KR20210021593A - T cell-antigen coupler with various structural optimizations - Google Patents

Abstract

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

Description

T cell-antigen coupler with various structural optimizations

Cross reference

This application is for U.S. Provisional Application No. 62/699,173 filed on July 17, 2018, U.S. Provisional Application No. 62/703,037 filed on July 25, 2018, and U.S. Provisional Application No. 62 filed on November 29, 2018. /773,120, U.S. Provisional Application No. 62/826,853 filed March 29, 2019, U.S. Provisional Application No. 62/828,879 filed April 3, 2019, U.S. Provisional Application No. filed April 26, 2019 62/839,235, U.S. Regular Application No. 16/442,274 filed June 14, 2019, and U.S. Provisional Application No. 62/874,426 filed July 15, 2019, each of which is in its entirety Is incorporated herein by reference.

Sequence list

This application contains a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy was created on July 17, 2019, was named 55247704601_SL.txt and has a size of 131,072 bytes.

summary

In certain embodiments, disclosed herein is a nucleic acid sequence encoding a CD19 trifunctional T cell-antigen coupler (CD19-TAC). In some embodiments, the nucleic acid sequence encoding the CD19 trifunctional T cell-antigen coupler (CD19-TAC) comprises (a) a first polynucleotide encoding a ligand that selectively binds to the CD19 antigen. In some embodiments, the nucleic acid sequence encoding the CD19 trifunctional T cell-antigen coupler (CD19-TAC) comprises (b) a second polynucleotide encoding a UCHT1 ligand that binds CD3. In some embodiments, the nucleic acid sequence encoding a CD19 trifunctional T cell-antigen coupler (CD19-TAC) comprises (c) a third polynucleotide encoding a TCR signaling domain polypeptide comprising a cytosolic domain and a transmembrane domain. Include. In some embodiments, the components encoded by the first, second and/or third polynucleotide are linked in any suitable manner, such as in any suitable order, and/or including any suitable linker(s). . In some embodiments, the component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused directly to each other or linked by at least one linker. In some embodiments, the ligand that selectively binds to the CD19 antigen is a single chain variable fragment (scFv). In some embodiments, the ligand that selectively binds the CD19 antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of SEQ ID NO: 36, Or an amino acid sequence with 100% sequence identity. In some embodiments, the UCHT1 ligand is a single chain antibody. In some embodiments, the UCHT1 ligand comprises the Y182T mutation (SEQ ID NO: 72). In some embodiments, the UCHT1 ligand is a humanized variant of UCHT1 (huUCHT1) ligand (SEQ ID NO: 44). In some embodiments, the UCHT1 ligand is a humanized variant of UCHT1 comprising the Y177T mutation (huUCHT1 (Y177T)) (SEQ ID NO: 46). In some embodiments, the UCHT1 ligand is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least with SEQ ID NO: 14, SEQ ID NO: 72, SEQ ID NO: 44, or SEQ ID NO: 46 98%, at least 99%, or 100% sequence identity. In some embodiments, the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain. In some embodiments, the third polynucleotide is 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 with SEQ ID NO: 18. It encodes a polypeptide comprising an amino acid sequence having identity. In some embodiments, the component encoded by (a) and the component encoded by (c) are fused to the component encoded by (b). In some embodiments, the component encoded by (b) and the component encoded by (c) are fused to the component encoded by (a). In some embodiments, at least one linker connects the component encoded by (a) to the component encoded by (b). In some embodiments, the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helical structure, or a short helical structure. In some embodiments, the at least one linker is SEQ ID NO: 12 (G _{4 S flexible linker (“G 4} S” disclosed as SEQ ID NO: 73)), SEQ ID NO: 32 (large protein domain), SEQ ID NO: 30 (long helical structure ), or 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 with SEQ ID NO: 28 (short helical structure). It includes an amino acid sequence having. In some embodiments, CD3 is of the TCR complex on the cell expressing the second polynucleotide. In some embodiments, binding of CD3 induces activation of a cell expressing the second polynucleotide. In some embodiments, the CD19-TAC is 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 with SEQ ID NO: 63. It includes a nucleic acid sequence having. In some embodiments, the CD19-TAC is 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 with SEQ ID NO: 64. It includes an amino acid sequence having. In some embodiments, the nucleic acid sequence does not encode an auxiliary stimulatory domain. In some embodiments, the nucleic acid sequence does not encode an activation domain.

In certain embodiments, (a) a nucleic acid sequence disclosed herein (eg, a nucleic acid sequence encoding CD19-TAC); And (b) a promoter that is functional in a mammalian cell is disclosed herein.

In certain embodiments, disclosed herein are T cells comprising a nucleic acid sequence disclosed herein (eg, a nucleic acid sequence encoding CD19-TAC).

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

In certain embodiments, as a method of treating a cancer expressing CD19 in an individual in need thereof, a pharmaceutical composition disclosed herein (e.g., CD19 trifunctional T cell-antigen coupler (CD19-TAC A method comprising administering to an individual a pharmaceutical composition comprising a T cell comprising any of the nucleic acid sequences described herein, such as any of the nucleic acid sequences or sequences described herein, encoding) is disclosed herein. In some embodiments, the cancer is a B cell malignant tumor. In some embodiments, the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma. In some embodiments, the pharmaceutical composition is administered into the carotid artery, subcutaneously, intradermally, intratumorally, intranodically, intrathecally, intramuscularly, intravenously or intraperitoneally.

In certain embodiments, (a) a first polynucleotide encoding a target specific ligand; (b) a second polynucleotide encoding a ligand that binds to the protein associated with the TCR complex; And (c) a nucleic acid sequence encoding a trifunctional T cell-antigen coupler (Tri-TAC) comprising a third polynucleotide encoding a T cell receptor signal transduction domain polypeptide, wherein the ligand binding to the protein related to the TCR complex is Nucleic acid sequences are disclosed herein which are selected from OKT3, F6A or L2K. In some embodiments, the component encoded by (a), the component encoded by (b) and the component encoded by (c) are directly fused or linked by at least one linker. In some embodiments, the component encoded by (a) and the component encoded by (b) are fused directly and linked by a linker to the component encoded by (c). In some 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 some embodiments, at least one linker is G ₄ S Flexible linkers (SEQ ID NO: 73), large protein domains, long helical structures, or short helical structures. In some embodiments, the at least one linker is SEQ ID NO: 12 (G _{4 S flexible linker (“G 4} S” disclosed as SEQ ID NO: 73)), SEQ ID NO: 32 (large protein domain), SEQ ID NO: 30 (long helical structure ), or 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 with SEQ ID NO: 28 (short helical structure). Has an amino acid sequence. In some embodiments, the ligand that binds to the protein associated with the TCR complex is OKT3. In some embodiments, the ligand that binds to the protein associated with the TCR complex is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of SEQ ID NO: 22. , Or an amino acid sequence with 100% sequence identity. In some embodiments, the ligand that binds to the protein associated with the TCR complex is F6A. In some embodiments, the ligand that binds to the protein associated with the TCR complex is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of SEQ ID NO: 24. , Or an amino acid sequence with 100% sequence identity. In some embodiments, the ligand that binds to the protein associated with the TCR complex is L2K. In some embodiments, the ligand that binds to the protein associated with the TCR complex is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of SEQ ID NO: 26. , Or an amino acid sequence with 100% sequence identity. In some embodiments, the protein associated with the TCR complex is CD3. In some embodiments, target specific ligands selectively bind to tumor antigens. In some embodiments, the target specific ligand is a designed ankyrin repeat (DARPin) polypeptide, or a single chain variable fragment (scFv). In some embodiments, the target specific ligand selectively binds to the CD19 antigen, HER2 antigen, or BCMA antigen. In some embodiments, the target-specific ligand selectively binds to the HER-2 antigen, and trastuzumab, Pertuzumab, Lapatinib, Neratinib, ado-trastuzumab M. Tanshin (Ado-trastuzmab Emtansine), Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab. In some embodiments, the target specific ligand selectively binds to the BCMA antigen and comprises an antigen binding domain of an antibody selected from Belantamab mafodotin, and GSK2857916. In some embodiments, the target specific ligand is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 36, SEQ ID NO: 8, or SEQ ID NO: 34. , At least 99%, or 100% sequence identity. In some embodiments, the T cell receptor signaling domain polypeptide comprises a cytosolic domain and a transmembrane domain. In some embodiments, the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain, or the cytosolic domain is a CD8 cytosolic domain and the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the nucleic acid sequence further comprises a leader sequence. In some embodiments, the leader sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50 It includes an amino acid sequence with 99%, or 100% sequence identity. In some embodiments, CD3 is of the TCR complex on the cell expressing the second polynucleotide. In some embodiments, binding of CD3 induces activation of a cell expressing the second polynucleotide. In some embodiments, the Tri-TAC is at least 80%, at least 85% with 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, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the Tri-TAC is at least 80%, at least 85% with 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, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the nucleic acid sequence does not encode an auxiliary stimulatory domain. In some embodiments, the nucleic acid sequence does not encode an activation domain.

In certain embodiments, (a) a first polynucleotide encoding a target specific ligand; (b) a second polynucleotide encoding a ligand that binds to the protein associated with the TCR complex; And (c) a third polynucleotide encoding a T cell receptor signal transduction domain polypeptide, the nucleic acid sequence encoding a trifunctional T cell-antigen coupler (Tri-TAC), wherein the nucleic acid sequence further comprises a leader sequence, , The component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused directly to each other, or are linked by at least one linker, disclosed herein. do. In some embodiments, target specific ligands selectively bind to tumor antigens. In some embodiments, the target specific ligand is a designed ankyrin repeat (DARPin) polypeptide, or single chain variable fragment (scFv). In some embodiments, the target specific ligand selectively binds to the CD19 antigen, HER2 antigen, or BCMA antigen. In some embodiments, the target-specific ligand selectively binds to the HER-2 antigen and trastuzumab, pertuzumab, lapatinib, neratinib, ado-trastuzumab emtansine, gankotamab, margetuximab, thymi Gutuzumab, and an antigen binding domain of an antibody selected from ertumaksomab. In some embodiments, the target specific ligand selectively binds to the BCMA antigen and comprises an antigen binding domain of an antibody selected from belantamab mapodotin, and GSK2857916. In some embodiments, the target specific ligand is at least 80%, at least 85%, at least 90%, at least 95%, at least 96% with SEQ ID NO: 36, SEQ ID NO: 8, SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO: 54. , At least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the ligand that binds to the protein associated with the TCR complex is selected from UCHT1, UCHT1 (Y182T), huUCHT1, huUCHT1 (Y177T), OKT3, F6A, or L2K. In some embodiments, the ligand that binds to the protein associated with the TCR complex is at least 80%, at least 85 with 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. %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the protein associated with the TCR complex is CD3. In some embodiments, the T cell receptor signaling domain polypeptide comprises a cytosolic domain and a transmembrane domain. In some embodiments, the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain, or the cytosolic domain is a CD8 cytosolic domain and the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the leader sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50 It includes an amino acid sequence with 99%, or 100% sequence identity. In some embodiments, the component encoded by (a) and the component encoded by (b) are fused directly and linked by a linker to the component encoded by (c). In some 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 some embodiments, the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helical structure, or a short helical structure. In some embodiments, the at least one linker is SEQ ID NO: 12 (G _{4 S flexible linker (“G 4} S” disclosed as SEQ ID NO: 73)), SEQ ID NO: 32 (large protein domain), SEQ ID NO: 30 (long helical structure ), or 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 with SEQ ID NO: 28 (short helical structure). Has an amino acid sequence. In some embodiments, CD3 is of the TCR complex on the cell expressing the second polynucleotide. In some embodiments, binding of CD3 induces activation of a cell expressing the second polynucleotide. In some embodiments, the Tri-TAC is at least 80%, at least 85% with 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, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the Tri-TAC is at least 80%, at least 85% with 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, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the nucleic acid sequence does not encode an auxiliary stimulatory domain. In some 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, (a) a nucleic acid sequence disclosed herein; And (b) a promoter that is functional in a mammalian cell is disclosed herein.

In certain embodiments, disclosed herein are T cells comprising the nucleic acid sequences disclosed herein.

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

In certain embodiments, disclosed is a method of treating cancer in an individual in need thereof comprising administering to the individual a pharmaceutical composition disclosed herein. In some embodiments, the subject is a mammal. In some embodiments, the cancer is a solid or liquid cancer. In some embodiments, the cancer is lung cancer, breast cancer, multiple myeloma, glioblastoma, gastric cancer, ovarian cancer, gastric cancer, colorectal cancer, urinary tract cancer, endometrial cancer, or colon cancer. In some embodiments, the cancer comprises CD19 expressing cancer cells. In some embodiments, the cancer is a B cell malignant tumor. In some embodiments, the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma. In some embodiments, the cancer comprises HER-2 expressing cancer cells. In some embodiments, the cancer is breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, or gastric cancer. In some embodiments, the cancer comprises BCMA expressing cancer cells. In some embodiments, the cancer is leukemia, lymphoma, or multiple myeloma. In some embodiments, the pharmaceutical composition is administered to the subject by carotid artery, subcutaneous, intradermal, intratumoral, intranodal, intrathecal, intramuscular, intravenous, or intraperitoneal. In some embodiments, the pharmaceutical composition is in unit dosage form. In some embodiments, the pharmaceutical composition comprises about 0.5-2 x 10 ⁹ T cells. In some embodiments, the pharmaceutical composition is administered daily, weekly, biweekly, monthly, bimonthly or annually.

In certain embodiments, (a) a first polynucleotide encoding a ligand that selectively binds to the CD19 antigen; (b) a second polynucleotide encoding a humanized variant of UCHT1 (huUCHT1) (huUCHT1(Y177T)) ligand comprising the Y177T mutation that binds CD3; And (c) a CD19 trifunctional T cell-antigen coupler (CD19-TAC) comprising a third polynucleotide encoding a polypeptide comprising a CD4 cytosolic domain and a CD4 transmembrane domain, wherein (a) Nucleic acid sequences are disclosed herein wherein the ligand encoded by, 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 some embodiments, the nucleic acid sequence does not encode an auxiliary stimulatory domain, an activation domain, or both an auxiliary stimulating domain and an activation domain. In some embodiments, the ligand that selectively binds to the CD19 antigen is a single chain variable fragment (scFv). In some embodiments, the ligand that selectively binds the CD19 antigen comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 36. In some embodiments, the ligand that selectively binds to the CD19 antigen comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the huUCHT1(Y177T) ligand is a single chain antibody. In some embodiments, the huUCHT1(Y177T) ligand comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 46. In some embodiments, the huUCHT1(Y177T) ligand comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the third polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 18.

