

100 105 110

Thr Tyr Phe Cys Gln Asn Thr Leu Pro Trp Thr Phe Ala Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser

145 150 155 160

Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys

165 170 175

Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln

180 185 190

Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr Lys

195 200 205

Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

210 215 220

Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr

225 230 235 240

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly

245 250 255

Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Phe Ser Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Met Ser Arg Gly Ser

290 295 300

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

305 310 315 320

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp

325 330 335

Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu

340 345 350

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Val

355 360 365

Asp Ala Ile Gly Phe Thr Pro Leu His Leu Ala Ala Phe Ile Gly His

370 375 380

Leu Glu Ile Ala Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala

385 390 395 400

Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Gly Asn Gly

405 410 415

Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn Glu Gln Lys Leu

420 425 430

Ile Ser Glu Glu Asp Leu Asn Val Asp Gly Gly Gly Gly Ser Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Ser

450 455 460

Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser

465 470 475 480

Thr Pro Val Gln Pro Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly

485 490 495

Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg

500 505 510

His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln Ile Lys Arg Leu Leu

515 520 525

Ser Glu Lys Lys Thr Cys Gln Cys Pro His Arg Phe Gln Lys Thr Cys

530 535 540

Ser Pro Ile

545

<210> 5

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 5

atggatttcc aggtccagat tttctccttc ctgctgattt ccgcaagcgt catt 54

<210> 6

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 6

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile

<210> 7

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 7

atgtcacggg gctccgacct gggcaaaaag ctgctggagg ccgctagggc cgggcaggac 60

gatgaagtga gaatcctgat ggccaacggg gctgacgtga atgctaagga tgagtacggc 120

ctgacccccc tgtatctggc tacagcacac ggccatctgg agatcgtgga agtcctgctg 180

aaaaacggag ccgacgtgaa tgcagtcgat gccattgggt tcactcctct gcacctggca 240

gcctttatcg gacatctgga gattgcagaa gtgctgctga agcacggcgc tgacgtgaac 300

gcacaggata agttcggaaa aaccgctttt gacatcagca ttggcaacgg aaatgaagac 360

ctggctgaaa tcctgcagaa actgaat 387

<210> 8

<211> 129

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 8

Met Ser Arg Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg

1 5 10 15

Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp

20 25 30

Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala Thr

35 40 45

Ala His Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala

50 55 60

Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His Leu Ala

65 70 75 80

Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val Leu Leu Lys His Gly

85 90 95

Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile

100 105 110

Ser Ile Gly Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu

115 120 125

Asn

<210> 9

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 9

gaacagaaac tgattagcga agaagacctg 30

<210> 10

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 10

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 11

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 11

aaccccgggg gaggaggagg gagcggggga ggaggcagcg gcgggggagg ctctggagga 60

ggagggagcg gatcc 75

<210> 12

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 12

Asn Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

1 5 10 15

Gly Ser Gly Gly Gly Gly Ser Gly Ser

20 25

<210> 13

<211> 750

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 13

atggacatcc agatgactca gaccacaagc tccctgtctg caagtctggg cgaccgggtg 60

acaatctcct gcagagcctc tcaggatatt aggaactacc tgaattggta tcagcagaaa 120

cctgatggca cagtcaagct gctgatctac tataccagcc ggctgcactc aggcgtgcca 180

agcaaattct caggaagcgg ctccgggact gactactccc tgaccatctc taacctggag 240

caggaagata ttgctaccta tttctgccag cagggcaata cactgccctg gacttttgcc 300

ggaggcacca aactggagat caagggggga ggcgggagtg gaggcggggg atcaggagga 360

ggaggcagcg gaggaggagg gtccgaggtc cagctgcagc agagcggacc agaactggtg 420

aagcccggag caagtatgaa aatctcctgt aaggcctcag gatacagctt caccggctat 480

acaatgaact gggtgaaaca gtcccatggc aagaacctgg aatggatggg gctgattaat 540

ccttacaaag gcgtcagcac ctataatcag aagtttaaag acaaggccac actgactgtg 600

gataagtcta gttcaaccgc ttacatggag ctgctgtccc tgacatctga agacagtgcc 660

gtgtactatt gtgctcggtc tggctactat ggggacagtg attggtactt cgatgtctgg 720

ggacagggca ctaccctgac cgtgttttct 750

<210> 14

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 14

Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu

1 5 10 15

Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn

20 25 30

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu

35 40 45

Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu

65 70 75 80

Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

85 90 95

Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

130 135 140

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

145 150 155 160

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met

165 170 175

Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

180 185 190

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

195 200 205

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

225 230 235 240

Gly Gln Gly Thr Thr Leu Thr Val Phe Ser

245 250

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 15

actagtggcg gaggaggatc actcgag 27

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 16

Thr Ser Gly Gly Gly Gly Ser Leu Glu

1 5

<210> 17

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 17

agcggacagg tgctgctgga atccaatatc aaagtcctgc ccacttggtc tacccccgtg 60

cagcctatgg ctctgattgt gctgggagga gtcgcaggac tgctgctgtt tatcgggctg 120

ggaattttct tttgcgtgcg ctgccggcac cggagaaggc aggccgagcg catgagccag 180

atcaagcgac tgctgagcga gaagaaaacc tgtcagtgtc cccatagatt ccagaagacc 240

tgttcaccca tt 252

<210> 18

<211> 84

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 18

Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp

1 5 10 15

Ser Thr Pro Val Gln Pro Met Ala Leu Ile Val Leu Gly Gly Val Ala

20 25 30

Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys

35 40 45

Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln Ile Lys Arg Leu

50 55 60

Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His Arg Phe Gln Lys Thr

65 70 75 80

Cys Ser Pro Ile

<210> 19

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 19

agcggacagg tgctgctgga atccaatatc aaagtcctgc ccacttggtc tacccccgtg 60

cagcct 66

<210> 20

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 20

Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp

1 5 10 15

Ser Thr Pro Val Gln Pro

20

<210> 21

<211> 769

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 21

atggccgaca tcgtgctgac acagagcccc gccatcatgt ctgccagccc tggcgagaaa 60

gtgaccatga cctgtagcgc cagcagcagc gtgtcctaca tgaactggta tcagcagaag 120

tccggcacca gccccaagcg gtggatctac gacacaagca agctggcctc tggcgtgccc 180

gcccacttta gaggctctgg cagcggcaca agctacagcc tgaccatcag cggcatggaa 240

gccgaggatg ccgccaccta ctactgccag cagtggtcca gcaacccctt cacctttggc 300

tccggcacaa agctggaaat caaccgggcc gacaccgccc ctacaggcgg cggaggatct 360

ggcggaggcg gatctggggg cggaggaagt ggggggggag gatctatggc tcaggtgcag 420

ctgcagcagt ctggcgccga actggctaga cctggcgcct ccgtgaagat gagctgcaag 480

gccagcggct acaccttcac ccggtacacc atgcactggg tcaagcagag gcctggacag 540

ggcctggaat ggatcggcta catcaacccc agccggggct acaccaacta caaccagaag 600

ttcaaggaca aggccaccct gaccaccgac aagagcagca gcaccgccta catgcagctg 660

tcctccctga ccagcgagga cagcgccgtg tactactgcg cccggtacta cgacgaccac 720

tactccctgg actactgggg ccagggcacc acactgaccg tgtctagta 769

<210> 22

<211> 256

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 22

Met Ala Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser

1 5 10 15

Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser

20 25 30

Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp

35 40 45

Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro

85 90 95

Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Thr

100 105 110

Ala Pro Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Met Ala Gln Val Gln Leu Gln Gln Ser