In some embodiments, the third polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the at least one linker is a G ₄ S flexible linker, a large protein domain, a long helical structure, or a short helical structure. In some embodiments, the at least one linker comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28. In some 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 some embodiments, CD3 is expressed on cells expressing the second polynucleotide. In some 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 some 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 some embodiments, the CD19-TAC comprises the sequence of SEQ ID NO: 63 or SEQ ID NO: 64. In certain embodiments, (a) a nucleic acid sequence disclosed herein; And (b) a promoter that is functional in a mammalian cell is disclosed herein. In certain embodiments, disclosed herein are compositions comprising the vectors disclosed herein and excipients. In certain embodiments, disclosed herein are polypeptides encoded by the nucleic acid sequences disclosed herein.

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

In certain embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61, Disclosed herein is a nucleic acid sequence encoding a BCMA trifunctional T cell-antigen coupler (BCMA-TAC) comprising a nucleic acid sequence having at least 99%, or 100% sequence identity. In some embodiments, BCMA-TAC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% with SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62, Amino acid sequences with at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the nucleic acid sequence does not encode an auxiliary stimulatory domain. In some embodiments, the nucleic acid sequence does not encode an activation domain.

The novel features of the invention are specifically set forth in the appended claims. The features and advantages of the present invention will be better understood with reference to the following detailed description and the following accompanying drawings, which illustrate exemplary embodiments in which the principles of the invention are employed:
1A is a schematic diagram of natural T cell activation.
[Fig. 1b] is a schematic diagram of CAR-based T cell activation.
[Fig. 1c] is a schematic diagram of T cell activation based on a trifunctional T cell-antigen coupler (Tri-TAC).
[Fig. 1D] is a schematic diagram of natural T cell activation.
1E is a schematic diagram of CAR-based T cell activation.
[Fig. 1f] is a schematic diagram of Tri-TAC-based T cell activation.
[Fig. 2A] is a schematic diagram of the Tri-TAC configuration in which the UCHT1 domain is the center between the transmembrane domain (TM) and the antigen-binding domain.
[Fig. 2B] is a schematic diagram of a Tri-TAC configuration in which the UCHT1 domain is N-terminal and the antigen binding domain and the transmembrane domain follow.
[Fig. 2c] is a schematic diagram of a Tri-TAC molecule having a general antigen-binding domain and a UCHT1 domain.
[Fig. 3A] is a schematic diagram of a Tri-TAC molecule having a general antigen-binding domain.
[Fig. 3B] is a schematic diagram of Tri-TAC having an anti-HER-2 DARPin antigen binding domain.
[Fig. 3C] is a schematic diagram of Tri-TAC having an anti-CD19 scFv antigen binding domain.
[Fig. 3D] is a schematic diagram of Tri-TAC having an anti-BCMA scFv antigen binding domain.
[Fig. 3E] is a schematic diagram of a Tri-TAC molecule having an anti-HER-2 DARPin antigen binding domain.
[Fig. 3f] is a schematic diagram of a Tri-TAC molecule having an anti-BCMA scFv antigen binding domain.
[Fig. 4A] to [Fig. 4D] illustrate T cells engineered with a Tri-TAC or CD28-based CAR for HER-2 using DARPin. Figure 4a illustrates the surface expression of Tri-TAC and CAR compared to T cells that do not express chimeric receptors. [Fig. 4B] illustrates the growth of three cell populations. Figures 4c to 4d illustrate the percentage of engineered cells that are positive for various T cell activation markers after stimulation with antigen.
[Fig. 5] exemplifies a model of the structure of the CD19-TAC protein.
6A to 6J illustrate receptor surface expression and activation of various anti-HER-2 DARPin Tri-TAC controls. T cells were engineered with a Tri-TAC variant without targeting element (-DARPin), a Tri-TAC variant without UCHT1 (-UCHT1) or full-length Tri-TAC. [FIG. 6A], [FIG. 6D], [FIG. 6G] illustrate T cell transduction and Her2 binding ability (left); [FIG. 6B], [FIG. 6E], [FIG. 6H] illustrate degranulation (middle), and [FIG. 6C], [FIG. 6F], [FIG. 6i] illustrate cytokine production (right side). [Fig. 6j] exemplifies that only full-length anti-HER-2 DARPin Tri-TAC can induce a cytotoxic response.
7A to 7C illustrate 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 CAR. 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] exemplifies the change in tumor growth on the day of T cell injection (day 35). [Fig. 7B] illustrates the change in body weight, which is a measure of toxicity in the same mouse. [Fig. 7c] exemplifies the cytokine concentration in mouse serum on the 7th day after T cell injection.
[Fig. 8A] to [Fig. 8H] illustrate Tri-TAC designed as various alternatives to the UCHT1 scFv-CD3 mobilization domain. Figure 8a provides a schematic diagram of a TAC receptor construct using anti-HER-2 DARPin corresponding to UCHT1 or OKT3 anti-CD3 scFv. [Fig. 8B] exemplifies HER-2 TAC surface expression of CD8+ NGFR+ (left) or CD4+ NGFR+ T cells (right). [Fig. 8c] and [Fig. 8c1] exemplify cytokine production by HER-2 specific TAC-T cells stimulated with antigen-positive SK-OV-3 tumor cells. [Fig. 8D] exemplifies the killing of SK-OV-3 tumor cells by HER-2 TAC and vector control (a vector containing only tNGFR) T cells. Vector control T cells (circles) are compared against HER-2 specific TAC-T cells bearing UCHT1 (square) or OKT3 (triangle). 8E provides a schematic diagram of a TAC receptor construct using anti-CD19 scFvs matched with huUCHT1, F6A, or L2K anti-CD3 scFvs. Figure 8f illustrates the surface expression of CD19-TAC in CD8+ NGFR+ (left) or CD4+ NGFR+ T cells (right). [Fig. 8G] and [Fig. 8G1] exemplify cytokine production by CD19-specific TAC-T cells stimulated with antigen-positive Raji tumor cells. Compare cytokine producing cells from TAC-T cells bearing huUCHT1 (square), F6A (triangle) or L2K (diamond). [Fig. 8H] exemplifies the killing of NALM-6 tumor cells by CD19 TAC and vector control (a vector bearing only tNGFR) T cells. Vector control T cells (circles) are compared against CD19 specific TAC-T cells bearing huUCHT1 (square), F6A (triangle), or L2K (diamond).
9A to 9H illustrate the effects of various anti-CD3 scFvs on TCR surface expression. 9A and 9E illustrate TCR surface expression of T cells engineered with a control vector (tNGFR), UCHT1, or OKT3 TAC variant. [Fig. 9b] and [Fig. 9f] illustrate that T cells engineered with OKT3-TAC significantly reduced TCR surface expression compared to UCHT1-TAC. [Fig. 9c] and [Fig. 9g] illustrate TCR surface expression of T cells engineered with a control vector (tNGFR), huUCHT1, F6A or L2K TAC variants. [FIG. 9D] and [FIG. 9H] illustrate that T cells engineered with L2K TAC significantly reduced TCR surface expression compared to huUCHT1-TAC.
[FIG. 10A] to [FIG. 10B] illustrate linker domain variants. The domain that connects the antigen binding domain to the TCR mobilization domain is called a linker domain. [Fig. 10A] provides a schematic diagram of TAC variants with different linker domains: (i) flexible linker, (ii) large domain linker (consisting of domains 3 and 4 derived from extracellular CD4 domains), (iii) A long helical coupler, and (iv) a short helical coupler. [Fig. 10b] provides exemplary amino acid sequences of the domains shown in [Fig. 10a] (SEQ ID NOs: 69, 28, 30, and 32, respectively, in the order shown)
Figures 11A-E illustrate exemplary in vitro parameters of CD19 TAC engineered with different linker variants. [Fig. 11A] illustrates the surface expression of TAC variants in both CD4 and CD8 cells. [Fig. 11B] exemplifies the surface expression of a TAC comprising a flexible linker to a TAC comprising a helical or large domain linker. 11C depicts total transduction of TACs containing alternative linkers to flexible linkers. [Fig. 11D] and [Fig. 11E] illustrate the relative cellular reactivity to antigen-positive Raji cells.
Figure 12a illustrates the in vitro cytotoxicity of BCMA Tri-TAC variants engineered with different linkers. 12B illustrates in vivo tumor control of the BCMA Tri-TAC variant engineered with a flexible connector compared to a short helical connector.
13A to 13C illustrate the properties of CD8α Tri-TAC scFv anti-HER-2 and CD8α Tri-TAC DARPin anti-HER-2. [Fig. 13A] and [Fig. 13C] illustrate surface expression. [Fig. 13b] illustrates cytokine production.
[Fig. 14A] to [Fig. 14D] provide schematic diagrams of CD8 Tri-TAC variants. Anti-HER-2-DARPin is used as an exemplary antigen binding domain and UCHT1 CD3 recruitment domain is used as an exemplary recruitment domain. 14A illustrates a Tri-TAC comprising a CD4 transmembrane and cytosolic domain (left) and a similar region of the CD8α/CD8β heterodimer (right). The key areas for co-receptor functionality (arginine rich domain and CXCP motif) are highlighted. 14B is a schematic diagram of a CD8α Tri-TAC containing a cysteine to serine mutation, and a CD8α cytosolic domain to ensure monomeric receptor distribution. 14C is a schematic diagram of a CD8α+Rβ Tri-TAC comprising a cysteine to serine mutation to ensure monomeric receptor distribution, and a chimeric CD8α cytosolic domain in which the CD8α arginine rich region is replaced by a CD8β arginine rich region. [Fig. 14D] is a CD8β comprising a chimeric CD8β cytosolic domain in which a CD8α CXCP domain containing a Lck binding motif and a CD8α CXCP domain including a Lck binding motif is added to the C-terminus of the CD8β cytosol domain to ensure monomeric receptor distribution. It is a schematic diagram of +Lck Tri-TAC.
15A to 15E illustrate the in vitro characterization of CD8 Tri-TAC variants against the prototype Tri-TAC containing the CD4 region. [FIG. 15A] to [FIG. 15B] illustrate the surface expression of the CD8-Tri TAC variant against the prototype Tri-TAC. Figure 15c illustrates the in vitro cytotoxicity of CD8-Tri TAC variants co-cultured with LOX IMVI (HER-2 negative) or A549, SKOV3, SKBR3 or MBA MB 231 (HER-2 positive). 15D illustrates cell division of T cells engineered with CD8 Tri-TAC variants or prototype Tri-TAC. [Fig. 15E] exemplifies TCR surface expression of engineered T cells containing CD8 Tri-TAC variants or prototype Tri-TAC.
[Fig. 16] illustrates various Tri-TACs.
[Fig. 17] exemplifies the TAC-CD19 insert in the pCCL lentiviral vector. 17 illustrates the various domains of TAC-CD19 (CD8a leader, FMC63 scFv, Myc tag, huUCHT1 Y177T mutant and truncated CD4 fixed co-receptor domain).
18 illustrates the in vivo efficacy of TAC-CD19 generated from different donors.
19A to 19C illustrate in vitro examples of TAC-CD19 cytotoxicity against tumor lines. [Fig. 19A] NALM-6 (acute lymphoblastic leukemia), [Fig. 19B] Jeko-1 (mantle cell lymphoma) and [Fig. 19C] Raji (Bucket's lymphoma).
[Fig. 19D] illustrates a schematic diagram of three different in vivo tumor models in NRG mice.
[Fig. 19E] to [Fig. 19G] show CD19 in NALM-6 (acute lymphoblastic leukemia) (Fig. 19E), Jeko-1 (mantle cell lymphoma) (Fig. 19F), and Raji (Bucket lymphoma) (Fig. 19G). -Illustrates the in vivo efficacy of TAC.
[Fig. 20A] Illustrates the experimental setup of TAC-CD19 treated mice bearing NALM-6 tumors. After successful treatment, mice are rechallenged with either NALM-6 (CD19 positive) or KMS11 (CD19 negative) tumor cells.
[Figure 20B] illustrates the in vivo efficacy of mice treated with TAC-CD19.
21A illustrates an experimental design to evaluate the dosage regimen and the effect of administration on efficacy and cell expansion.
21B illustrates the in vivo survival rate of NALM-6-bearing mice treated with single or divided doses of TAC-CD19.
[Fig. 22A] to [Fig. 22B] exemplify experimental settings and data regarding expansion of TAC-CD19 in vivo after administration of divided doses. 22A illustrates the gating strategy used to identify T cells in mouse blood. [Fig. 22B] exemplifies the in vivo results of T cell expansion in blood.
23A to 23C illustrate long-term in vivo studies of TAC-CD19 in mice. 23A illustrates the experimental protocol of NALM-6 bearing mice treated with various controls and TAC-CD19 at two dose levels. 23B illustrates the in vivo efficacy of the control versus the TAC-CD19 treatment group at two dose levels. 23C illustrates the long-term survival rate of mice treated with low-dose TAC-CD19.
Fig. 24 illustrates the results of clinical chemical analysis from mice treated with TAC-CD19 or untransduced T cells.
Fig. 25 illustrates human cytokines released from mouse blood after treatment with TAC-CD19 or untransduced T cells.
[Fig. 26A] to [Fig. 26C] illustrate the efficacy of BCMA-TAC in different configurations. [Fig. 26A] illustrates the experimental design. 26B illustrates various control and test products. Figure 26c illustrates the in vivo efficacy of various TAC constructs. 26A to 26C disclose “G ₄ S” as SEQ ID NO: 73.
Fig. 27 illustrates that TAC proliferates when it encounters an antigen on a cell, but does not proliferate when the antigen is presented to artificial beads. However, CARs proliferate regardless of whether the antigen is present on the beads or cells.
[Fig. 28A] to [Fig. 28B] exemplify that TAC engineered T cells expand in vivo and provide long-term protection to show cell persistence in a myeloma model. [Fig. 28A] to [Fig. 28B] illustrate 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 engraftment, mice were treated with BCMA TAC-T cells (bearing firefly luciferase). TAC-T cells are greatly expanded after administration. It is associated with tumor regression. Treated mice were resistant to tumor re-administration showing long-term persistence of TAC-T cells.
[Fig. 29] exemplifies human cytokines released from mouse blood after treatment with TAC-CD19 or untransduced T cells.
Figure 30 illustrates a representative histogram of TAC receptor surface expression in CD4 and CD8 engineered T cells. Cells were engineered with muIgG HER2 TAC (EF1α promoter), huIgG HER2 TAC (MSCV promoter) and muIgG HER2 TAC (MSCV promoter). After manipulation, T cells were stained with a TAC specific reagent and measured using flow cytometry. All constructs show similar levels of surface expression, and EFla induced expression is higher compared to the MSCV construct.
[Fig. 31] exemplifies the relative percentage of T cells expressing TNFα, IFNγ or IL-2 after co-culture with OVCAR3 (HER2 positive) or LOX IMVI (HER2 negative) cells. T cells were engineered with muIgG HER2 TAC (EF1α promoter), TAC construct without HER2 binding domain (Δ binding TAC; EF1α promoter), huIgG HER2 TAC (MSCV promoter) or muIgG HER2 TAC (MSCV promoter). When co-cultured with LOX-IMVI (HER2 ^neg ), the Δbinding TAC control and HER2 TAC cells did not show significant cytokine expression. All HER2 TAC engineered constructs co-cultured with OVCAR3 (HER2 ^pos ) show similar ability to produce cytokines, whereas control T cells engineered with Δ binding TAC do not show significant cytokine production.
Figure 32 illustrates the in vivo efficacy of TAC engineered T cells in the OVCAR3 solid tumor model. T cells were engineered with Δ-binding TAC (EF1α promoter), muIgG HER2 TAC (EF1α promoter), huIgG HER2 TAC (MSCV promoter) or muIgG HER2 TAC (MSCV promoter). Mice were inoculated subcutaneously with OVCAR3 (HER-2 positive) tumors. They grew to a size of about 100 mm ^3. Then, mice were treated with HER2 TAC engineered or Δ-binding TAC control T cells at 48 hour intervals via tail vein injection. Tumor progression was measured and followed every other week. Δ binding TAC did not show tumor control or tumor regression. All HER2 TAC engineered T cells were shown to significantly reduce tumor progression, including tumor regression compared to control mice. All HER2 TAC engineered T cells showed similar anti-tumor activity.

Cancer is a major health problem, with over 150,000 cancer cases expected to be diagnosed in Canada alone. Patients with early stage disease are sometimes effectively treated with conventional therapies (surgery, radiation, chemotherapy), but few options are available for patients with advanced disease, and these options are generally palliative in nature.

Active immunotherapy offers an option for patients who attempt to use the patient's immune system to clear the tumor and have failed conventional therapy. Typically, this therapy involves injecting a large number of tumor-specific T cells into the patient. This approach has been successfully demonstrated in early clinical trials for a number of diseases including melanoma, myeloma, leukemia, lymphoma and synovial sarcoma. As a specific example, several clinical studies have demonstrated that immunotherapy with T cells is therapeutic in patients with advanced melanoma, confirming the usefulness of this approach. In addition, patients suffering from chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) were effectively treated and cured by T cell immunotherapy.

The main problem facing the clinical application of adoptive T cell therapy is the source of T cells. Typically, T cells isolated from tumor bearing patients are cultured in large numbers ex vivo and re-administered to the patient to induce a strong anti-tumor immune response. Tumor specificity is achieved by one of the following: (i) isolating naturally occurring tumor specific T cells from the patient; Or (ii) engineering bulk T cells from peripheral blood to express tumor specific receptors. Naturally occurring tumor-specific T cells are rare, and isolating these cells in therapeutic amounts from cancer patients is a laborious and costly procedure. In contrast, it is becoming more efficient to manipulate readily available peripheral T cells with tumor-specific receptors through genetic manipulation. Technology has been developed for this clinically viable manipulation process, and several clinical trials have demonstrated the viability and efficacy of genetically engineered T cells for cancer treatment.

Up to this point, most engineered T cell therapies involving genetic modification of T cells have resulted in (i) forced expression of the T cell receptor (TCR); Or (ii) a chimeric antigen receptor (CAR) specific for an antigenic target on the tumor. To date, chimeric antigen receptors used to manipulate T cells include (i) targeting domains, generally single chain variable fragments (scFv); (ii) a transmembrane domain; And (iii) a cytosolic domain comprising signal transduction elements from T cell receptors and related proteins. These chimeric antigen receptors have also been referred to as “T-body” or “Chimeric Immune Receptor (CIR)”, but most researchers now use the term “CAR.” One of the CAR approaches One advantage is that any patient's immune cells can be targeted to any desired target in a major histocompatibility complex (MHC) independent manner, which is of interest because MHC presentation is often defective in tumor cells. Is dragging.

CAR is considered from a modular perspective, and scientists have spent considerable time investigating the impact of various cytoplasmic signaling domains on CAR function. Conventional CARs generally contain two main components: (i) a CD3 zeta cytoplasmic domain containing an immunotyrosine activation motif (ITAM) important for T cell activation; And (ii) a component of costimulatory receptors that trigger important survival pathways such as the Akt pathway.

The first generation CAR used a single signaling domain from CD3ζ or FcεRIγ. The second generation CAR bound the signaling domain of CD3ζ with the cytoplasmic domain of a co-stimulatory receptor from the CD28 or TNFR receptor family. Most of the CAR engineered T cells currently being tested in clinical practice use second-generation CARs in which CD3ζ is bound to the cytoplasmic domain of CD28 or CD137. These second generation CARs demonstrated anti-tumor activity in CD19 positive tumors. The third-generation CAR has bound multiple auxiliary stimulating domains, but there is concern that the third-generation CAR may lose antigen specificity.

CAR engineered T cells have shown considerable potential in clinical applications, but they rely on synthetic methods to replace the natural activation signals provided by the T cell receptor (TCR). Because this synthetic receptor does not transmit all of the signaling components associated with TCR (e.g., CD3γ, CD3δ, ITAM on CD3ε), whether T cells are optimally activated by CAR, or that CAR activation results in T cell differentiation (e.g., into memory). It is not clear how it affects the progress of In addition, since the CAR signaling domain is immediately disconnected from the natural regulatory partner due to the nature of the CAR structure, there is an intrinsic risk that the CAR can lead to low levels of constitutive activation, which can lead to off-target toxicity. Thus, the synthetic nature of the prototype CAR can interfere with standard mechanisms that limit TCR activation, and can often support the severe toxicity associated with the therapeutic dose of existing CAR T cells.

Given these limitations, it is desirable to divert T cells to attack tumors through native TCRs. To this end, a class of recombinant proteins called "Bispecific T-cell Engagers" (BiTE) was created. These proteins cross-link the T cell TCR receptor with the target antigen using a bispecific antibody fragment. This induces efficient T cell activation, leading to cytotoxicity. Similarly, bispecific antibodies have been generated that achieve this goal, and some scientists have simply linked anti-CD3 antibodies to tumor specific antibodies using chemical linkage. Although these bispecific proteins have shown some activity in vitro, GMP production, short biological half-life, and limited bioavailability present serious problems for the successful use of these molecules in cancer treatment. In addition, since these molecules do not bind to the TCR co-receptors (CD8 and CD4), they do not properly reproduce natural TCR signaling.

In view of the above, there is still a need for a chimeric receptor with improved activity and safety.

An alternative chimeric receptor called the trifunctional T cell-antigen coupler (Tri-TAC or TAC) receptor has been developed, which uses a separate biology to direct T cells to attack tumors. CARs are fully synthetic receptors that link together the components of the T cell receptor (TCR) signaling complex, while the TAC receptor places TCRs on tumor targets and reproduces the native TCR signaling structure. For example, in some embodiments, the TACs disclosed herein activate natural major histocompatibility complex (MHC) signaling through the T cell receptor (TCR) while maintaining MHC unrestricted targeting. In addition, the TACs disclosed herein recruit T cell receptors (TCRs) in combination with coreceptor stimulation. Moreover, in some embodiments, Tri-TAC disclosed herein exhibits improved activity and safety.

Specific term

The term “T cell” as used herein refers to a type of lymphocyte that plays a central role in cell mediated immunity. T cells, also called T lymphocytes, have a T cell receptor (TCR) on the cell surface that differentiates them from other lymphocytes such as B cells and natural killer cells. There are several subpopulations of T cells with distinct functions including, but not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells and natural killer T cells.

The term “T cell-antigen coupler” or TAC is used interchangeably with “trifunctional T cell-antigen coupler” or Tri-TAC, which, when expressed in T cells, facilitates targeting of T cells to specific antigens. To an engineered nucleic acid construct or polypeptide that is helpful. In some 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.

The terms “polynucleotide” and/or “nucleic acid sequence” and/or “nucleic acid” as used herein refer to a sequence of nucleosides or nucleotide monomers consisting of base, sugar and inter-sugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequence of the present application may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. Sequences can also include modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; And xanthine and hypoxanthine. The nucleic acids of the present disclosure can be isolated from biological organisms, formed by laboratory methods of genetic recombination, or obtained by chemical synthesis or other known protocols for nucleic acid production.

As used herein, the term "isolated polynucleotide" or "isolated nucleic acid sequence" refers to a cellular material or culture medium when produced by recombinant DNA technology, or a chemical precursor, or other chemical substance when chemically synthesized. Refers to nucleic acids without The isolated nucleic acid is also substantially free of sequences that are naturally flanked by the nucleic acid from which the nucleic acid is derived (ie, sequences located at the 5'and 3'ends of the nucleic acid). The term “nucleic acid” is intended to include DNA and RNA and is double stranded or single stranded and refers to the sense or antisense strand. In addition, the term “nucleic acid” includes complementary nucleic acid sequences.

The term “recombinant nucleic acid” or “engineered nucleic acid” as used herein refers to a nucleic acid or polynucleotide that is not found in a biological organism. For example, recombinant nucleic acids can be formed by laboratory methods of genetic recombination (eg, molecular cloning) to produce sequences not otherwise found in nature. Recombinant nucleic acids may also be produced by chemical synthesis or other known protocols for nucleic acid production.

As used herein, the term “polypeptide” or “protein” describes an amino acid. Polypeptides or proteins of the present disclosure are generally peptides describing amino acids. The term protein as used herein also describes a large molecule comprising one or more amino acids, and in some embodiments is a fragment or domain of a protein or a full-length protein. Also, as used herein, the term protein refers to an amino acid that has been processed and folded into a linear or functional protein of amino acids. The protein structure is divided into four distinct levels: (1) the primary structure representing the amino acid sequence of the polypeptide chain, (2) the regular local partial structure of the polypeptide backbone chain, such as 2 representing the α-helix and β-sheet. A secondary structure, (3) a tertiary structure representing a three-dimensional structure in the case of a monomeric and multimeric protein molecule, and (4) a quaternary structure representing the three-dimensional structure forming an aggregation of two or more individual polypeptide chains acting as a single functional unit. rescue. Proteins of the present disclosure, in some embodiments, by isolating and purifying them from cells from which the proteins are naturally produced, by enzymatic (e.g., proteolytic) cleavage, and/or by removing a protein or fragment of the present disclosure. It is obtained recombinantly by expression of the encoding nucleic acid. In some embodiments, proteins and/or fragments of the present disclosure are obtained by chemical synthesis or other known protocols for producing 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 technology, or chemical precursors or other chemical substances 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. Antibodies can be derived from recombinant sources and/or produced in transgenic animals. As used herein, the term "antibody fragment" refers to, without limitation, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific It is intended to include enemy antibody fragments and domain antibodies.

As used herein, the term “vector” refers to a polynucleotide used to deliver a nucleic acid into a cell. In some embodiments, the vector is an expression vector comprising an expression control sequence (eg, 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, phages, cosmids and viruses.

The term “tumor antigen” or “tumor associated antigen” as used herein refers to an antigenic substance produced in tumor cells that elicit an immune response in the host (eg, provided by an MHC complex). In some embodiments, the tumor antigen is on the surface of the tumor cells.

The term “T cell receptor” or TCR as used herein refers to a complex of intrinsic membrane proteins that participate in the activation of T cells in response to antigen binding. TCRs are disulfide-linked membrane-fixed heterodimers consisting of highly variable alpha (α) and beta (β) chains that are generally expressed as part of a complex with a constant CD3 (differentiation cluster 3) chain molecule. T cells expressing this receptor are referred to as α:β (or αβ) T cells, but a small number of T cells express alternative receptors formed by variable gamma (γ) and delta (δ) chains called γδ T cells. . CD3 is a protein complex composed of four distinct chains. In mammals, the complex comprises a CD3γ chain, a CD3δ chain, two CD3ε chains and two CD3ζ chains.

As used herein, the term “transmembrane and cytosolic domain” refers to a polypeptide comprising the transmembrane domain and cytosolic domain of a protein associated with a T cell receptor (TCR) complex. In some embodiments, such transmembrane and cytosolic domains may include, but are not limited to, protein domains that (a) bind to lipid rafts and/or (b) bind to Lck.

As used herein, “TCR coreceptor” refers to a molecule that assists the T cell receptor (TCR) in communication with antigen presenting cells, and can be considered as part of the first signal that induces the activation of TCR. Examples of TCR co-receptors include, but are not limited to, CD4, LAG3, and CD8.

As used herein, a “TCR co-stimulator” refers to a molecule that enhances the response of T cells to an antigen and can be considered as a second signal that induces activation of TCR. Examples of TCR co-stimulants include ligands that specifically bind to CD83, ICOS, CD27, CD28, 4-1BB (CD 137), OX40 (CD134), CD30, CD40, lymphocyte function related antigen 1 (LFA-1), and CD2. , CD7, LIGHT, NKG2C, and B7-H3. .

As used herein, “TCR adjuvant inhibitor” or “checkpoint receptor” refers to a molecule that inhibits the response of T cells to an antigen. Examples of TCR adjuvant inhibitors include, but are not limited to, PD-1, TIM3, LAG-3, TIGIT, BTLA, CD160, and CD37.

The terms “recipient”, “subject”, “subject”, “host” and “patient” are used interchangeably herein and, in some embodiments, any mammalian subject in need of diagnosis, treatment, or therapy, particularly human Refers to. "Mammals" for therapeutic purposes include humans, livestock and farm animals, laboratory, zoo, sports or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. It refers to all animals classified as mammals, including. In some embodiments, the mammal is human. Neither of these terms requires medical supervision.