130 135 140

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

145 150 155 160

Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

165 170 175

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

180 185 190

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

195 200 205

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

210 215 220

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

225 230 235 240

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

245 250 255

<210> 23

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 23

cagaccgtgg tgacccagga gcccagcctg accgtgagcc ccggcggcac cgtgaccctg 60

acctgcggca gcagcaccgg cgccgtgacc agcggctact accccaactg ggtgcagcag 120

aagcccggcc aggcccccag gggcctgatc ggcggcacca agttcctggc ccccggcacc 180

cccgccaggt tcagcggcag cctgctgggc ggcaaggccg ccctgaccct gagcggcgtg 240

cagcccgagg acgaggccga gtactactgc gccctgtggt acagcaacag gtgggtgttc 300

ggcggcggca ccaagctgac cgtgctgggc ggcggcggca gcggcggcgg cggcagcggc 360

ggcggcggca gcgaggtgca gctgctggag agcggcggcg gcctggtgca gcccggcggc 420

agcctgaagc tgagctgcgc cgccagcggc ttcaccttca acatctacgc catgaactgg 480

gtgaggcagg cccccggcaa gggcctggag tgggtggcca ggatcaggag caagtacaac 540

aactacgcca cctactacgc cgacagcgtg aagagcaggt tcaccatcag cagggacgac 600

agcaagaaca ccgcctacct gcagatgaac aacctgaaga ccgaggacac cgccgtgtac 660

tactgcgtga ggcacggcaa cttcggcaac agctacgtga gcttcttcgc ctactggggc 720

cagggcaccc tggtgaccgt gagcagc 747

<210> 24

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 24

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Tyr Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ile Tyr Ala Met Asn Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg

165 170 175

Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Ser

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

195 200 205

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

210 215 220

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Phe Phe Ala Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser

245

<210> 25

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 25

gacatccagc tgacccagag ccccgccatc atgagcgcca gccccggcga gaaggtgacc 60

atgacctgca gggccagcag cagcgtgagc tacatgaact ggtaccagca gaagagcggc 120

accagcccca agaggtggat ctacgacacc agcaaggtgg ccagcggcgt gccctacagg 180

ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcagcat ggaggccgag 240

gacgccgcca cctactactg ccagcagtgg agcagcaacc ccctgacctt cggcgccggc 300

accaagctgg agctgaaggg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 360

agcgacatca agctgcagca gagcggcgcc gagctggcca ggcccggcgc cagcgtgaag 420

atgagctgca agaccagcgg ctacaccttc accaggtaca ccatgcactg ggtgaagcag 480

aggcccggcc agggcctgga gtggatcggc tacatcaacc ccagcagggg ctacaccaac 540

tacaaccaga agttcaagga caaggccacc ctgaccaccg acaagagcag cagcaccgcc 600

tacatgcagc tgagcagcct gaccagcgag gacagcgccg tgtactactg cgccaggtac 660

tacgacgacc actactgcct ggactactgg ggccagggca ccaccctgac cgtgagcagc 720

<210> 26

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 26

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser

115 120 125

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

130 135 140

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

145 150 155 160

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

165 170 175

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

180 185 190

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

195 200 205

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

210 215 220

Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

225 230 235 240

<210> 27

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 27

gccgaagcag cagcaaagga ggccgcagcg aaggaagcag ctgcgaaggc c 51

<210> 28

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 28

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Ala

<210> 29

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 29

gccgaggcag ctgcaaagga agctgcggcg aaggaggccg cagcgaaaga agcagcggca 60

aaagaagcag ccgccaaagc c 81

<210> 30

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 30

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

20 25

<210> 31

<211> 576

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 31

atcgtagtgt tggcatttca aaaagcgtct agcatcgtct ataagaagga aggtgaacaa 60

gtcgagtttt ctttccccct tgcatttacg gtggaaaagc ttacgggtag cggcgagctg 120

tggtggcaag ctgaacgggc ttcaagctca aaatcttgga ttacttttga cttgaagaac 180

aaagaggtga gtgtcaaaag agttactcag gacccaaagc ttcaaatggg gaagaaactt 240

ccgctgcacc tgacgttgcc tcaggccctg cctcaatatg ccggctcagg caatctgacc 300

ctcgcgctgg aagctaagac cggaaaattg caccaggaag tcaatttggt tgtgatgcgc 360

gccactcagc tccaaaaaaa tctcacttgc gaggtatggg ggcctacgag cccaaaactt 420

atgctgtctt tgaagcttga aaacaaggaa gcgaaagttt ctaagcgcga gaaagcggta 480

tgggttttga atcctgaggc tggaatgtgg caatgcctcc tgagcgatag cgggcaggtg 540

ctgttggaga gcaacatcaa ggttttgcca gcagcc 576

<210> 32

<211> 192

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 32

Ile Val Val Leu Ala Phe Gln Lys Ala Ser Ser Ile Val Tyr Lys Lys

1 5 10 15

Glu Gly Glu Gln Val Glu Phe Ser Phe Pro Leu Ala Phe Thr Val Glu

20 25 30

Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser

35 40 45

Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu Val Ser

50 55 60

Val Lys Arg Val Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys Leu

65 70 75 80

Pro Leu His Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr Ala Gly Ser

85 90 95

Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys Leu His Gln

100 105 110

Glu Val Asn Leu Val Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu

115 120 125

Thr Cys Glu Val Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu

130 135 140

Lys Leu Glu Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val

145 150 155 160

Trp Val Leu Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp

165 170 175

Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Ala Ala

180 185 190

<210> 33

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 33

atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60

agagacatcg tgctgaccca gagccccccc agcctggcca tgtctctggg caagagagcc 120

accatcagct gccgggccag cgagagcgtg accatcctgg gcagccacct gatccactgg 180

tatcagcaga agcccggcca gccccccacc ctgctgatcc agctcgccag caatgtgcag 240

accggcgtgc ccgccagatt cagcggcagc ggcagcagaa ccgacttcac cctgaccatc 300

gaccccgtgg aagaggacga cgtggccgtg tactactgcc tgcagagccg gaccatcccc 360

cggacctttg gcggaggcac caaactggaa atcaagggca gcaccagcgg ctccggcaag 420

cctggctctg gcgagggcag cacaaaggga cagattcagc tggtgcagag cggccctgag 480

ctgaagaaac ccggcgagac agtgaagatc agctgcaagg cctccggcta caccttcacc 540

gactacagca tcaactgggt gaaaagagcc cctggcaagg gcctgaagtg gatgggctgg 600

atcaacaccg agacaagaga gcccgcctac gcctacgact tccggggcag attcgccttc 660

agcctggaaa ccagcgccag caccgcctac ctgcagatca acaacctgaa gtacgaggac 720

accgccacct acttttgcgc cctggactac agctacgcca tggactactg gggccagggc 780

accagcgtga ccgtgtccag c 801

<210> 34

<211> 267

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 34

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu

20 25 30

Ala Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu

35 40 45

Ser Val Thr Ile Leu Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln

65 70 75 80

Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe

85 90 95

Thr Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr

100 105 110

Cys Leu Gln Ser Arg Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly

130 135 140

Glu Gly Ser Thr Lys Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu

145 150 155 160

Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly

165 170 175

Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly

180 185 190

Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro

195 200 205

Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr

210 215 220

Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp

225 230 235 240

Thr Ala Thr Tyr Phe Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

260 265

<210> 35

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 35

gatatccaga tgactcagac gacctcatca ttgtccgcca gtttggggga cagggttaca 60

atatcctgcc gggcgagcca agacatcagt aaatatctta attggtacca gcagaaacca 120

gatggtacag taaaacttct tatctaccac acctctcggc tccactctgg ggttccctct 180

aggttcagtg gtagtgggtc aggcaccgac tacagcctta cgataagcaa cttggaacag 240

gaggatatcg caacttactt ctgccaacag ggaaataccc tgccttacac gttcggtgga 300

ggcactaaac tggagatcac tgggtcaacc tctggtagcg gtaagcctgg ctccggcgaa 360

ggctccacaa agggtgaggt gaaactccaa gagtcaggtc ccggtttggt agccccctca 420

caaagtttgt cagttacttg taccgtaagc ggcgtttccc tgcccgatta cggtgtgagc 480

tggataaggc agccaccgag aaaaggtctt gaatggctgg gagtgatctg ggggtctgag 540

acaacgtatt acaactcagc tcttaagagc aggcttacga tcattaaaga taacagcaaa 600

tctcaagtgt tcctcaaaat gaatagcctt caaactgatg atactgccat ctattattgt 660

gctaagcatt attactatgg cggcagttac gcaatggatt attgggggca aggtacctca 720

gtcactgtaa gcagc 735

<210> 36

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 36

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 37

<211> 237

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 37

ctcgagctga ggcccgaggc ttctagacct gctgccggcg gagccgtgca caccagaggc 60

ctggacttcg ccagcgacat ctacatctgg gcccctctgg ccggcacctg tggcgtgctg 120

ctgctgagcc tggtcatcac cctgtactgc aaccaccgga accggcggag agtgtgcaag 180

tgccccagac ccgtggtcaa gagcggcgac aagcccagcc tgagcgccag atacgtg 237

<210> 38

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 38

Leu Glu Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val

1 5 10 15

His Thr Arg Gly Leu Asp Phe Ala Ser Asp Ile Tyr Ile Trp Ala Pro

20 25 30

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

35 40 45

Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro

50 55 60

Val Val Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

65 70 75

<210> 39

<211> 234

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 39

ctcgagctga ggcccgaggc ttctagacct gctgccggcg gagccgtgca caccagaggc 60

ctggacttcg ccagcgacat ctacatctgg gcccctctgg ccggcacctg tggcgtgctg 120

ctgctgagcc tggtcatcac cctgtacctg tgctgcagac ggcggagagt gtgcaagtgc 180

cccagacccg tggtcaagag cggcgacaag cccagcctga gcgccagata cgtg 234

<210> 40

<211> 78

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 40

Leu Glu Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val

1 5 10 15

His Thr Arg Gly Leu Asp Phe Ala Ser Asp Ile Tyr Ile Trp Ala Pro

20 25 30

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

35 40 45

Tyr Leu Cys Cys Arg Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val

50 55 60

Val Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

65 70 75

<210> 41

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 41

ctcgagaaga agtccaccct gaagaaacgg gtgtcccggc tgcccagacc cgagacacag 60

aagggccccc tgagcagccc tatcaccctg ggactgctgg tggccggcgt gctggtgctg 120

ctggtgtctc tgggagtggc catccacctg tgctgccggc ggagaagggc ctgcaagtgc 180

cccagactgc ggttcatgaa gcagttctac aag 213

<210> 42

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 42

Leu Glu Lys Lys Ser Thr Leu Lys Lys Arg Val Ser Arg Leu Pro Arg

1 5 10 15

Pro Glu Thr Gln Lys Gly Pro Leu Ser Ser Pro Ile Thr Leu Gly Leu

20 25 30

Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly Val Ala Ile

35 40 45

His Leu Cys Cys Arg Arg Arg Arg Ala Cys Lys Cys Pro Arg Leu Arg

50 55 60

Phe Met Lys Gln Phe Tyr Lys

65 70

<210> 43

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 43

atggatatcc agatgaccca gtccccgagc tccctgtccg cctctgtggg cgatagggtc 60

accatcacct gccgtgccag tcaggacatc cgtaattatc tgaactggta tcaacagaaa 120

ccaggaaaag ctccgaaact actgatttac tatacctccc gcctggagtc tggagtccct 180

tctcgcttct ctggttctgg ttctgggacg gattacactc tgaccatcag cagtctgcaa 240

ccggaagact tcgcaactta ttactgtcag caaggtaata ctctgccgtg gacgttcgga 300

cagggcacca aggtggagat caaaggcggc ggcggaagtg gaggaggagg ctcaggcgga 360

ggagggagcg aggttcagct ggtggagtct ggcggtggcc tggtgcagcc agggggctca 420

ctccgtttgt cctgtgcagc ttctggctac tcctttaccg gctacactat gaactgggtg 480

cgtcaggccc caggtaaggg cctggaatgg gttgcactga ttaatcctta taaaggtgtt 540

agtacctaca accagaagtt caaggaccgt ttcactataa gcgtagataa atccaaaaac 600

acagcctacc tgcaaatgaa cagcctgcgt gctgaggaca ctgccgtcta ttattgtgct 660

agaagcggat actacggcga tagtgactgg tattttgacg tgtggggtca aggaaccctg 720

gtcaccgtct cctcg 735

<210> 44

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 44

Met Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn

20 25 30

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro

165 170 175

Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr

180 185 190

Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr

210 215 220

Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser

245

<210> 45

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 45

atggatatcc agatgaccca gtccccgagc tccctgtccg cctctgtggg cgatagggtc 60

accatcacct gccgtgccag tcaggacatc cgtaattatc tgaactggta tcaacagaaa 120

ccaggaaaag ctccgaaact actgatttac tatacctccc gcctggagtc tggagtccct 180

tctcgcttct ctggttctgg ttctgggacg gattacactc tgaccatcag cagtctgcaa 240

ccggaagact tcgcaactta ttactgtcag caaggtaata ctctgccgtg gacgttcgga 300

cagggcacca aggtggagat caaaggcggc ggcggaagtg gaggaggagg ctcaggcgga 360

ggagggagcg aggttcagct ggtggagtct ggcggtggcc tggtgcagcc agggggctca 420

ctccgtttgt cctgtgcagc ttctggctac tcctttaccg gctacactat gaactgggtg 480

cgtcaggccc caggtaaggg cctggaatgg gttgcactga ttaatcctac caaaggtgtt 540

agtacctaca accagaagtt caaggaccgt ttcactataa gcgtagataa atccaaaaac 600

acagcctacc tgcaaatgaa cagcctgcgt gctgaggaca ctgccgtcta ttattgtgct 660

agaagcggat actacggcga tagtgactgg tattttgacg tgtggggtca aggaaccctg 720

gtcaccgtct cctcg 735

<210> 46

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 46

Met Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn

20 25 30

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro

165 170 175

Thr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr

180 185 190

Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr

210 215 220

Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser

245

<210> 47

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 47

atggagaccc ccgcccagct gctgttcctg ctgctgctgt ggctgcccga caccaccggc 60

<210> 48

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 48

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly

20

<210> 49

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 49

atggctttgc ctgtcacggc tcttctgctc cctctggccc tgcttctgca cgcggcgcga 60

ccc 63

<210> 50

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 50

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 51

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 51

gaggtgcagc tggtggagtc tggaggaggc ctggtgcagc ctggcggctc cctgaggctg 60

tcttgcgcag caagcggctt caacatctac tatagctaca tgcactgggt gcgccaggcc 120

cctggcaagg gcctggagtg ggtggcctcc atctctccat actatggcta cacctcctat 180

gccgactctg tgaagggccg gtttacaatc agcgccgata cctccaagaa cacagcctat 240

ctgcagatga attccctgag ggcagaggac accgccgtgt actattgcgc cagacacggc 300

tacgccctgg attattgggg ccagggcacc ctggtgacag tgagctccgg cagcacatcc 360

ggatctggca agccaggctc tggagaggga agcaccaagg gcgacatcca gatgacacag 420

tccccatcta gcctgagcgc ctccgtgggc gatagggtga ccatcacatg tcgcgcctct 480

cagagcgtgt cctctgccgt ggcatggtac cagcagaagc ccggcaaggc ccctaagctg 540

ctgatctaca gcgccagctc cctgtattcc ggcgtgcctt ctcggttctc cggctctaga 600

agcggcaccg actttaccct gacaatctct agcctgcagc ccgaggattt cgccacatac 660

tattgtcagc agagcgtgtg ggtgggctac tccctgatca cctttggcca gggcacaaag 720

gtggagatca ag 732

<210> 52

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 52

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 Asn Ile Tyr Tyr Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 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 His Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly

115 120 125

Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

145 150 155 160

Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Ser Val Trp Val Gly Tyr Ser Leu Ile Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys

<210> 53

<211> 732

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 53

gacatccaga tgacacagtc cccaagctcc ctgtccgcct ctgtgggcga tagggtgacc 60

atcacatgca gggcaagcca gtccgtgtct agcgccgtgg catggtacca gcagaagccc 120

ggcaaggccc ctaagctgct gatctacagc gcctcctctc tgtattccgg cgtgccatct 180

cggttctctg gcagcagatc cggcaccgac tttaccctga caatcagctc cctgcagccc 240

gaggatttcg ccacatacta ttgccagcag agcgtgtggg tgggctactc cctgatcacc 300

tttggccagg gcacaaaggt ggagatcaag ggatctacca gcggatccgg caagcctggc 360

agcggagagg gatccacaaa gggagaggtg cagctggtgg agtctggagg aggcctggtg 420

cagcctggcg gctctctgag gctgagctgt gcagcatccg gcttcaacat ctactatagc 480

tacatgcact gggtgcgcca ggcccccggc aagggcctgg agtgggtggc ctctatcagc 540

ccttactatg gctacacctc ttatgccgac agcgtgaagg gccggtttac aatctccgcc 600

gatacctcta agaacacagc ctatctgcag atgaattccc tgagggcaga ggacaccgcc 660

gtgtactatt gtgccagaca cggctacgcc ctggattatt ggggccaggg caccctggtg 720

acagtgtcta gc 732

<210> 54

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 54

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Trp Val Gly Tyr

85 90 95

Ser Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser

100 105 110

Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly

115 120 125

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

130 135 140

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Tyr Tyr Ser

145 150 155 160

Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

165 170 175

Ala Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val

180 185 190

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg His Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ser

<210> 55

<211> 1905

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 55

atggccctgc cagtgaccgc cctgctgctg ccactggccc tgctgctgca cgccgccaga 60

cccgaggtgc agctggtgga gtctggagga ggcctggtgc agcctggcgg ctccctgagg 120

ctgtcttgcg cagcaagcgg cttcaacatc tactatagct acatgcactg ggtgcgccag 180

gcccctggca agggcctgga gtgggtggcc tccatctctc catactatgg ctacacctcc 240

tatgccgact ctgtgaaggg ccggtttaca atcagcgccg atacctccaa gaacacagcc 300

tatctgcaga tgaattccct gagggcagag gacaccgccg tgtactattg cgccagacac 360

ggctacgccc tggattattg gggccagggc accctggtga cagtgagctc cggcagcaca 420

tccggatctg gcaagccagg ctctggagag ggaagcacca agggcgacat ccagatgaca 480

cagtccccat ctagcctgag cgcctccgtg ggcgataggg tgaccatcac atgtcgcgcc 540

tctcagagcg tgtcctctgc cgtggcatgg taccagcaga agcccggcaa ggcccctaag 600

ctgctgatct acagcgccag ctccctgtat tccggcgtgc cttctcggtt ctccggctct 660

agaagcggca ccgactttac cctgacaatc tctagcctgc agcccgagga tttcgccaca 720

tactattgtc agcagagcgt gtgggtgggc tactccctga tcacctttgg ccagggcaca 780

aaggtggaga tcaaggagca gaagctgatc agcgaggagg acctgaatcc cggggccgaa 840

gcagcagcaa aggaggccgc agcgaaggaa gcagctgcga aggccggatc catggatatc 900

cagatgaccc agtccccgag ctccctgtcc gcctctgtgg gcgatagggt caccatcacc 960

tgccgtgcca gtcaggacat ccgtaattat ctgaactggt atcaacagaa accaggaaaa 1020

gctccgaaac tactgattta ctatacctcc cgcctggagt ctggagtccc ttctcgcttc 1080

tctggttctg gttctgggac ggattacact ctgaccatca gcagtctgca accggaagac 1140

ttcgcaactt attactgtca gcaaggtaat actctgccgt ggacgttcgg acagggcacc 1200

aaggtggaga tcaaaggcgg cggcggaagt ggaggaggag gctcaggcgg aggagggagc 1260

gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 1320

tcctgtgcag cttctggcta ctcctttacc ggctacacta tgaactgggt gcgtcaggcc 1380

ccaggtaagg gcctggaatg ggttgcactg attaatcctt ataaaggtgt tagtacctac 1440

aaccagaagt tcaaggaccg tttcactata agcgtagata aatccaaaaa cacagcctac 1500

ctgcaaatga acagcctgcg tgctgaggac actgccgtct attattgtgc tagaagcgga 1560

tactacggcg atagtgactg gtattttgac gtgtggggtc aaggaaccct ggtcaccgtc 1620

tcctcgacta gtggcggagg aggatcactc gagagcggac aggtgctgct ggaatccaat 1680

atcaaagtcc tgcccacttg gtctaccccc gtgcagccta tggctctgat tgtgctggga 1740

ggagtcgcag gactgctgct gtttatcggg ctgggaattt tcttttgcgt gcgctgccgg 1800

caccggagaa ggcaggccga gcgcatgagc cagatcaagc gactgctgag cgagaagaaa 1860

acctgtcagt gtccccatag attccagaag acctgttcac ccatt 1905

<210> 56

<211> 635

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 56

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Asn Ile Tyr Tyr Ser Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

85 90 95

Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Ala Leu Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly

130 135 140

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser

195 200 205

Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Ser Val Trp Val Gly Tyr Ser Leu Ile Thr Phe