As used herein, the terms “treatment”, “treating”, and the like, in some embodiments refer to administering an agent or performing a procedure for the purpose of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of affecting partial or complete cure for a disease and/or symptom of a disease. As used herein, “treatment” may include treatment of a disease or disorder (eg, cancer) in a mammal, particularly a human, and includes: (a) the disease or symptom of a disease is a disease Preventing it from occurring in a subject that may be susceptible to, but has not yet been diagnosed with, a disease (including, for example, a disease associated with or may be caused by a primary disease); (b) inhibiting the disease, ie preventing its occurrence; And (c) alleviating the disease, ie causing regression of the disease. Treatment can mean any indication of success in the treatment or amelioration or prevention of cancer. This includes the relief; ease; Decreased symptoms; Or a disease condition that the patient can better tolerate; Delay in the rate of regression or decline; Or all objective or subjective parameters, such as a reduction in the weakening of the final point of regression. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of the physician's examination. Thus, the term “treatment” includes administration of a compound or agent of the present invention to prevent, delay, alleviate, arrest or inhibit the occurrence of a symptom or condition associated with a disease (eg, cancer). The term “therapeutic effect” refers to the reduction, elimination, or prevention of a disease, symptom of a disease, or side effect of a disease in a subject.

As used herein, the singular forms “a”, “and” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “antibody” includes multiple antibodies, and in some embodiments reference to “antibody” includes multiple antibodies, and the like.

As used herein, all numerical values or numerical ranges include all integers within or encompassing such ranges and values or fractions of integers within or encompassing such ranges, unless the context clearly indicates otherwise. Thus, for example, references to the range of 90 to 100% are 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, This includes 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, reference to a range of 1 to 5,000 times is 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.

As used herein, a “about” number includes a number and means a range between 10% below and 10% above the number. A “about” range refers to a range 10% lower than the lower limit of the range and 10% higher than the upper limit of the range.

"Percent identity (%)" refers to the degree to which two sequences (nucleotides or amino acids) have the same residue at the same position in the alignment. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" means the% identity of the amino acid sequence to SEQ ID NO: Y, and describes that the residue X% of the amino acid sequence is identical to the residue of the sequence disclosed in SEQ ID NO: Y. do. Typically, computer programs are used for these calculations. Exemplary programs for comparing and aligning sequence pairs are 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 "selective binding" refers to a molecule (e.g., a protein such as a target binding ligand of a TAC) than another molecule (e.g., HER-2, BCMA, or CD19). Target antigen).

T cell-antigen coupler ( Tri -TAC or TAC)

In certain embodiments, disclosed herein are nucleic acids encoding a trifunctional T cell-antigen coupler (Tri-TAC). In some 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 the TCR complex; And (c) a third polynucleotide encoding a transmembrane domain and a cytosolic domain. In some embodiments, the nucleic acid encoding Tri-TAC does not encode a secondary stimulation domain. In some embodiments, the nucleic acid encoding Tri-TAC does not encode a secondary activation domain.

Target specific ligand

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 some embodiments, a target specific ligand binds an antigen on a target cell. In some embodiments, the target cells include cancer, hematologic malignancies, large B cell lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high grade B cell lymphoma, or large B cell lymphoma arising from follicular lymphoma. Cells associated with a disease state, but not limited thereto. In some embodiments, the target cell is a tumor cell. In some embodiments, the target specific ligand binds to a tumor antigen or a tumor associated antigen on a tumor cell. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the tumor antigen, when proteinaceous, is a sequence from 8 or more amino acids to the entire protein. In some embodiments, the tumor antigen is any number of amino acids from 8 to full-length protein comprising at least one antigenic fragment of a full-length protein presented in a Major Histocompatibility Complex (MHC). Examples of tumor antigens include CD19, HER-2 (erbB-2), B-cell maturation antigen (BCMA), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), and CA. -125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), prostate-specific antigen (PSA), glioma-related antigen , β-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF , Prostase, PAP, NY-ESO-1, LAGE-1a, p53, prosteine, PSMA, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1) ), ELF2M, neutrophil elastase, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In some embodiments, target specific ligands are antibodies and fragments thereof that bind target cells and/or antigens, e.g., single chain antibodies such as single chain antibodies (scFv), single domain antibodies, peptides, peptidomimetics, Proteins, glycoproteins, or proteoglycans include, but are not limited to. In some embodiments, the target specific ligand is a designed ankyrin repeat protein (DARPin), lectin, notin, centirin, anticalin, or a naturally occurring ligand such as a growth factor, enzyme substrate, receptor or binding protein for a tumor antigen. Including but not limited to. In some embodiments, target specific ligands include non-protein compounds that bind to target cells and/or antigens, including, but not limited to, carbohydrates, lipids, nucleic acids, or small molecules. In some embodiments, the target specific ligand is a designed ankyrin repeat (DARPin) that targets specific cells and/or antigens. In some embodiments, the target specific ligand is a single chain variable fragment (ScFv) that targets a specific cell and/or antigen.

In some embodiments, the tumor antigen is a HER-2 antigen. In some embodiments, the HER-2 specific ligand is trastuzumab, pertuzumab, lapatinib, neratinib, ado-trastuzumab emtansine, gankotamab, margetuximab, timigutuzumab, and ertumaxomab. And the antigen binding domain of the antibody selected from. In some embodiments, the target specific ligand is DARPin that selectively binds to the HER-2 (erbB-2) antigen. In some embodiments, the target specific ligand is DARPin that specifically binds to the HER-2 (erbB-2) antigen. In some embodiments, the DARPin targeting HER-2 (erb-2) comprises SEQ ID NO: 7 or SEQ ID NO: 8.

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

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

In some embodiments, the tumor antigen is a BCMA antigen. In some embodiments, the BCMA specific ligand comprises an antigen binding domain of an antibody selected from belantamab mapodotin, and GSK2857916. In some embodiments, the target specific ligand is a scFv that selectively binds BCMA. In some embodiments, the target specific ligand is a scFv that specifically binds BCMA. In some embodiments, the scFv that binds BCMA comprises SEQ ID NO: 33 or SEQ ID NO: 34.

In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 33. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 33. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 33. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 33. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33. In some embodiments, the first polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 33.

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

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

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

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

TCR Ligand that binds to the complex

In some embodiments, the TAC comprises a ligand that binds to a protein associated with the TCR complex. In some embodiments, the ligand that binds to the protein associated with the TCR complex comprises a substance that directly or indirectly binds to the protein of TCR. In some embodiments, the ligand that binds to the protein associated with the TCR complex comprises a substance that selectively binds to the protein of TCR. In some embodiments, the ligand that binds to the protein associated with the TCR complex comprises a substance that specifically binds to the protein of TCR. Proteins related to TCR include, but are not limited to, TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3γ chain, CD3δ chain and CD3ε chain. In some embodiments, the ligand that binds to the protein associated with the TCR complex is a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, a CD3γ chain, a CD3δ chain, and/or Or an antibody against the CD3ε chain. In some embodiments, the protein associated with the TCR complex is CD3. In some embodiments, the protein associated with the TCR complex is CD3ε. Examples of CD3 antibodies include, but are not limited to: In some embodiments, the antibody that binds CD3 is a single chain antibody, eg, a single chain variable fragment (scFv). In some embodiments, the ligand that binds the TCR is an anti-CD3 antibody, or a fragment thereof, such as muromonab, otelixizumab, teplizumab, visilizumab, CD3-12, MEM-57, 4D10A6, CD3D, or TR66.

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

In some embodiments, the ligand that binds the TCR complex is UCHT1, or a variant thereof. In some embodiments, the ligand that binds the TCR complex is UCHT1 (SEQ ID NO: 13, SEQ ID NO: 14, or a homologue thereof). In some embodiments, the UCHT1 ligand binds to CD3. In some embodiments, the UCHT1 ligand selectively binds to CD3. In some embodiments, the UCHT1 ligand specifically binds to CD3. In some embodiments, the UCHT1 ligand binds CD3ε. In some embodiments, the UCHT1 ligand selectively binds to CD3ε. In some embodiments, the UCHT1 ligand specifically binds CD3ε. In some embodiments, the UCHT1 ligand is encoded by SEQ ID NO: 13. In some embodiments, the UCHT1 ligand comprises SEQ ID NO: 14. In some embodiments, the UCHT1 ligand is mutated. In some embodiments, the UCHT1 ligand comprises the Y182T mutation (also referred to as UCHT1 (Y182T)) (SEQ ID NO: 71 and SEQ ID NO: 72). In some embodiments, the UCHT1 (Y182T) ligand binds CD3. In some embodiments, the UCHT1 (Y182T) ligand selectively binds to CD3. In some embodiments, the UCHT1 (Y182T) ligand specifically binds CD3. In some embodiments, the UCHT1 (Y182T) ligand binds CD3ε. In some embodiments, the UCHT1(Y182T) ligand selectively binds CD3ε. In some embodiments, the UCHT1 (Y182T) ligand specifically binds CD3ε. In some embodiments, the UCHT1 (Y182T) ligand is encoded by SEQ ID NO: 71. In some embodiments, the UCHT1 (Y182T) ligand comprises SEQ ID NO: 72. In some embodiments, the ligand that binds the TCR complex is humanized UCHT1 (huUCHT1). In some embodiments, the ligand that binds the TCR complex is huUCHT1 (SEQ ID NO: 43, SEQ ID NO: 44, or a homolog thereof). In some embodiments, the huUCHT1 ligand binds CD3. In some embodiments, the huUCHT1 ligand selectively binds to CD3. In some embodiments, the huUCHT1 ligand specifically binds CD3. In some embodiments, the huUCHT1 ligand binds CD3ε. In some embodiments, the huUCHT1 ligand selectively binds CD3ε. In some embodiments, the huUCHT1 ligand specifically binds CD3ε. In some embodiments, the huUCHT1 ligand is encoded by SEQ ID NO: 43. In some embodiments, the huUCHT1 ligand comprises SEQ ID NO: 44. In some embodiments, huUCHT1 has a Y177T mutation (also referred to as huUCHT1 (Y177T)) (SEQ ID NO: 45 and SEQ ID NO: 46). In some embodiments, the huUCHT1(Y177T) ligand binds CD3. In some embodiments, the huUCHT1(Y177T) ligand selectively binds to CD3. In some embodiments, the huUCHT1(Y177T) ligand specifically binds CD3. In some embodiments, the huUCHT1(Y177T) ligand binds CD3ε. In some embodiments, the huUCHT1(Y177T) ligand selectively binds CD3ε. In some embodiments, the huUCHT1(Y177T) ligand specifically binds CD3ε. In some embodiments, the huUCHT1(Y177T) ligand is encoded by SEQ ID NO: 45. In some embodiments, the huUCHT1 ligand comprises SEQ ID NO: 46.

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

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

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

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

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

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

In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 45. In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 45. In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 45. In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 45. In some embodiments, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 45.

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

In some embodiments, the ligand that binds CD3 is OKT3. In some embodiments, the OKT3 ligand binds to CD3. In some embodiments, the OKT3 ligand selectively binds to CD3. In some embodiments, the OKT3 ligand specifically binds CD3. In some embodiments, the OKT3 ligand binds CD3ε. In some embodiments, the OKT3 ligand selectively binds to CD3ε. In some embodiments, the OKT3 ligand specifically binds CD3ε. In some embodiments, the murine OKT3 ligand is encoded by SEQ ID NO: 21. In some embodiments, the OKT3 ligand comprises SEQ ID NO: 22.

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

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

In some embodiments, the ligand that binds CD3 is F6A. In some embodiments, the F6A ligand binds CD3. In some embodiments, the F6A ligand selectively binds to CD3. In some embodiments, the F6A ligand specifically binds CD3. In some embodiments, the F6A ligand binds CD3ε. In some embodiments, the F6A ligand selectively binds CD3ε. In some embodiments, the F6A ligand specifically binds CD3ε. In some embodiments, the murine F6A ligand is encoded by SEQ ID NO: 23. In some embodiments, the F6A ligand comprises SEQ ID NO: 24.

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

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

In some embodiments, the ligand that binds CD3 is L2K. In some embodiments, the L2K ligand binds to CD3. In some embodiments, the L2K ligand selectively binds to CD3. In some embodiments, the L2K ligand specifically binds to CD3. In some embodiments, the L2K ligand binds CD3ε. In some embodiments, the L2K ligand selectively binds to CD3ε. In some embodiments, the L2K ligand specifically binds CD3ε. In some embodiments, the murine L2K ligand is encoded by SEQ ID NO: 25. In some embodiments, the L2K ligand comprises SEQ ID NO: 26.

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

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

Transmembrane domain and Cytosol domain

In some embodiments, the T cell-antigen coupler comprises a T cell receptor signaling domain polypeptide. In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain. In some embodiments, the TCR signaling domain polypeptide comprises a cytosolic domain. In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and a cytosolic domain. In some embodiments, the cytosolic domain and the transmembrane domain are optionally linked by a linker. In some embodiments, the T cell receptor signaling domain polypeptide comprises a TCR coreceptor domain. In some embodiments, the T cell receptor signaling domain polypeptide does not comprise a TCR co-stimulatory domain. In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytosolic domain of a TCR co-receptor. In some embodiments, the TCR coreceptor is CD4, CD8, LAG3, or a chimeric variant thereof.

In some embodiments, the TCR coreceptor is CD4. In some embodiments, the TCR signal transduction domain polypeptide comprises the transmembrane and cytosolic domains of the CD4 coreceptor encoded by SEQ ID NO: 17. In some embodiments, the TCR signaling domain polypeptide comprises the transmembrane and cytosolic domains of a CD4 coreceptor comprising SEQ ID NO: 18.

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

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

In some embodiments, the TCR coreceptor is CD8. In some embodiments, the TCR coreceptor is CD8α. In some embodiments, the TCR signal transduction domain polypeptide comprises the transmembrane and cytosolic domains of the CD8α coreceptor encoded by SEQ ID NO: 37. In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane and cytoplasmic domain of a CD8α coreceptor comprising SEQ ID NO: 38.

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

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

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

In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 39. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 39. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 39. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 39. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 39. In some embodiments, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 39.

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

In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 41. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 41. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 41. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the third polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 41. In some embodiments, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 41.

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

In some embodiments, the T cell receptor signaling domain polypeptide comprises a TCR co-stimulatory domain. In some embodiments, the TCR co-stimulatory agent is ICOS. In some embodiments, the TCR co-stimulatory agent is CD27. In some embodiments, the TCR co-stimulatory agent is CD28. In some embodiments, the TCR co-stimulatory agent is 4-1BB (CD137). In some embodiments, the TCR co-stimulatory agent is OX40 (CD134). In some embodiments, the TCR co-stimulatory agent is CD30. In some embodiments, the TCR co-stimulatory agent is CD40. In some embodiments, the TCR co-stimulatory agent is lymphocyte function related antigen 1 (LFA-1). In some embodiments, the TCR co-stimulatory agent is CD2. In some embodiments, the TCR co-stimulatory agent is CD7. In some embodiments, the TCR auxiliary stimulator is LIGHT. In some embodiments, the TCR co-stimulatory agent is NKG2C. In some embodiments, the TCR co-stimulatory agent is B7-H3. In some embodiments, the TCR co-stimulatory agent is a ligand that specifically binds CD83.

In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytosolic domain of a TCR auxiliary inhibitor. In some embodiments, the TCR adjuvant inhibitor is PD-1. In some embodiments, the TCR adjuvant inhibitor is TIM3. In some embodiments, the TCR adjuvant inhibitor is LAG-3. In some embodiments, the TCR adjuvant inhibitor is TIGIT. In some embodiments, the TCR adjuvant inhibitor is BTLA. In some embodiments, the TCR adjuvant inhibitor is CD160. In some embodiments, the TCR adjuvant inhibitor is CD37.

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

Linker, coupler , And configuration

In some embodiments, a nucleic acid disclosed herein comprises (1) a first polynucleotide encoding a target specific ligand; (2) a second polynucleotide encoding a ligand that binds to the TCR complex; (3) The sequence of the third polynucleotide encoding the transmembrane domain and the cytosolic domain. In some embodiments, a nucleic acid disclosed herein comprises (1) a first polynucleotide encoding a target specific ligand; (2) a second polynucleotide encoding a ligand that binds to the TCR complex; (3) the sequence of the third polynucleotide encoding the transmembrane domain and the cytosolic domain, wherein the sequence is from 5'end to 3'end. In some embodiments, a nucleic acid disclosed herein comprises (1) a first polynucleotide encoding a target specific ligand; (2) a second polynucleotide encoding a ligand that binds to the TCR complex; (3) the sequence of the third polynucleotide encoding the transmembrane domain and the cytosolic domain, where the sequence is from 3'end to 5'end. In some embodiments, a nucleic acid described herein comprises (1) a first polynucleotide encoding a ligand that binds to the TCR complex; (2) a second polynucleotide encoding a target specific ligand; (3) The sequence of the third polynucleotide encoding the transmembrane domain and the cytosolic domain. In some embodiments, a nucleic acid described herein comprises (1) a first polynucleotide encoding a ligand that binds to the TCR complex; (2) a second polynucleotide encoding a target specific ligand; (3) the sequence of the third polynucleotide encoding the transmembrane domain and the cytosolic domain, wherein the sequence is from 5'end to 3'end. In some embodiments, a nucleic acid described herein comprises (1) a first polynucleotide encoding a ligand that binds to the TCR complex; (2) a second polynucleotide encoding a target specific ligand; (3) the sequence of the third polynucleotide encoding the transmembrane domain and the cytosolic domain, where the sequence is from 3'end to 5'end.

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

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

In some embodiments, the peptide linker comprises a G ₄ S ₃ linker (SEQ ID NO: 74). In some embodiments, the peptide linker comprises SEQ ID NO: 11, 12, 15, 16, 19, 20, or a variant or fragment thereof.

In some embodiments, a peptide linker that connects a target specific ligand to a ligand that binds to a TCR complex (e.g., UCHT1) is known as a linker to distinguish this protein domain from other linkers in Tri-TAC. The connector is of any size. In some embodiments, the linker between the ligand binding to the TCR complex and the target specific ligand is a short spiral comprising SEQ ID NO: 28. In some embodiments, the linker between the ligand binding to the TCR complex and the target specific ligand is a short spiral encoded by SEQ ID NO: 27. In some embodiments, the linker between the ligand binding to the TCR complex and the target specific ligand is a long spiral comprising SEQ ID NO: 30. In some embodiments, the linker between the ligand binding to the TCR complex and the target specific ligand is a long spiral encoded by SEQ ID NO: 29. In some embodiments, the linker between the ligand binding to the TCR complex and the target specific ligand is a large domain comprising SEQ ID NO:32. In some embodiments, the linker between the ligand that binds the TCR complex and the target specific ligand is a large domain encoded by SEQ ID NO: 31.

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

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

Tri-TAC is contemplated to exist in various configurations and combinations of (a) a target specific ligand, (b) a ligand that binds to the TCR complex, and (c) a TCR signaling domain as disclosed herein.

In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) a single chain antibody that binds to CD3ε (scFv), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) UCHT1, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) OKT3, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) F6A, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) L2K, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor.

In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) DARPin, (b) UCHT1, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises (a) DARPin, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) DARPin, (b) huUCHT1, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises (a) DARPin, (b) huUCHT1 (Y177T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) DARPin, (b) OKT3, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises (a) DARPin, (b) F6A, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) DARPin, (b) L2K, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor.

In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) UCHT1, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises (a) scFv, (b) UCHT1 (Y182T), and (c) transmembrane and cytosolic domains of the CD4 co-receptor. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) huUCHT1, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises (a) scFv, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) OKT3, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) F6A, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) L2K, and (c) CD4 coreceptors.

In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) UCHT1, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) OKT3, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) F6A, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) L2K, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor.

In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) UCHT1, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) UCHT1 (Y182T), and (c) transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) OKT3, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) F6A, and (c) transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) L2K, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor.

In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) OKT3, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) F6A, and (c) the transmembrane and cytosolic domains of the CD4 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) L2K, and (c) the transmembrane and cytosolic domains of a CD4 coreceptor.

In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) a single chain antibody that binds to CD3ε (scFv), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) UCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) OKT3, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) F6A, and (c) the transmembrane and cytosolic domains of a CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) L2K, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor.

In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) DARPin, (b) UCHT1, and (c) CD4 coreceptors. In some embodiments, Tri-TAC comprises (a) DARPin, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) DARPin, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) DARPin, (b) huUCHT1 (Y177T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) DARPin, (b) OKT3, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) DARPin, (b) F6A, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) DARPin, (b) L2K, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor.

In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) UCHT1, and (c) CD8 coreceptors. In some embodiments, Tri-TAC comprises (a) scFv, (b) UCHT1 (Y182T), and (c) transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) huUCHT1, and (c) CD8 coreceptors. In some embodiments, Tri-TAC comprises (a) scFv, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) OKT3, and (c) CD8 coreceptors. In some embodiments, Tri-TAC comprises the transmembrane and cytosolic domains of (a) scFv, (b) F6A, and (c) CD8 coreceptors. In some embodiments, Tri-TAC comprises (a) scFv, (b) L2K, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor.

In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) UCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) UCHT1(Y182T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a target specific ligand, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) OKT3, and (c) the transmembrane and cytosolic domains of a CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) F6A, and (c) the transmembrane and cytosolic domains of a CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a HER-2 specific DARPin, (b) L2K, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor.

In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) UCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) UCHT1 (Y182T), and (c) transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) huUCHT1, and (c) transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) huUCHT1(Y177T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) OKT3, and (c) transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) F6A, and (c) transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) BCMA specific ScFv, (b) L2K, and (c) transmembrane and cytosolic domains of the CD8 coreceptor.

In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) UCHT1 (Y182T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) huUCHT1 (Y177T), and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) OKT3, and (c) the transmembrane and cytosolic domains of a CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) F6A, and (c) the transmembrane and cytosolic domains of the CD8 coreceptor. In some embodiments, Tri-TAC comprises (a) a CD19 specific ScFv, (b) L2K, and (c) the transmembrane and cytosolic domains of a CD8 coreceptor.

In some embodiments, Tri-TAC pulls CD3 and TCR into the lipid raft region of the membrane and brings Lck close to the TCR, which is similar to native MHC binding.

In some 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), in sequence:

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

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

iii) Myc tag (SEQ ID NOs: 9 and 10)

iv) linker (SEQ ID NOs: 11 and 12)

v) UCHT1 (SEQ ID NOs: 13 and 14)

vi) linker (SEQ ID NOs: 15 and 16)

vii) CD4 (SEQ ID NOs: 17 and 18).

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

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

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

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

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

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

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

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

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

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

Polypeptides and Vector Constructs

In certain embodiments, disclosed herein are polypeptides encoded by nucleic acid sequences as disclosed herein. Also disclosed herein are vectors comprising a nucleic acid sequence as disclosed herein. In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is functional in mammalian cells. Promoters, which are DNA regions that initiate transcription of specific nucleic acid sequences, are well known in the art. “A functional promoter in a mammalian cell” refers to a promoter that drives the expression of an associated nucleic acid sequence in a mammalian cell. A promoter that drives the expression of a nucleic acid sequence is said to be "operably linked" to the nucleic acid sequence.

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

In certain embodiments, disclosed herein are polynucleotides included in a vector to provide a vector construct, also referred to herein as a vector. In some 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 the protein associated with the TCR complex;

c. A third polynucleotide encoding a T cell receptor signaling domain polypeptide; And

d. Functional promoter in mammalian cells.

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

In some embodiments, the ligand that binds to the protein associated with the TCR complex is UCHT1, humanized UCHT1 (huUCHT1), OKT3, F6A, or L2K. In some embodiments, the ligand that binds to the protein associated with the TCR complex is UCHT1, or a variant thereof. In some embodiments, the ligand that binds to the protein associated with the TCR complex is UCHT1 and is encoded by SEQ ID NO: 13. In some embodiments, the ligand that binds the protein associated with the TCR complex is UCHT1 and comprises SEQ ID NO: 14. In some embodiments, the UCHT1 ligand that binds the protein associated with the TCR complex has the Y182T mutation (UCHT1 (Y182T)) and is encoded by SEQ ID NO: 71. In some embodiments, the ligand that binds the protein associated with the TCR complex is UCHT1 (Y182T) and comprises SEQ ID NO: 72. In some embodiments, the ligand that binds to the protein associated with the TCR complex is humanized UCHT1 (huUCHT1), or a variant thereof. In some embodiments, the ligand that binds the protein associated with the TCR complex is humanized UCHT1 (huUCHT1) and is encoded by SEQ ID NO: 43. In some embodiments, the ligand that binds the protein associated with the TCR complex is huUCHT1 and comprises SEQ ID NO: 44. In some embodiments, the huUCHT1 ligand that binds the protein associated with the TCR complex has the Y177T mutation (huUCHT1 (Y177T)) and is encoded by SEQ ID NO: 45. In some embodiments, the ligand that binds the protein associated with the TCR complex is huUCHT1 (Y177T) and comprises SEQ ID NO: 46.

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

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

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

In some embodiments, the protein associated with the TCR complex is CD3. In some embodiments, the protein associated with the TCR complex is CD3ε.

In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytosolic domain of a TCR co-receptor. In some embodiments, the TCR coreceptor is CD4, CD8, LAG3, or a chimeric variant thereof.

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

In some embodiments, useful vectors include vectors derived from lentivirus, Murine Stem Cell Viruse (MSCV), poxvirus, oncoretrovirus, adenovirus, and adeno-associated virus. Other useful transfer vectors include vectors derived from herpes simplex virus, transposon, vaccinia virus, human papilloma virus, simian immunodeficiency virus, HTLV, human foam-forming virus and variants thereof. Additional useful vectors include vectors derived from spumavirus, mammalian type B retrovirus, mammalian type C retrovirus, avian type C retrovirus, mammalian type D retrovirus and HTLV/BLV type retrovirus. One example of a lentiviral vector useful in the disclosed compositions and methods is the pCCL4 vector.

In some embodiments, the nucleic acid is a recombinant or engineered nucleic acid. In some embodiments, the first, second and/or third polynucleotide is a recombinant or engineered polynucleotide. In some embodiments, 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 coupler. In some embodiments, the nucleic acid encodes a polypeptide.

In some embodiments, modifications are made to polynucleotide sequences comprising the vector sequences and polypeptide sequences disclosed herein. Modifications include substitutions, insertions or deletions of nucleotides or amino acids, or alteration of the relative position 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-TAC disclosed herein. In some embodiments, T cells express Tri-TAC disclosed herein. Further disclosed herein are T cells transduced or transfected with a vector comprising a T cell-antigen coupler or Tri-TAC. In some embodiments, the T cell is an isolated T cell.

In some embodiments, human T cells engineered to express Tri-TAC display functionality equivalent to a conventional CAR in vitro. In some embodiments, T cells engineered with Tri-TAC exhibit superior functionality in vitro than conventional CARs. In some embodiments, disclosed herein are human T cells engineered with Tri-TAC that exhibit improved safety compared to conventional CARs. In some embodiments, human T cells engineered to express Tri-TAC show improved safety compared to conventional CARs.

In some embodiments, T cells include, but are not limited to, blood (e.g., peripheral blood mononuclear cells), bone marrow, thymus tissue, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, spleen tissue, or tumor. Are obtained from a number of sources. In some embodiments, the T cell is an autologous T cell. In some embodiments, T cells are obtained from a cell line of T cells. In some embodiments, the T cells are obtained from a donor (allogeneic T cells). In some embodiments, T cells are obtained from pluripotent stem cells induced or by differentiation of embryonic or adult stem cells. In some embodiments, regardless of the source of T cells, the T cells are modified to lack the expression of endogenous TCR and/or permanently or temporarily lack the expression of MHC/HLA molecules (universal donor T cells). In some embodiments, the T cells are autologous to the subject. In some embodiments, the cells are allogeneic, syngeneic, or heterologous to the subject.

In some embodiments, once obtained, T cells are selectively enriched in vitro. In some embodiments, the cell population is enriched by positive or negative selection. Additionally, T cells are optionally frozen or cryopreserved and then thawed later.

In some embodiments, the T cell is activated and/or expanded before or after introducing Tri-TAC into the T cell. In some embodiments, T cells are expanded by contact with a surface to which an agent that stimulates a CD3/TCR complex related signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cell is attached. In some embodiments, T cells are expanded by contact with one or more soluble agents that stimulate CD3/TCR complex signaling and co-stimulatory molecule signaling.