245 250 255

Gly Gln Gly Thr Lys Val Glu Ile Lys Glu Gln Lys Leu Ile Ser Glu

260 265 270

Glu Asp Leu Asn Pro Gly Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala

275 280 285

Lys Glu Ala Ala Ala Lys Ala Gly Ser Met Asp Ile Gln Met Thr Gln

290 295 300

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

305 310 315 320

Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln

325 330 335

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu

340 345 350

Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

355 360 365

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

370 375 380

Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr

385 390 395 400

Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

420 425 430

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser

435 440 445

Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

450 455 460

Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr

465 470 475 480

Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys

485 490 495

Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

500 505 510

Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr

515 520 525

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Ser

530 535 540

Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu Ser Asn

545 550 555 560

Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu

565 570 575

Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly

580 585 590

Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg

595 600 605

Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys

610 615 620

Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

625 630 635

<210> 57

<211> 1905

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 57

atggccctgc cagtgaccgc cctgctgctg ccactggccc tgctgctgca cgccgcccgg 60

cctgacatcc agatgacaca gtccccaagc tccctgtccg cctctgtggg cgatagggtg 120

accatcacat gcagggcaag ccagtccgtg tctagcgccg tggcatggta ccagcagaag 180

cccggcaagg cccctaagct gctgatctac agcgcctcct ctctgtattc cggcgtgcca 240

tctcggttct ctggcagcag atccggcacc gactttaccc tgacaatcag ctccctgcag 300

cccgaggatt tcgccacata ctattgccag cagagcgtgt gggtgggcta ctccctgatc 360

acctttggcc agggcacaaa ggtggagatc aagggatcta ccagcggatc cggcaagcct 420

ggcagcggag agggatccac aaagggagag gtgcagctgg tggagtctgg aggaggcctg 480

gtgcagcctg gcggctctct gaggctgagc tgtgcagcat ccggcttcaa catctactat 540

agctacatgc actgggtgcg ccaggccccc ggcaagggcc tggagtgggt ggcctctatc 600

agcccttact atggctacac ctcttatgcc gacagcgtga agggccggtt tacaatctcc 660

gccgatacct ctaagaacac agcctatctg cagatgaatt ccctgagggc agaggacacc 720

gccgtgtact attgtgccag acacggctac gccctggatt attggggcca gggcaccctg 780

gtgacagtgt ctagcgagca gaagctgatc agcgaggagg acctgaatcc cggggccgaa 840

gcagcagcaa aggaggccgc agcgaaggaa gcagctgcga aggccggatc catggatatc 900

cagatgaccc agtccccgag ctccctgtcc gcctctgtgg gcgatagggt caccatcacc 960

tgccgtgcca gtcaggacat ccgtaattat ctgaactggt atcaacagaa accaggaaaa 1020

gctccgaaac tactgattta ctatacctcc cgcctggagt ctggagtccc ttctcgcttc 1080

tctggttctg gttctgggac ggattacact ctgaccatca gcagtctgca accggaagac 1140

ttcgcaactt attactgtca gcaaggtaat actctgccgt ggacgttcgg acagggcacc 1200

aaggtggaga tcaaaggcgg cggcggaagt ggaggaggag gctcaggcgg aggagggagc 1260

gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 1320

tcctgtgcag cttctggcta ctcctttacc ggctacacta tgaactgggt gcgtcaggcc 1380

ccaggtaagg gcctggaatg ggttgcactg attaatcctt ataaaggtgt tagtacctac 1440

aaccagaagt tcaaggaccg tttcactata agcgtagata aatccaaaaa cacagcctac 1500

ctgcaaatga acagcctgcg tgctgaggac actgccgtct attattgtgc tagaagcgga 1560

tactacggcg atagtgactg gtattttgac gtgtggggtc aaggaaccct ggtcaccgtc 1620

tcctcgacta gtggcggagg aggatcactc gagagcggac aggtgctgct ggaatccaat 1680

atcaaagtcc tgcccacttg gtctaccccc gtgcagccta tggctctgat tgtgctggga 1740

ggagtcgcag gactgctgct gtttatcggg ctgggaattt tcttttgcgt gcgctgccgg 1800

caccggagaa ggcaggccga gcgcatgagc cagatcaagc gactgctgag cgagaagaaa 1860

acctgtcagt gtccccatag attccagaag acctgttcac ccatt 1905

<210> 58

<211> 635

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 58

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln

35 40 45

Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

100 105 110

Val Trp Val Gly Tyr Ser Leu Ile Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu

130 135 140

Gly Ser Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

165 170 175

Asn Ile Tyr Tyr Ser Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys

180 185 190

Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser

195 200 205

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

210 215 220

Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Ala Leu Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Leu Val Thr Val Ser Ser Glu Gln Lys Leu Ile Ser Glu

260 265 270

Glu Asp Leu Asn Pro Gly Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala

275 280 285

Lys Glu Ala Ala Ala Lys Ala Gly Ser Met Asp Ile Gln Met Thr Gln

290 295 300

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

305 310 315 320

Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln

325 330 335

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu

340 345 350

Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

355 360 365

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

370 375 380

Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr

385 390 395 400

Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

420 425 430

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser

435 440 445

Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

450 455 460

Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr

465 470 475 480

Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys

485 490 495

Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

500 505 510

Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr

515 520 525

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Ser

530 535 540

Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu Ser Asn

545 550 555 560

Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu

565 570 575

Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly

580 585 590

Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg

595 600 605

Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys

610 615 620

Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

625 630 635

<210> 59

<211> 1914

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 59

atggccctgc cagtgaccgc cctgctgctg ccactggccc tgctgctgca cgccgccaga 60

cccgaggtgc agctggtgga gtctggagga ggcctggtgc agcctggcgg ctccctgagg 120

ctgtcttgcg cagcaagcgg cttcaacatc tactatagct acatgcactg ggtgcgccag 180

gcccctggca agggcctgga gtgggtggcc tccatctctc catactatgg ctacacctcc 240

tatgccgact ctgtgaaggg ccggtttaca atcagcgccg atacctccaa gaacacagcc 300

tatctgcaga tgaattccct gagggcagag gacaccgccg tgtactattg cgccagacac 360

ggctacgccc tggattattg gggccagggc accctggtga cagtgagctc cggcagcaca 420

tccggatctg gcaagccagg ctctggagag ggaagcacca agggcgacat ccagatgaca 480

cagtccccat ctagcctgag cgcctccgtg ggcgataggg tgaccatcac atgtcgcgcc 540

tctcagagcg tgtcctctgc cgtggcatgg taccagcaga agcccggcaa ggcccctaag 600

ctgctgatct acagcgccag ctccctgtat tccggcgtgc cttctcggtt ctccggctct 660

agaagcggca ccgactttac cctgacaatc tctagcctgc agcccgagga tttcgccaca 720

tactattgtc agcagagcgt gtgggtgggc tactccctga tcacctttgg ccagggcaca 780

aaggtggaga tcaaggagca gaagctgatc agcgaggagg acctgaatcc cgggggagga 840

ggagggagcg ggggaggagg cagcggcggg ggaggctctg gaggaggagg gagcggatcc 900

atggatatcc agatgaccca gtccccgagc tccctgtccg cctctgtggg cgatagggtc 960

accatcacct gccgtgccag tcaggacatc cgtaattatc tgaactggta tcaacagaaa 1020

ccaggaaaag ctccgaaact actgatttac tatacctccc gcctggagtc tggagtccct 1080

tctcgcttct ctggttctgg ttctgggacg gattacactc tgaccatcag cagtctgcaa 1140

ccggaagact tcgcaactta ttactgtcag caaggtaata ctctgccgtg gacgttcgga 1200

cagggcacca aggtggagat caaaggcggc ggcggaagtg gaggaggagg ctcaggcgga 1260

ggagggagcg aggttcagct ggtggagtct ggcggtggcc tggtgcagcc agggggctca 1320

ctccgtttgt cctgtgcagc ttctggctac tcctttaccg gctacactat gaactgggtg 1380

cgtcaggccc caggtaaggg cctggaatgg gttgcactga ttaatcctta taaaggtgtt 1440

agtacctaca accagaagtt caaggaccgt ttcactataa gcgtagataa atccaaaaac 1500

acagcctacc tgcaaatgaa cagcctgcgt gctgaggaca ctgccgtcta ttattgtgct 1560

agaagcggat actacggcga tagtgactgg tattttgacg tgtggggtca aggaaccctg 1620

gtcaccgtct cctcgactag tggcggagga ggatcactcg agagcggaca ggtgctgctg 1680

gaatccaata tcaaagtcct gcccacttgg tctacccccg tgcagcctat ggctctgatt 1740

gtgctgggag gagtcgcagg actgctgctg tttatcgggc tgggaatttt cttttgcgtg 1800

cgctgccggc accggagaag gcaggccgag cgcatgagcc agatcaagcg actgctgagc 1860

gagaagaaaa cctgtcagtg tccccataga ttccagaaga cctgttcacc catt 1914

<210> 60

<211> 638

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 60

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Asn Ile Tyr Tyr Ser Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

85 90 95

Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Ala Leu Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly

130 135 140

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser

195 200 205

Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Ser Val Trp Val Gly Tyr Ser Leu Ile Thr Phe

245 250 255

Gly Gln Gly Thr Lys Val Glu Ile Lys Glu Gln Lys Leu Ile Ser Glu

260 265 270

Glu Asp Leu Asn Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Met Asp Ile Gln

290 295 300

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

305 310 315 320

Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp

325 330 335

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr

340 345 350

Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

355 360 365

Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

370 375 380

Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly

385 390 395 400

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

420 425 430

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

435 440 445

Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro

450 455 460

Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val

465 470 475 480

Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp

485 490 495

Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

500 505 510

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser

515 520 525

Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

530 535 540

Ser Thr Ser Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu

545 550 555 560

Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro

565 570 575

Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile

580 585 590

Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln

595 600 605

Ala Glu Arg Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr

610 615 620

Cys Gln Cys Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

625 630 635

<210> 61

<211> 1914

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 61

atggccctgc cagtgaccgc cctgctgctg ccactggccc tgctgctgca cgccgcccgg 60

cctgacatcc agatgacaca gtccccaagc tccctgtccg cctctgtggg cgatagggtg 120

accatcacat gcagggcaag ccagtccgtg tctagcgccg tggcatggta ccagcagaag 180

cccggcaagg cccctaagct gctgatctac agcgcctcct ctctgtattc cggcgtgcca 240

tctcggttct ctggcagcag atccggcacc gactttaccc tgacaatcag ctccctgcag 300

cccgaggatt tcgccacata ctattgccag cagagcgtgt gggtgggcta ctccctgatc 360

acctttggcc agggcacaaa ggtggagatc aagggatcta ccagcggatc cggcaagcct 420

ggcagcggag agggatccac aaagggagag gtgcagctgg tggagtctgg aggaggcctg 480

gtgcagcctg gcggctctct gaggctgagc tgtgcagcat ccggcttcaa catctactat 540

agctacatgc actgggtgcg ccaggccccc ggcaagggcc tggagtgggt ggcctctatc 600

agcccttact atggctacac ctcttatgcc gacagcgtga agggccggtt tacaatctcc 660

gccgatacct ctaagaacac agcctatctg cagatgaatt ccctgagggc agaggacacc 720

gccgtgtact attgtgccag acacggctac gccctggatt attggggcca gggcaccctg 780

gtgacagtgt ctagcgagca gaagctgatc agcgaggagg acctgaatcc cgggggagga 840

ggagggagcg ggggaggagg cagcggcggg ggaggctctg gaggaggagg gagcggatcc 900

atggatatcc agatgaccca gtccccgagc tccctgtccg cctctgtggg cgatagggtc 960

accatcacct gccgtgccag tcaggacatc cgtaattatc tgaactggta tcaacagaaa 1020

ccaggaaaag ctccgaaact actgatttac tatacctccc gcctggagtc tggagtccct 1080

tctcgcttct ctggttctgg ttctgggacg gattacactc tgaccatcag cagtctgcaa 1140

ccggaagact tcgcaactta ttactgtcag caaggtaata ctctgccgtg gacgttcgga 1200

cagggcacca aggtggagat caaaggcggc ggcggaagtg gaggaggagg ctcaggcgga 1260

ggagggagcg aggttcagct ggtggagtct ggcggtggcc tggtgcagcc agggggctca 1320

ctccgtttgt cctgtgcagc ttctggctac tcctttaccg gctacactat gaactgggtg 1380

cgtcaggccc caggtaaggg cctggaatgg gttgcactga ttaatcctta taaaggtgtt 1440

agtacctaca accagaagtt caaggaccgt ttcactataa gcgtagataa atccaaaaac 1500

acagcctacc tgcaaatgaa cagcctgcgt gctgaggaca ctgccgtcta ttattgtgct 1560

agaagcggat actacggcga tagtgactgg tattttgacg tgtggggtca aggaaccctg 1620

gtcaccgtct cctcgactag tggcggagga ggatcactcg agagcggaca ggtgctgctg 1680

gaatccaata tcaaagtcct gcccacttgg tctacccccg tgcagcctat ggctctgatt 1740

gtgctgggag gagtcgcagg actgctgctg tttatcgggc tgggaatttt cttttgcgtg 1800

cgctgccggc accggagaag gcaggccgag cgcatgagcc agatcaagcg actgctgagc 1860

gagaagaaaa cctgtcagtg tccccataga ttccagaaga cctgttcacc catt 1914

<210> 62

<211> 638

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 62

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln

35 40 45

Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

100 105 110

Val Trp Val Gly Tyr Ser Leu Ile Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu

130 135 140

Gly Ser Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

165 170 175

Asn Ile Tyr Tyr Ser Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys

180 185 190

Gly Leu Glu Trp Val Ala Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser

195 200 205

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

210 215 220

Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Ala Leu Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Leu Val Thr Val Ser Ser Glu Gln Lys Leu Ile Ser Glu

260 265 270

Glu Asp Leu Asn Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Met Asp Ile Gln

290 295 300

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

305 310 315 320

Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp

325 330 335

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr

340 345 350

Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

355 360 365

Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

370 375 380

Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly

385 390 395 400

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

420 425 430

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

435 440 445

Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro

450 455 460

Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val

465 470 475 480

Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp

485 490 495

Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

500 505 510

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser

515 520 525

Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

530 535 540

Ser Thr Ser Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu

545 550 555 560

Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro

565 570 575

Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile

580 585 590

Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln

595 600 605

Ala Glu Arg Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr

610 615 620

Cys Gln Cys Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

625 630 635

<210> 63

<211> 1917

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 63

atggctttgc ctgtcacggc tcttctgctc cctctggccc tgcttctgca cgcggcgcga 60

cccgatatcc agatgactca gacgacctca tcattgtccg ccagtttggg ggacagggtt 120

acaatatcct gccgggcgag ccaagacatc agtaaatatc ttaattggta ccagcagaaa 180

ccagatggta cagtaaaact tcttatctac cacacctctc ggctccactc tggggttccc 240

tctaggttca gtggtagtgg gtcaggcacc gactacagcc ttacgataag caacttggaa 300

caggaggata tcgcaactta cttctgccaa cagggaaata ccctgcctta cacgttcggt 360

ggaggcacta aactggagat cactgggtca acctctggta gcggtaagcc tggctccggc 420

gaaggctcca caaagggtga ggtgaaactc caagagtcag gtcccggttt ggtagccccc 480

tcacaaagtt tgtcagttac ttgtaccgta agcggcgttt ccctgcccga ttacggtgtg 540

agctggataa ggcagccacc gagaaaaggt cttgaatggc tgggagtgat ctgggggtct 600

gagacaacgt attacaactc agctcttaag agcaggctta cgatcattaa agataacagc 660

aaatctcaag tgttcctcaa aatgaatagc cttcaaactg atgatactgc catctattat 720

tgtgctaagc attattacta tggcggcagt tacgcaatgg attattgggg gcaaggtacc 780

tcagtcactg taagcagcga acagaagctc atttctgaag aagacctcaa ccccggaggg 840

ggagggggaa gtgggggagg gggtagtggt ggcggaggat caggcggggg gggatcagga 900

tccatggata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 960

gtcaccatca cctgccgtgc cagtcaggac atccgtaatt atctgaactg gtatcaacag 1020

aaaccaggaa aagctccgaa actactgatt tactatacct cccgcctgga gtctggagtc 1080

ccttctcgct tctctggttc tggttctggg acggattaca ctctgaccat cagcagtctg 1140

caaccggaag acttcgcaac ttattactgt cagcaaggta atactctgcc gtggacgttc 1200

ggacagggca ccaaggtgga gatcaaaggc ggcggcggaa gtggaggagg aggctcaggc 1260

ggaggaggga gcgaggttca gctggtggag tctggcggtg gcctggtgca gccagggggc 1320

tcactccgtt tgtcctgtgc agcttctggc tactccttta ccggctacac tatgaactgg 1380

gtgcgtcagg ccccaggtaa gggcctggaa tgggttgcac tgattaatcc taccaaaggt 1440

gttagtacct acaaccagaa gttcaaggac cgtttcacta taagcgtaga taaatccaaa 1500

aacacagcct acctgcaaat gaacagcctg cgtgctgagg acactgccgt ctattattgt 1560

gctagaagcg gatactacgg cgatagtgac tggtattttg acgtgtgggg tcaaggaacc 1620

ctggtcaccg tctcctcgac tagtggcgga ggaggatcac tcgagagcgg acaggtgctg 1680

ctggaatcca atatcaaagt cctgcccact tggtctaccc ccgtgcagcc tatggctctg 1740

attgtgctgg gaggagtcgc aggactgctg ctgtttatcg ggctgggaat tttcttttgc 1800

gtgcgctgcc ggcaccggag aaggcaggcc gagcgcatga gccagatcaa gcgactgctg 1860

agcgagaaga aaacctgtca gtgtccccat agattccaga agacctgttc acccatt 1917

<210> 64

<211> 639

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 64

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

130 135 140

Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro

145 150 155 160

Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro

165 170 175

Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu

180 185 190

Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala

195 200 205

Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val

210 215 220

Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr

225 230 235 240

Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Gln Lys Leu Ile Ser

260 265 270

Glu Glu Asp Leu Asn Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Met Asp Ile