In some embodiments, T cells are transduced or transfected with a nucleic acid sequence. Transduced or transfected T cells express the protein encoded by the transfected or transduced nucleic acid sequence. Nucleic acids can be introduced into cells by physical, chemical or biological means. Physical means include, but are not limited to, microinjection, electroporation, particle 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 into T cells. Viral vectors include vectors derived from lentivirus, murine stem cell virus (MSCV), poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus. The vector optionally includes a promoter (eg, CMV promoter, eF1a promoter, or MSCV promoter) that induces expression of the transduced nucleic acid molecule in T cells.

Any suitable assay is used to confirm the presence and/or expression of the transduced nucleic acid sequence and/or the polypeptide encoded by the nucleic acid in the T cell. Assays include, but are not limited to, Southern and Northern blotting, RT-PCR and PCR, ELISA, Western blotting, and flow cytometry.

T cells expressing TAC have increased T cell activation in the presence of an antigen compared to T cells that do not express TAC and/or compared to T cells expressing a conventional CAR. Increased T cell activation includes, but is limited to, increased tumor cell line death, increased cytokine production, increased cell degradation, increased degranulation and/or increased expression of activating markers such as CD107α, IFNγ, IL2 or TNFα. It is confirmed by numerous methods that do not. In some embodiments, the increase is measured in individual cells or cell populations.

As used herein, the term “increased” or “increasing” refers to TAC compared to a T cell or T cell population that does not express TAC cells and/or to a T cell or T cell population expressing a conventional CAR. It refers to an increase of at least 1%, 2%, 5%, 10%, 25%, 50%, 100% or 200% in a cell or T cell population expressing the cell.

Pharmaceutical composition

In certain embodiments, a pharmaceutical composition comprising an engineered T cell disclosed herein (transduced with TAC and/or expressing TAC), and a pharmaceutically acceptable carrier is disclosed. Pharmaceutically possible carriers include buffers such as neutral buffered saline and phosphate buffered saline; Carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; Amino acids such as polypeptide or glycine; Antioxidants; Chelating agents such as EDTA or glutathione; Adjuvants (eg, aluminum hydroxide); Or preservatives, but is not limited thereto. In some embodiments, 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 dosage and frequency of administration are determined by factors such as the patient's condition, and the type and severity of the patient's disease, but the appropriate dosage is determined by clinical trials. When “immunologically effective amount”, “anti-tumor effective amount”, “tumor inhibitory effective amount” or “therapeutic amount” is indicated, the exact amount of the composition of the invention administered is age, body weight, tumor size, degree of infection or metastasis, and patient It is determined by the doctor in consideration of individual differences in the state of the (subject).

In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are 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 10 cells per kg body weight. ⁸ cells, 10 ⁶ to 10 ⁷ cells per kg body weight, or 10 ⁵ to 10 ⁶ cells per kg body weight, including all integer values within that range. In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of greater than ^{10 1 cells per kg body weight.} In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of less than ^{10 15 cells per kg body weight.}

In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are 0.5 x 10 ⁶ cells, 2 x 10 ⁶ cells, 4 x 10 ⁶ cells, 5 x 10 ⁶ cells, 1.2 x 10 ⁷ cells, 2 x 10 ⁷ cells, 5 x10 ⁷ cells, 2 x10 ⁸ cells, 5 x10 ⁸ cells, 2 x10 ⁹ cells, 0.5-2000 x10 ⁶ cells, 0.5-2 x10 ⁶ cells, 0.5-2 x10 ^{7 cells} Cells, 0.5-2 x 10 ⁸ cells, or 0.5-2 x 10 ⁹ cells (including all integer values within that range) are administered at a dose.

In some embodiments, the T cell composition is administered multiple times at such dosages. In some embodiments, the dosage is administered once or several times, such as daily, weekly, biweekly, or monthly, hourly, or upon recurrence, exacerbation, or progression of the cancer being treated. In some embodiments, cells are administered using infusion techniques commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

Pharmaceutical compositions include, for example, endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, mixed human serum , Bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria, fungi, mycoplasma, substantially free of contaminants, selected from the group consisting of IL-2 and IL-7. For example, there are no detectable levels of contaminants.

In some embodiments, after administering the engineered T cells disclosed herein to a subject, blood is redrawn (or apheresis is performed), T cells therefrom are activated and reinjected into the patient with engineered T cells. . In some embodiments, this process is performed several times every few weeks. T cells are activated from 10 cc to 400 cc blood collection. T cells are activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc blood collection.

Modified/engineered T cells and/or pharmaceutical compositions are administered by methods including, but not limited to, aerosol inhalation, injection, infusion, ingestion, blood transfusion, or implantation or transplantation. Modified T cells and/or pharmaceutical compositions are carotid, subcutaneous, intradermal, intratumoral, intranodal, intrathecal, intramuscular, intravenous (iv) injection, intravenous (iv) injection, or intraperitoneal It is administered to the subject. The modified/engineered T cells and/or pharmaceutical compositions thereof are administered to the patient by intradermal or subcutaneous injection. Modified/engineered T cells and/or pharmaceutical compositions thereof are i.v. It is administered by injection. The modified/engineered T cells and/or pharmaceutical compositions thereof are injected directly into the tumor, lymph node, or site of infection.

Modified/engineered T cells and/or pharmaceutical compositions are about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

Modified/engineered T cells and/or pharmaceutical compositions up to about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or more than 500 mL.

Modified/engineered T cells and/or pharmaceutical compositions are at least about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, It is administered in volumes of 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

The pharmaceutical composition is prepared by a known method per se for the preparation of a pharmaceutically acceptable composition to be administered to a subject, so that an effective amount of T cells is combined with a pharmaceutically acceptable carrier in a mixture. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20 ^th ed., Mack Publishing Company, Easton, Pa., USA, 2000. On this basis, the composition, which is proprietary, is described) However, it includes solutions of substances contained in a buffered solution having an appropriate pH and isobaric pressure combined with one or more pharmaceutically acceptable carriers or diluents and having a physiological fluid.

Suitable pharmaceutically acceptable carriers include essentially chemically inert and non-toxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers are water, saline, glycerol solution, N-(1(2,3-dioleyyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleylphosphotidyl-ethanolamine (DOPE) and liposomes. In some embodiments, such compositions contain a therapeutically effective amount of the compound along with an appropriate amount of a carrier to provide a form for direct administration to a patient.

Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injection solutions or suspensions, which make the composition substantially compatible with the tissues or blood of the intended recipient, such as antioxidants, buffers, bacteriostatic agents and It may contain additional solutes. Other ingredients that may be present in such compositions include, for example, water, surfactants (eg Tween), alcohols, polyols, glycerin and vegetable oils. Extemporaneous injection solutions and suspensions can be prepared as sterile powders, granules, tablets, or concentrated solutions or suspensions.

The pharmaceutical compositions disclosed herein are formulated in a variety of forms and administered by a number of different means. The pharmaceutical preparations are administered orally, rectally or parenterally as a preparation containing an acceptable carrier, adjuvant, and vehicle, if necessary. As used herein, the term “parenteral” includes subcutaneous, intravenous, intramuscular, or intrasternal injection and infusion techniques. Administration may be by injection including intraarterial, intracardiac, intraventricular, intradermal, intraduodenal, intrathecal, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous Includes infusion, inhalation and transdermal, transmucosal, sublingual, buccal and topical (epicutaneous, skin, enema, eye drops, ear drops, nasal, vaginal) administration. In some exemplary embodiments, the route of administration is via injection, such as intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

Liquid formulations include oral formulations, intravenous formulations, intranasal formulations, ocular formulations, ear formulations, aerosols and the like. In certain embodiments, combinations of various agents are administered. In certain embodiments, the composition is formulated for a delayed release profile.

Treatment and method of use

In certain embodiments, a method of using the Tri-TAC disclosed herein in the treatment of cancer in an individual in need thereof is disclosed. In some embodiments, a target specific ligand of a TAC disclosed herein binds a tumor antigen or a tumor associated antigen on a tumor cell. In some embodiments, a target specific ligand of a TAC disclosed herein selectively binds to a tumor antigen or a tumor associated antigen on a tumor cell. In some embodiments, a target specific ligand of a TAC disclosed herein specifically binds a tumor antigen or a tumor associated antigen on a tumor cell. In some embodiments, the target antigen is a tumor antigen. Examples of tumor antigens include CD19, HER-2 (erbB-2), B cell maturation antigen (BCMA), alpha fetal protein (AFP), cancer embryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen ( ETA), tyrosinase, melanoma-related antigen (MAGE), prostate specific antigen (PSA), glioma-related antigen, β-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase Reverse transcriptase, RU1, RU2(AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, PAP, NY-ESO-1, LAGE-1a, p53, prosteine, PSMA, survivor Bin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin, but It is not limited thereto.

In certain embodiments, herein is a method of treating a cancer expressing a target antigen in an individual in need thereof, comprising administering to the subject an engineered T cell disclosed herein. Is initiated. In some embodiments, the target antigen is CD19. In some embodiments, a method of treating a cancer expressing CD19 in an individual in need of treatment for a cancer expressing CD19 comprises administering to the individual an engineered T cell comprising a TAC comprising a CD19 targeting ligand. . In some embodiments, examples of cancers treated by TAC comprising a CD19 targeting ligand include, but are not limited to, B cell malignancies. In some embodiments, examples of cancers treated by TAC comprising a CD19 targeting ligand include, but are not limited to, B cell lymphoma, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, examples of cancers treated by TAC comprising a CD19 targeting ligand include, but are not limited to, non-Hodgkin's lymphoma (NHL).

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

In some embodiments, the target antigen is BCMA. In some embodiments, the method of treating a cancer in which the cancer cell expresses BCMA in an individual in need of cancer treatment in which the cancer cell expresses BCMA comprises an engineered T cell comprising a TAC comprising a BCMA-2 targeting ligand to the individual. And administering. In some embodiments, examples of cancers treated by TAC comprising a BCMA targeting ligand include, but are not limited to, leukemia, lymphoma, and multiple myeloma.

Further disclosed herein is the use of engineered T cells disclosed herein in the manufacture of a medicament for treating cancer in an individual in need thereof. Also disclosed herein is the use of a mixture of T cells comprising different populations of modified cells with or without modified and unmodified cells or with or without unmodified cells. One of skill in the art will understand that the therapeutic amount of modified T cells need not be essentially homogeneous.

In some embodiments, the engineered T cells disclosed herein are part of a combination therapy. In some embodiments, the therapeutic effect of the disclosure herein is evaluated multiple times. In some embodiments, patients are stratified based on response to the treatment disclosed herein. In some embodiments, the treatment effect determines entry to the test.

In some embodiments, cancers treated with engineered T cells comprising any of the TACs disclosed herein include any form of neoplastic disease. In some embodiments, examples of cancers treated include breast cancer, lung cancer and leukemia, such as mixed lineage leukemia (MLL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), but It is not limited thereto. In some embodiments, examples of cancer to be treated include, but are limited to, large B cell lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, high grade B cell lymphoma, or large B cell lymphoma arising from follicular lymphoma. It doesn't work. Other cancers include carcinoma, blastoma, melanoma, sarcoma, hematologic cancer, lymphoid malignancies, benign and malignant tumors, and malignant tumors. In some embodiments, the cancer comprises a non-solid tumor or a solid tumor. In some embodiments, the cancer to be treated includes tumors that have not been vascularized, or tumors that have not yet been substantially vascularized, as well as vascularized tumors. In some embodiments, the cancer is or comprises a solid tumor. In some embodiments, the cancer is liquid cancer or comprises a liquid tumor. In some embodiments, the cancer is lung cancer, breast cancer, colon cancer, multiple myeloma, glioblastoma, gastrointestinal cancer, ovarian cancer, gastric cancer, colorectal cancer, urinary tract cancer, endometrial cancer, or melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is urinary tract epithelial cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is melanoma.

[Table 1]

Example

The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. This example, together with the methods described herein, is representative of the presently preferred embodiments, is exemplary, and is not intended as a limitation on the scope of the invention. Those skilled in the art will come up with variations and other uses therein which are included within the spirit of the invention as defined by the scope of the claims.

Example One. Tri -Specialization of TAC technology

An overview of the Tri-TAC technology is provided in [Figs. 1A] to [Fig. 1C].

1A shows an example of CD8 T cell activation based on co-assembly of different receptors and their related protein partners. Initially, the major histocompatibility complex I presents the antigen (helix). It is recognized by a T cell receptor (TCR) complex that can bind to an antigen. The TCR complex contains several individual subunits. The α/β domain can directly interact with the antigen presented to MHC-I. The α/β domains then interact with several other domains (ε, γ, δ and ζ), all of which participate in T cell activation through various intracellular activation domains. The TCR complex interacts with MHC-I simultaneously with the CD8 coreceptor. The CD8 coreceptor binds to MHC-1 in an antigen-independent manner. CD8 directly interacts with Lck, a protein kinase important for activation of the TCR receptor complex. The CD8 and Lck interactions also ensure relevance with lipid raft (membrane portion) microdomains that are supposed to construct and encapsulate other related signaling moieties (dark spheres). Then the subsequent activation phase leads to CD28 mobilization. When this interaction cascade occurs several times simultaneously, T cells can be activated and exert a cytotoxic effect.

1B provides an overview of the chimeric antigen receptor (CAR). CAR seeks to recreate 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 directly with the antigen of choice using specific binding domains. Shown here is ankyrin repeat protein (DARPin). It is thought that these multiple interactions occurring simultaneously lead to T cell activation.

[Figure 1c] is an overview of Tri-TAC technology that mimics the natural activation process. Tri-TAC was developed to better reproduce natural signaling through TCR while maintaining MHC unrestricted targeting. T cell activation occurs after ligation of MHC by TCR and T cell co-receptors (CD4 or CD8), which simultaneously binds to a conserved region within the MHC molecule. The co-receptor is located within the "lipid raft", a membrane microdomain that is particularly important for the formation of the TCR signal complex. In addition to ensuring accurate microdomain localization of the TCR activation complex, this co-receptor binds directly to Lck, a protein kinase important for T cell activation. None of the conventional chimeric receptors or bifunctional proteins are involved in the coreceptor molecule or Lck. Molecules were generated in which the transmembrane and intracellular regions of the CD4 co-receptor that each localized to the lipid raft and bound to Lck were fused to a single chain antibody (UCHT1; SEQ ID NOs: 13, 14 and their homologues) that binds to CD3. This construct is designed to pull the CD3 molecule and TCR into the region of the lipid raft and bring Lck into the proximity of the TCR, which is similar to natural MHC binding. To target this receptor, the designed ankyrin repeat (DARPin) was ligated to the CD4-UCHT1 chimera to generate a trifunctional T cell-antigen coupler (Tri-TAC). In this example, DARPin was specific for the proto-oncogene HER-2 (erbB-2).