290 295 300

Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

305 310 315 320

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn

325 330 335

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr

340 345 350

Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

355 360 365

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

370 375 380

Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe

385 390 395 400

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

405 410 415

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

420 425 430

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

435 440 445

Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala

450 455 460

Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Thr Lys Gly

465 470 475 480

Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val

485 490 495

Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

500 505 510

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp

515 520 525

Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

530 535 540

Ser Ser Thr Ser Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu

545 550 555 560

Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln

565 570 575

Pro Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe

580 585 590

Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg

595 600 605

Gln Ala Glu Arg Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys

610 615 620

Thr Cys Gln Cys Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

625 630 635

<210> 65

<211> 1566

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 65

atggagaccc ccgcccagct gctgttcctg ctgctgctgt ggctgcccga caccaccggc 60

atgtcacggg gctccgacct gggcaaaaag ctgctggagg ccgctagggc cgggcaggac 120

gatgaagtga gaatcctgat ggccaacggg gctgacgtga atgctaagga tgagtacggc 180

ctgacccccc tgtatctggc tacagcacac ggccatctgg agatcgtgga agtcctgctg 240

aaaaacggag ccgacgtgaa tgcagtcgat gccattgggt tcactcctct gcacctggca 300

gcctttatcg gacatctgga gattgcagaa gtgctgctga agcacggcgc tgacgtgaac 360

gcacaggata agttcggaaa aaccgctttt gacatcagca ttggcaacgg aaatgaagac 420

ctggctgaaa tcctgcagaa actgaatgaa cagaaactga ttagcgaaga agacctgaac 480

cccgggggag gaggagggag cgggggagga ggcagcggcg ggggaggctc tggaggagga 540

gggagcggat ccatggatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 600

ggcgataggg tcaccatcac ctgccgtgcc agtcaggaca tccgtaatta tctgaactgg 660

tatcaacaga aaccaggaaa agctccgaaa ctactgattt actatacctc ccgcctggag 720

tctggagtcc cttctcgctt ctctggttct ggttctggga cggattacac tctgaccatc 780

agcagtctgc aaccggaaga cttcgcaact tattactgtc agcaaggtaa tactctgccg 840

tggacgttcg gacagggcac caaggtggag atcaaaggcg gcggcggaag tggaggagga 900

ggctcaggcg gaggagggag cgaggttcag ctggtggagt ctggcggtgg cctggtgcag 960

ccagggggct cactccgttt gtcctgtgca gcttctggct actcctttac cggctacact 1020

atgaactggg tgcgtcaggc cccaggtaag ggcctggaat gggttgcact gattaatcct 1080

tataaaggtg ttagtaccta caaccagaag ttcaaggacc gtttcactat aagcgtagat 1140

aaatccaaaa acacagccta cctgcaaatg aacagcctgc gtgctgagga cactgccgtc 1200

tattattgtg ctagaagcgg atactacggc gatagtgact ggtattttga cgtgtggggt 1260

caaggaaccc tggtcaccgt ctcctcgact agtggcggag gaggatcact cgagagcgga 1320

caggtgctgc tggaatccaa tatcaaagtc ctgcccactt ggtctacccc cgtgcagcct 1380

atggctctga ttgtgctggg aggagtcgca ggactgctgc tgtttatcgg gctgggaatt 1440

ttcttttgcg tgcgctgccg gcaccggaga aggcaggccg agcgcatgag ccagatcaag 1500

cgactgctga gcgagaagaa aacctgtcag tgtccccata gattccagaa gacctgttca 1560

cccatt 1566

<210> 66

<211> 522

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 66

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Met Ser Arg Gly Ser Asp Leu Gly Lys Lys Leu Leu

20 25 30

Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala

35 40 45

Asn Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu

50 55 60

Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu Leu

65 70 75 80

Lys Asn Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr Pro

85 90 95

Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val Leu

100 105 110

Leu Lys His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr

115 120 125

Ala Phe Asp Ile Ser Ile Gly Asn Gly Asn Glu Asp Leu Ala Glu Ile

130 135 140

Leu Gln Lys Leu Asn Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn

145 150 155 160

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

165 170 175

Ser Gly Gly Gly Gly Ser Gly Ser Met Asp Ile Gln Met Thr Gln Ser

180 185 190

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

195 200 205

Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

210 215 220

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu

225 230 235 240

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

245 250 255

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

260 265 270

Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys

275 280 285

Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

325 330 335

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn

355 360 365

Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

405 410 415

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Ser Gly

420 425 430

Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu Ser Asn Ile

435 440 445

Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile

450 455 460

Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile

465 470 475 480

Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met

485 490 495

Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro

500 505 510

His Arg Phe Gln Lys Thr Cys Ser Pro Ile

515 520

<210> 67

<211> 1569

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 67

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgatgtcac ggggctccga cctgggcaaa aagctgctgg aggccgctag ggccgggcag 120

gacgatgaag tgagaatcct gatggccaac ggggctgacg tgaatgctaa ggatgagtac 180

ggcctgaccc ccctgtatct ggctacagca cacggccatc tggagatcgt ggaagtcctg 240

ctgaaaaacg gagccgacgt gaatgcagtc gatgccattg ggttcactcc tctgcacctg 300

gcagccttta tcggacatct ggagattgca gaagtgctgc tgaagcacgg cgctgacgtg 360

aacgcacagg ataagttcgg aaaaaccgct tttgacatca gcattggcaa cggaaatgaa 420

gacctggctg aaatcctgca gaaactgaat gaacagaaac tgattagcga agaagacctg 480

aaccccgggg gaggaggagg gagcggggga ggaggcagcg gcgggggagg ctctggagga 540

ggagggagcg gatccatgga tatccagatg acccagtccc cgagctccct gtccgcctct 600

gtgggcgata gggtcaccat cacctgccgt gccagtcagg acatccgtaa ttatctgaac 660

tggtatcaac agaaaccagg aaaagctccg aaactactga tttactatac ctcccgcctg 720

gagtctggag tcccttctcg cttctctggt tctggttctg ggacggatta cactctgacc 780

atcagcagtc tgcaaccgga agacttcgca acttattact gtcagcaagg taatactctg 840

ccgtggacgt tcggacaggg caccaaggtg gagatcaaag gcggcggcgg aagtggagga 900

ggaggctcag gcggaggagg gagcgaggtt cagctggtgg agtctggcgg tggcctggtg 960

cagccagggg gctcactccg tttgtcctgt gcagcttctg gctactcctt taccggctac 1020

actatgaact gggtgcgtca ggccccaggt aagggcctgg aatgggttgc actgattaat 1080

ccttataaag gtgttagtac ctacaaccag aagttcaagg accgtttcac tataagcgta 1140

gataaatcca aaaacacagc ctacctgcaa atgaacagcc tgcgtgctga ggacactgcc 1200

gtctattatt gtgctagaag cggatactac ggcgatagtg actggtattt tgacgtgtgg 1260

ggtcaaggaa ccctggtcac cgtctcctcg actagtggcg gaggaggatc actcgagagc 1320

ggacaggtgc tgctggaatc caatatcaaa gtcctgccca cttggtctac ccccgtgcag 1380

cctatggctc tgattgtgct gggaggagtc gcaggactgc tgctgtttat cgggctggga 1440

attttctttt gcgtgcgctg ccggcaccgg agaaggcagg ccgagcgcat gagccagatc 1500

aagcgactgc tgagcgagaa gaaaacctgt cagtgtcccc atagattcca gaagacctgt 1560

tcacccatt 1569

<210> 68

<211> 523

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 68

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Met Ser Arg Gly Ser Asp Leu Gly Lys Lys Leu