Several Tri-TAC configurations are possible (Figs. 2A and 2B). In configuration 1 (FIG. 2A), the antigen binding domain is located at the N-terminus, and is linked to the CD3 ligand binding domain and then to the coreceptor domain. In configuration 2 (FIG. 2B ), the CD3 ligand binding domain is located at the N-terminus, and is linked to the antigen binding domain, which in turn is linked to the coreceptor domain.

Several classes of ligand binding domains can be incorporated into the Tri-TAC molecule (Figures 3A-3D). Examples herein include Configuration 1 Tri-TAC (Figure 3A), Tri-TAC with HER-2 specific DARPin (Figure 3B), Tri-TAC with CD19 specific scFv (Figure 3C), and BCMA specific It shows a general schematic of Tri-TAC with scFv (Figure 3D).

Figures 4a to 4d illustrate the functionality of Tri-TAC with HER-2 specific DARPin. Human T cells were engineered to express either the Tri-TAC disclosed herein or an existing CAR with the same DARPin. It was confirmed in all aspects that T cells engineered with Tri-TAC exhibit at least equivalent functionality to conventional CARs. Interestingly, with regard to two parameters (TNF-α production and CD107a mobilization), Tri-TAC was observed to be more active than conventional CARs in some circumstances.

Figure 4a shows the surface expression of anti-HER-2 DARPin CAR and anti-HER-2 DARPin Tri-TAC compared to control T cells. Chimeric receptors were detected by incubation with recombinant HER-2. Anti-HER-2 DARPin Tri-TAC was well expressed on the surface of engineered T cells. [Figure 4b] shows the growth of engineered T cell culture. T cells were activated with anti-CD3/anti-CD28 Dynabeads and engineered with a lentivirus encoding Tri-TAC, CAR or not (control) receptors. After 2 weeks, CAR and control cultures grew to similar numbers while Tri-TAC cultures grew slightly slower. [Fig. 4c] and [Fig. 4d] show the functional properties of the engineered T cells. T cells engineered to express Tri-TAC or CAR carrying HER-2 DARPin were stimulated with plate-bound antigen. T cells engineered to express Tri-TAC and CAR were able to detail all of the functions measured (TNF-α production, IFN-γ production and CD107a mobilization, FIGS. 3C and 3D). T cells engineered with Tri-TAC showed an elevated frequency of CD107a positive cells after stimulation compared to T cells engineered with CAR (FIG. 3D ), suggesting that the cytotoxicity was elevated on a per cell basis.

[Fig. 6a] to [Fig. 6j] provide data confirming the importance of the ligand binding domain and the UCHT1 CD3 binding domain for Tri-TAC functionality. T cells are full-length Tri-TAC with HER-2 DARPin (Fig. 6G, Fig. 6H, Fig. 6I, bottom row), and DARPin-free Tri-TAC variants (Fig. 6A, Fig. 6B. Fig. 6C, top row). ), or a Tri-TAC variant without UCHT1 (Fig. 6D, Fig. 6E, Fig. 6F, middle row). All three engineered T cell populations were stimulated with HER-2-positive tumor cells. T cells engineered with full-length Tri-TAC were able to produce IFN-g, TNF- and IL-2 after stimulation, whereas the mutant did not produce any cytokines after stimulation. The three T cell populations were also co-cultured with D2F2/E2 cells (HER-2 expression) or D2F2 cells (HER-2 negative) at 4:1 effector: target (FIG. 6J ). T cells engineered with full-length Tri-TAC showed strong killing against D2F2/E2 cells, but not D2F2 cells. Other Tri-TAC variants without DARPin or UCHT1 showed no death.

[Fig. 7a] to [Fig. 7c] show the results of mice treated with vector control (NGFR), anti-HER-2 DARPin CAR or anti-HER-2 DARPin Tri-TAC. A xenograft mouse model was used. OVCAR-3 tumor cells were administered subcutaneously to mice and allowed to grow until ^{tumors reached 100-200 mm 3 size.} 7A shows the relative tumor progression normalized to tumor size on the treatment day. Anti-HER-2 DARPin Tri-TAC engineered T-cells drastically reduced the tumor volume, the control had no effect, and the CAR cells slowed tumor growth and showed a delayed reduction in tumor size. [Fig. 7b] exemplifies the relative change in body weight after T cell injection. 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 show significant weight loss with severe toxicity. [Fig. 7c] exemplifies the cytokine concentration in mouse serum on the 7th day after T cell injection. Cytokine levels were higher in CAR treated mice compared to Tri-TAC treated mice.

Example 2: UCHT1 Substitution is Tri -Affect TAC function give

[Fig. 8A]-[Fig. 8H] illustrate the functionality of Tri-TAC with an alternative CD3 binding domain. Domains are listed in [Fig. 8A] and [Fig. 8E]. Tri-TAC containing UCHT1 (Fig. 8b), OKT3 (Fig. 8b) and huUCHT1 (Fig. 8f) showed high surface expression, whereas Tri-TAC containing F6A (Fig. 8f) and L2K (Fig. 8f) more Showed low surface expression. Cells expressing Tri-TAC containing OKT3 showed low cytokine production (FIG. 8C, FIG. 8C1) and moderate cytotoxicity (FIG. 8D) upon Tri-TAC ligation. Cells expressing Tri-TAC containing F6A showed strong cytokine production (Fig. 8G, Fig. 8G1) and cytotoxicity (Fig. 8H) after Tri-TAC ligation. Cells expressing Tri-TAC containing L2K showed low cytokine production (Fig. 8G, Fig. 8G1) and moderate cytotoxicity (Fig. 8H).

[Fig. 9A] to [Fig. 9H] illustrate TCR surface expression on T cells engineered with different Tri-TAC variants shown in [Fig. 8A] and [Fig. 8E]. T cells engineered with Tri-TAC variants containing OKT3 (Figs. 9A, 9E and 9B, Fig. 9F) or L2K (Fig. 9C, Fig. 9G and Fig. 9D, Fig. 9H), respectively, are Tri-T cells containing UCHT1 or huUCHT1. -Showed lower TCR surface expression compared to T cells engineered with TAC. In contrast, T cells engineered with the Tri-TAC variant containing F6A did not show TCR downregulation compared to Tri-TAC bearing huUCHT1 (FIGS. 9C, 9G and 9D, 9H ). F6A substitution reduced the surface expression of the Tri-TAC receptor while maintaining moderate cytokine production and cytotoxicity. L2K substitution moderately reduced surface expression and reduced cytokine production, but maintained moderate cytotoxicity. OKT3 substitution resulted in high Tri-TAC surface expression, low cytokine production, and moderate cytotoxicity. These data indicate that Tri-TAC surface expression and T cell effector function are not essentially proportional and in some cases Tri-TAC domain substitution alters effector function independent of the level of surface expression. It is conceivable that TAC variants with reduced cytotoxicity and low surface expression may have value in certain clinical applications.

In many cases, scFv substitution weakened the ability of engineered T cells to produce IFN-γ, TNF-α, and IL-2, but engineered T cells retained their ability to kill target cells. Excessive cytokine production is associated with adverse events in the clinical setting, limiting current CAR technology to life-threatening diseases. The ability to modify TAC molecules to reduce cytokine production while maintaining moderate cytotoxicity will enable the creation of Tri-TAC receptors with the exact level of responsiveness required to meet clinical efficacy and safety.

The ability of Tri-TAC variants including OKT3 to inhibit TCR surface expression and cytokine production while maintaining cytotoxicity may be of great value in allogeneic situations where inhibition of TCR can suppress graft versus host disease.

These data demonstrate that the scFv substitution of UCHT1 affects the function of Tri-TAC. Further modifications will result in Tri-TACs useful for a variety of applications (eg, oncology, autoimmunity, allergy).

Example 3. TCR Introduction of various linkers connecting the ligand binding to the complex to the target binding ligand domain

[FIG. 10A] to [FIG. 10B] illustrate several TAC variants having different linkers connecting the ligand binding to the TCR complex and the target binding ligand domain. The flexible connector 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 stiffness but are less restrictive compared to large domain linkers.

[Fig. 11A] to [Fig. 11E] illustrate the effect of linker substitution on Tri-TAC surface expression, Tri-TAC transduction efficiency, and cytokine production during Tri-TAC ligation. [Fig. 11A] and [Fig. 11B] show that the helical linker improves surface expression and transduction efficiency when compared to the flexible linker, while the large domain linker improves the transduction efficiency but does not improve the surface expression. [Fig. 11D] and [Fig. 11E] exemplify cytokine production by cells expressing Tri-TAC having a short helical, long helical, or large domain linking group.

12A illustrates the improved in vitro cytotoxicity of T cells expressing Tri-TAC with short helical linkers. 12B illustrates improved in vivo tumor control of T cells expressing Tri-TAC with short helical linkers. Short helical connector was associated with high cytotoxicity in vitro and effective tumor control in vivo.

Example 4. CD8α /β Cytosol Introduction of the domain

[Fig. 13a] exemplifies the surface expression of anti-HER-2 scFv or [Fig. 13c] anti-HER-2 DARPin and corresponding CD8α Tri-TAC. 13B illustrates cytokine production by T cells expressing CD8α Tri-TAC corresponding to anti-HER-2 scFv or anti-HER-2 DARPin.

14A illustrates CD4 Tri-TAC monomer and CD8α/β heterodimer. The TCR co-receptor, CD4 and CD8 all possess functional domains that are important for co-receptor functionality. These regions include the arginine-rich region, which is assumed to be important for lipid raft binding, and the CXCP motif required for Lck binding. Unlike the monomer CD4, the CD8 co-receptor is a heterodimer composed of α and β subunits (Fig. 14A). Both the α and β CD8 subunits contain the arginine rich region, but only the α subunit contains the CXCP motif.

[Fig. 14B] to [Fig. 14D] provide schematic diagrams of a Tri-TAC variant comprising elements from the CD8 co-receptor shown in [Fig. 14A]. Cysteine, responsible for dimerization of CD8α and CD8β, was replaced by alanine in all CD8 Tri-TAC variants. 14B is a schematic diagram of a CD8α Tri-TAC comprising a CD8α cytosolic domain and a cysteine to serine mutation to ensure monomeric receptor distribution. 14C is a schematic diagram of a CD8α+Rβ Tri-TAC comprising a chimeric CD8α cytosolic domain in which a cysteine to serine mutation and a CD8α arginine rich region are replaced with a CD8β arginine rich region to ensure monomeric receptor distribution. [Fig. 14D] is a CD8β comprising a chimeric CD8β cytosolic domain in which a CD8α CXCP domain containing a Lck binding motif and a CD8α CXCP domain including a Lck binding motif is added to the C-terminus of the CD8β cytosol domain to ensure monomeric receptor distribution. It is a schematic diagram of +Lck Tri-TAC.

15A to 15D illustrate various phenotypic and functional properties of CD8 based Tri-TAC variants for prototype Tri-TAC. [FIG. 15A] to [FIG. 15B] illustrate the surface expression of the CD8-Tri-TAC variant against the prototype Tri-TAC. Surface expression was similar between the different Tri-TACs. 15C illustrates in vitro cytotoxicity of CD8-Tri TAC variants co-cultured with LOX IMVI (HER-2 negative) and A549, SKOV3, SKBR3, or MBA MB 231 (all HER-2 positive). All T cells engineered with Tri-TAC showed cytotoxicity. [Fig. 15D] exemplifies cell division of T cells engineered with CD8 Tri-TAC variant or prototype Tri-TAC (Fig. 15D). [Fig. 15E] exemplifies TCR surface expression of engineered T cells containing CD8 Tri-TAC variants or prototype Tri-TAC. All Tri-TAC variants had similar effects on TCR expression. The CD4 coreceptor showed good surface expression and functionality by both scFv and DARPin anti-HER-2, but the CD8α construct showed activity only with respect to the DARPin antigen binding domain. When testing different CD8α cytosolic domains, all constructs included reported key sequence attributes related to co-receptor functionality (arginine rich region and CXCP). All CD8α/β constructs showed similar performance when compared to the CD4 prototype. This highlights that the retention of certain biochemical properties, such as lipid raft affinity and Lck binding, is more important in determining Tri-TAC performance than certain cytosolic polypeptide sequences.

The growth of T cells engineered with CD8α+R(β) and CD8β+Lck Tri-TAC was significantly worse compared to that of T cells engineered with other mutations. Despite having a significant effect on growth, all of these Tri-TACs showed similar abilities to activate T cells. Reduced growth of CD8α+R(β) and CD8β+Lck Tri-TAC may be beneficial for certain applications where maximal T cell expansion is not desired.

Example 5. Development of CD19-TAC construct

[Figure 16] illustrates the phased development of the CD19-TAC construct. Several generations of lentiviral vectors are generated with various modifications of design elements to ensure CD19 specificity, proper TAC expression, and GMP grade lentiviral production. Each box represents a lentiviral vector and designates the three main design elements (A) antigen binding domain, (B) TCR/CD3 binding domain and (C) co-receptor domain. The shaded area represents the domain that was subject to transformation during the vector development process.

In the first step, the TAC comprises an HER-2 specific designed ankyrin repeat protein (DARPin), a murine UCHT1 CD3 specific scFv, and a flexible transmembrane and cytosolic CD4 polypeptide. TAC is cloned into the pCCL4 lentiviral vector.

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

The UCHT1 domain was replaced with a humanized form (huUCHT1) to reduce immunogenicity. This TAC construct showed a superior level of surface expression than the precursor.

To further improve receptor expression on the cell surface of T cells without compromising functionality, two distinct modifications were evaluated simultaneously. To increase short chain stabilization, the G ₄ S linker (SEQ ID NO: 73) used in the anti-CD19 scFv was replaced with a more structured Whitlow linker. Separately, the Y177T mutation was introduced into the huUCHT1 domain. Both strategies improved the expression of the TAC receptor and produced a receptor with both the Whitlow linker and the Y177T mutation.