20 25 30

Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met

35 40 45

Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro

50 55 60

Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu

65 70 75 80

Leu Lys Asn Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr

85 90 95

Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val

100 105 110

Leu Leu Lys His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys

115 120 125

Thr Ala Phe Asp Ile Ser Ile Gly Asn Gly Asn Glu Asp Leu Ala Glu

130 135 140

Ile Leu Gln Lys Leu Asn Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

145 150 155 160

Asn Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

165 170 175

Gly Ser Gly Gly Gly Gly Ser Gly Ser Met Asp Ile Gln Met Thr Gln

180 185 190

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

195 200 205

Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln

210 215 220

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu

225 230 235 240

Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

245 250 255

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

260 265 270

Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr

275 280 285

Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

305 310 315 320

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser

325 330 335

Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

340 345 350

Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr

355 360 365

Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys

370 375 380

Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

385 390 395 400

Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr

405 410 415

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Ser

420 425 430

Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu Ser Asn

435 440 445

Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu

450 455 460

Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly

465 470 475 480

Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg

485 490 495

Met Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys

500 505 510

Pro His Arg Phe Gln Lys Thr Cys Ser Pro Ile

515 520

<210> 69

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 69

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 70

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 70

ggaggaggag ggagcggggg aggaggcagc ggcgggggag gctctggagg aggagggagc 60

<210> 71

<211> 750

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 71

atggacatcc agatgactca gaccacaagc tccctgtctg caagtctggg cgaccgggtg 60

acaatctcct gcagagcctc tcaggatatt aggaactacc tgaattggta tcagcagaaa 120

cctgatggca cagtcaagct gctgatctac tataccagcc ggctgcactc aggcgtgcca 180

agcaaattct caggaagcgg ctccgggact gactactccc tgaccatctc taacctggag 240

caggaagata ttgctaccta tttctgccag cagggcaata cactgccctg gacttttgcc 300

ggaggcacca aactggagat caagggggga ggcgggagtg gaggcggggg atcaggagga 360

ggaggcagcg gaggaggagg gtccgaggtc cagctgcagc agagcggacc agaactggtg 420

aagcccggag caagtatgaa aatctcctgt aaggcctcag gatacagctt caccggctat 480

acaatgaact gggtgaaaca gtcccatggc aagaacctgg aatggatggg gctgattaat 540

cctaccaaag gcgtcagcac ctataatcag aagtttaaag acaaggccac actgactgtg 600

gataagtcta gttcaaccgc ttacatggag ctgctgtccc tgacatctga agacagtgcc 660

gtgtactatt gtgctcggtc tggctactat ggggacagtg attggtactt cgatgtctgg 720

ggacagggca ctaccctgac cgtgttttct 750

<210> 72

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 72

Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu

1 5 10 15

Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn

20 25 30

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu

35 40 45

Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu

65 70 75 80

Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

85 90 95

Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

130 135 140

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

145 150 155 160

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met

165 170 175

Gly Leu Ile Asn Pro Thr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

180 185 190

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

195 200 205

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

225 230 235 240

Gly Gln Gly Thr Thr Leu Thr Val Phe Ser

245 250

<210> 73

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 73

Gly Gly Gly Gly Ser

1 5

<210> 74

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic peptides

<400> 74

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 75

<211> 1560

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polynucleotides

<400> 75

atggatttcc aggtccagat tttctccttc ctgctgattt ccgcaagcgt cattatgtca 60

cggggctccg acctgggcaa aaagctgctg gaggccgcta gggccgggca ggacgatgaa 120

gtgagaatcc tgatggccaa cggggctgac gtgaatgcta aggatgagta cggcctgacc 180

cccctgtatc tggctacagc acacggccat ctggagatcg tggaagtcct gctgaaaaac 240

ggagccgacg tgaatgcagt cgatgccatt gggttcactc ctctgcacct ggcagccttt 300

atcggacatc tggagattgc agaagtgctg ctgaagcacg gcgctgacgt gaacgcacag 360

gataagttcg gaaaaaccgc ttttgacatc agcattggca acggaaatga agacctggct 420

gaaatcctgc agaaactgaa tgaacagaaa ctgattagcg aagaagacct gaaccccggg 480

ggaggaggag ggagcggggg aggaggcagc ggcgggggag gctctggagg aggagggagc 540

ggatccatgg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 600

agggtcacca tcacctgccg tgccagtcag gacatccgta attatctgaa ctggtatcaa 660

cagaaaccag gaaaagctcc gaaactactg atttactata cctcccgcct ggagtctgga 720

gtcccttctc gcttctctgg ttctggttct gggacggatt acactctgac catcagcagt 780

ctgcaaccgg aagacttcgc aacttattac tgtcagcaag gtaatactct gccgtggacg 840

ttcggacagg gcaccaaggt ggagatcaaa ggcggcggcg gaagtggagg aggaggctca 900

ggcggaggag ggagcgaggt tcagctggtg gagtctggcg gtggcctggt gcagccaggg 960

ggctcactcc gtttgtcctg tgcagcttct ggctactcct ttaccggcta cactatgaac 1020

tgggtgcgtc aggccccagg taagggcctg gaatgggttg cactgattaa tccttataaa 1080

ggtgttagta cctacaacca gaagttcaag gaccgtttca ctataagcgt agataaatcc 1140

aaaaacacag cctacctgca aatgaacagc ctgcgtgctg aggacactgc cgtctattat 1200

tgtgctagaa gcggatacta cggcgatagt gactggtatt ttgacgtgtg gggtcaagga 1260

accctggtca ccgtctcctc gactagtggc ggaggaggat cactcgagag cggacaggtg 1320

ctgctggaat ccaatatcaa agtcctgccc acttggtcta cccccgtgca gcctatggct 1380

ctgattgtgc tgggaggagt cgcaggactg ctgctgttta tcgggctggg aattttcttt 1440

tgcgtgcgct gccggcaccg gagaaggcag gccgagcgca tgagccagat caagcgactg 1500

ctgagcgaga agaaaacctg tcagtgtccc catagattcc agaagacctg ttcacccatt 1560

<210> 76

<211> 520

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence synthetic polypeptide

<400> 76

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala

20 25 30

Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly

35 40 45

Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu

50 55 60

Ala Thr Ala His Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn

65 70 75 80

Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His

85 90 95

Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val Leu Leu Lys

100 105 110

His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe

115 120 125

Asp Ile Ser Ile Gly Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln

130 135 140

Lys Leu Asn Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Pro Gly

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Gly Ser Met Asp Ile Gln Met Thr Gln Ser Pro Ser

180 185 190

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

195 200 205

Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

210 215 220

Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly

225 230 235 240

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

245 250 255

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

260 265 270

Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

275 280 285

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

305 310 315 320

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly

325 330 335

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

340 345 350

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

355 360 365

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

370 375 380

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

385 390 395 400

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Ser Gly Gly Gly

420 425 430

Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val

435 440 445

Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile Val Leu

450 455 460

Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe

465 470 475 480

Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln

485 490 495

Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His Arg

500 505 510

Phe Gln Lys Thr Cys Ser Pro Ile

515 520

<210> 77

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial sequence synthetic oligonucleotides

<400> 77

ggcggcggcg gaagtggagg aggaggctca ggcggaggag ggagc 45

Claims

1. A human epidermal growth factor receptor 2 (HER 2) T cell antigen conjugate (HER 2-TAC) polypeptide consisting of an amino acid sequence having 100% sequence identity to SEQ ID No. 66, SEQ ID No. 68 or SEQ ID No. 76.

2. A nucleic acid molecule encoding the HER2-TAC of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule has 100% sequence identity to SEQ ID No. 65, SEQ ID No. 67 or SEQ ID No. 75.

4. A vector construct comprising:

(a) The nucleic acid molecule of claim 2, and

(B) Promoters functional in mammalian cells.

5. The vector construct of claim 4, wherein the nucleic acid molecule has 100% sequence identity to SEQ ID NO. 65, SEQ ID NO. 67 or SEQ ID NO. 75.

6. A T cell comprising (a) HER2-TAC according to claim 1, (b) the nucleic acid molecule according to claim 2 or 3, or (c) the vector construct according to claim 4 or 5.

7. A pharmaceutical composition comprising the T cell of claim 6 and a pharmaceutically acceptable excipient.

8. Use of the T cell of claim 6 in the manufacture of a medicament for treating a HER2 expressing cancer in an individual in need thereof.