[Fig. 17] exemplifies the CD19-TAC insert in the pCCL lentiviral vector. The pCCL vector is characterized by a bidirectional promoter system in which ΔNGFR(hu) is under the control of the mCMV promoter and TAC expression is driven by the EF-1α promoter. ΔNGFR(hu) is truncated human CD271 (tumor necrosis factor receptor superfamily member 16) and has a transmembrane domain but no cytosolic signaling domain. The ΔNGFR(hu) expression product is used to quantify lentiviral transduction. The CD19-TAC#921 open reading frame has been expanded to show the main elements of the TAC construct: CD8α leader, FMC63 short chain (anti-CD19 scFv), human c-Myc tag, huUCHT1 (Y177T) and ΔCD4 domains. The huUCHT1 (Y177T) mutation was identified by examining a randomly introduced point mutation at the residue of the murine UCHT1 CD3 epsilon binding interface. In screening, the (Y177T) mutation was successfully identified. The (Y177T) mutation results in better Tri-TAC surface expression while maintaining T cell activation. ΔCD4 lacks four CD4 extracellular immunoglobulin-like domains and has an extracellular linker, transmembrane and cytosolic domain.

To generate a GMP grade lentiviral vector, the CD19-Tri-TAC construct was cloned into a new lentiviral vector under the control of the MSCV promoter. The CD19-Tri-TAC construct is identical to that shown in Fig. 17.

Example 6. Ability to Produce CD19-TAC Expressing T Cells from Different Donor Materials

Figure 18 illustrates the efficacy of CD19 TAC expressing T cells prepared from multiple donors. CD19-TAC expressing T cells were produced using T cells from three different donors and tested in the NALM-6 tumor model. Mice bearing established NALM-6 tumors were treated with a single dose of ^{4 x 10 6 CD19 TAC expressing T cells.} Control mice showed rapid tumor growth and all mice reached the endpoint by the end of the study. The T cell products of donors 1 & 2 brought full control in all mice. The T cell product of Donor 3 resulted in strong tumor control in all mice and showed long-term control in 2 of 4 treated mice. The study confirms that tumor rejection is achieved by CD19 TAC expressing T cells from several healthy donors. The results of the NALM-6 tumor model in Fig. 18 suggest that effective CD19 TAC is produced from several donor source materials.

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

To evaluate the ability of CD19-TAC to effectively bind to various CD19 positive cells, Tri-TAC engineered T cells were treated with NALM-6 (acute lymphoblastic leukemia), Raji (Buckett lymphoma) or Jeko-1 (mantle cell lymphoma). ) And co-cultured. NALM-6, Jeko-1, and Raji cells were engineered with enhanced firefly luciferase to track tumor burden in vitro and in live animals via bioluminescent imaging.

19A to 19C illustrate the killing of a tumor cell line by CD19-TAC-expressing T cells. The effect was dose dependent and increased with increasing effector to target (E:T) ratio. As a negative control, cells engineered with ΔTAC (no antigen binding domain) or untransduced T cells were used. These results demonstrate that CD19-TAC expressing T cells kill CD19 positive tumor cells.

[Fig. 19D] to [Fig. 19G] evaluate the efficacy of CD19-TAC in mice implanted with NALM-6 (acute lymphoblastic leukemia), Raji (Bucket's lymphoma) or Jeko-1 (mantle cell lymphoma) liquid tumors. Illustrate the design and results of in vivo studies. To induce NALM-6, Raji and Jeko-1 tumors, mice were inoculated with NALM-6, Raji, or Jeko-1 cells and received for 4 or 7 days, respectively, to allow engraftment of the tumor. On day 4 or 7, CD19-TAC expressing T cells were given by intravenous tail vein injection. Tumor burden was measured at weekly intervals, and data are plotted as average radiant luminance [p/s/cm^ 2/sr].

[Fig. 19E] to [Fig. 19G] show that CD19-TAC engineered T cells are tumor regression and long-term in NALM-6 (acute lymphoblastic leukemia), Raji (Bucket's lymphoma) or Jeko-1 (mantle cell lymphoma) liquid tumor. It exemplifies that it is effective in inducing tumor control.

The results of the NAML-6, Raji, or Jeko-1 tumor models in FIGS. 19A to 19G suggest that CD19-TAC is effective in various CD19 positive tumor models.

Example 8. CD19-TAC expressing T cell persistence and sustained tumor immunity.

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

ve) 마우스에 종양 세포를 동시 주입하고 음성 대조군으로 사용한다. 종양 부담은 발광 신호로 추적한다.[FIG. 20A] illustrates an experimental setup for measuring CD19-TAC persistence in mice. Mice were first inoculated with NALM-6 cells and treated with CD19-TAC after a 4 day engraftment period. All mice showed tumor regression and complete tumor control. 56 days after the initial treatment, mice were re-inoculated with either NALM-6 (CD19 positive) or KMS11 (CD19 negative) liquid tumors. Contactless in all cases (na

ve) Tumor cells were simultaneously injected into mice and used as a negative control. Tumor burden is tracked by luminescent signals.

[Fig. 20B]: Mice bearing established NALM-6 tumors were treated with CD19-TAC-expressing T cells provided as divided doses of a ^{total of 4 x 10 6 engineered cells.} As a control, a group of untreated animals was used. After ACT, the treated mice showed a persistent anti-tumor response. In contrast, control mice showed an exponential increase in tumor mass and reached a tumor burden-related endpoint. On day 56 after ACT, mice were re-administered with NALM-6 tumor cells (CD19 positive) or KMS11 tumor cells (CD19 negative). CD19-TAC treated mice remain protected from NALM-6 (CD19 positive) tumor cells, but not from KMS11 (CD19 negative) tumor cells.

The results of re-administration experiments in [Fig. 20A] and [Fig. 20B] suggest that CD19-TAC differentiates into long-lasting memory cells that retain anti-tumor properties in some cases.

Example 9. In vivo expansion and dose dependence of CD19-TAC expressing cells.

[Fig. 21] and [Fig. 22] illustrate the dose dependence, dose regimen (divided or single) and expansion of CD19-TAC expressing T cells in the NALM-6 cancer model. [Fig. 21A] illustrates the experimental design. Mice received a single dose of CD19-TAC expressing T cells on day 4 after tumor inoculation, or divided doses administered 7 days apart. Several CD19-TAC expressing T cell doses were tested: 0.5 x 10 ⁶ , 1 x 10 ⁶ , and 4 x 10 ⁶ cells. [Fig. 21B] Mice in the control group receive 4 x 10 ⁶ untransduced cells or frozen medium (vehicle control).

21B illustrates the survival rate of mice after NALM-6 injection and CD19-TAC injection. Dose-dependent promotion of survival was observed in both single dose and divided dose groups, with the highest single dose limiting tumor growth and promoting survival of mice.

22A illustrates the gating strategy used to evaluate T cell proliferation. Cells were selected based on anterior and lateral scattering for the lymphocyte population. Single cells were identified through the forward scattering area above the height gate. Live cells were identified through near-infrared gating. Human cells were identified through the hCD45 gate. The resulting cell subset was further divided into CD3 positive cells. Then, these cells were gated on CD4/CD8 and protein L. The staining strategy also included muCD45_1 to identify murine blood cells. CD19 was included to stain the NALM-6 cells.

[Fig. 22B] Expansion of T cells in mice after split-dose adoptive T cell transfer (ACT). After ACT, blood samples were regularly collected and analyzed through flow cytometry. Values were normalized to the total number of T cells present in blood after ACT1. Values were also normalized to the total number of CD45.1+ (murine) cells to account for differences in blood draws. T cells from mice treated with CD19-TAC engineered cells were shown to expand in recipient mice within about 1-2 weeks after the first ACT (FIG. 22B ). Cells that were not transduced did not expand (FIG. 22B ).

The results of the various doses, dose regimens (Figure 21B) and T cell counts (Figure 22B) show that the CD19-TAC efficacy is dose dependent, the engineered T cells expand in vivo, and this expansion is the result of CD19 positive tumor bearing animals. This suggests that it is specific for CD19-TAC engineered cells.

Example 10. In vivo efficacy, long-term efficacy and safety of CD19-TAC treatment

Figures 23 to 25 show long-term safety and efficacy (Figure 23) and the absence of any acute treatment-related toxicity (Figures 24 to 25).

[Fig. 23A] illustrates the experimental design. Mice were injected with 0.5 x 10 ⁶ enriched luciferase engineered NALM-6 cells and allowed to engraft for 4 days. The mice are then treated with two dose levels of CD19-TAC engineered T cells (4 and 12 x 10 ⁶ engineered cells) in a single dose administration. Then, tumor growth was tracked through periodic luminescence measurements. Mouse health was regularly assessed by examining mouse behavior and physical properties (grooming, motility, integrity of hair).

23B shows vehicle alone (frozen medium), unengineered control cells (total T cell volume equal to the total T cell volume of the best engineered treatment group) and 4 or 12×10 ⁶ engineered CD19-TAC engineered cells. The tumor burden through light emission after treatment with is illustrated. Both controls showed rapid tumor growth and no antitumor efficacy. The control dose delays tumor growth compared to vehicle alone, possibly due to competition between the high dose T cells and tumor cells for the engraftment environment. Engineered T cells show tumor regression in all cases. The high-dose treatment group shows complete tumor control in all cases. The 4 x 10 ⁶ treatment group showed 3 mice in full control, one with delayed tumor growth, and one with controlled but high tumor burden.

23C illustrates the overall survival rates of different treatment groups. In both vehicle and unengineered control mice, all mice die of tumors within 23 to 35 days each. In the case of high-dose CD19-TAC treatment, all mice show symptoms of GvHD and die of GvHD within 61 days. GvHD is a result of the mouse model itself and not treatment with modified T cells. Low-dose mice show survival of 3 mice by the end of the study at 90 days, one mouse dies of high tumor burden, and one mouse dies of GvHD.

Figures 24 and 25 show clinical chemistry parameters and cytokine levels from vehicle control, unengineered cells and CD19-TAC (4 and 12 x 10 ⁶ effective CD19-TAC engineered cells) treated mice. Illustratively. Mice were followed for 33 days while collecting blood on days 5, 12 and 33 after ACT. Only CD19-TAC treated mice survived for 33 days. Vehicle control mice died of tumor burden prior to collection of the third blood sample, and unmanipulated cells were sacrificed early on day 26, just before the mice reached tumor burden related endpoints. All blood samples were analyzed for several clinical chemistry parameters and cytokine levels.

[Fig. 24] exemplifies that the mice treated with CD19-TAC on days 5 and 12 do not show significantly higher parameters compared to the control group. All mice treated on day 33 show similar clinical chemistry parameters at the time of initial treatment. The exceptions are alanine aminotransferase (ALT) and aspartate aminotransferase (AST), where some mice undergo similar high levels as mice treated with unengineered cells sampled on day 26.

[Fig. 25] exemplifies cytokine reactions on days 5, 12 and 33. On day 5 after ACT, CD19-TAC control mice except for the control show an elevation in all cytokines tested. Cytokine increase is consistent with the inflammatory response of CD19-TAC engineered T cells that recognize and respond to antigen-positive NALM-6 tumor cells. Cytokine levels fall after an initial response up to day 12, which is then associated with induced tumor regression and generally low tumor burden. Between those treated with CD19-TAC on day 12, cytokine levels were similar or lower than unengineered T cells except for IL10. All mice treated with untransduced T cells or CD19-TAC engineered T cells at later stages show an increase in cytokines, possibly associated with the onset of GvHD. Also, reference is made to [Fig. 29] illustrating the cytokine reaction on days 5, 12, 26 and 33.

Long-term follow-up results of mice treated with CD19-TAC and their clinical chemistry profile demonstrate that engineered T cells are safe to use and show no signs of toxicity specifically induced by CD19-TAC manipulation. The results of cytokine studies show an initial inflammatory response associated with anti-tumor efficacy after all cytokine levels have been reduced, suggesting a controlled inflammatory response.

Example 11. Multiple BCMA Tri -TAC Mutant In vivo efficacy

26 illustrates in vivo efficacy studies of various BCMA Tri-TAC constructs. [Fig. 26A] illustrates the overall experimental design. One million luciferase engineered KMS11 (BCMA positive) tumor cells were left to engraft for 12 days. The mice were then treated with 4 million BCMA constructs and a single effective dose of the control (FIG. 26B ). Tumor burden was regularly assessed through luminescence measurements. Then, 1 million KMS11 cells were re-administered on day 25 after ACT to all mice showing tumor regression and tumor control.

[Fig. 26C]: After ACT, control mice showed rapid growth of tumor cells reaching tumor-related endpoints within 19 to 25 days. In contrast, all BCMA-TAC treated mice showed early tumor regression. Tumor control _{varied across constructs, with G 4} S (SEQ ID NO: 73) 3625VH-VL showing the lowest level of initial tumor control and short spiral 3625 VL-VH showing the highest level of initial tumor control. After re-administration, the majority of all constructs that maintained tumor control until day 25 remained protected from re-administration.

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

Example 12. TAC- Her2 In vivo

OVCAR3 solid tumors are inoculated on the back flank of the mouse. The tumor is established and allowed to grow to a size of ^{100 mm 3.} Then, mice were treated with tail vein injection of TAC-Her2 engineered T cells. The tumor volume is measured regularly.

Example 13. Clinical trial

A clinical study is conducted in which subjects 18 years of age or older suffering from CD19 positive diffuse large B cell lymphoma who have failed two or more prior therapy regimens including ASCT or who are not eligible for ASCT are treated with CD19-TAC expressing cells. This study is an open-label, single-group, phase 1/2 phase 2 clinical trial characterized by a dose escalation step to determine the maximum tolerated dose (MTD) or phase 2 recommended dose (RPh2D). In followed by an expansion cohort.

Upon enrollment, the subject undergoes leukocyte collection to obtain T cells for the production of CD19-TAC expressing T cells. Upon successful manufacture, the subject enters the treatment phase. This step involves intravenous (IV) administration of CD19-TAC expressing T cells after lymphatic chemotherapy with fludarabine and cyclophosphamide. After treatment with CD19-TAC expressing T cells, subjects enter post-treatment follow-up and are followed for safety, disease status, and survival for 2 years after the last administration of CD19-TAC expressing T cells. After study completion, subjects are followed for survival, long-term toxicity and viral vector safety in a separate long-term follow-up protocol for up to 15 years after the last dose of CD19-TAC expressing T cells.

Safety is assessed throughout the study in all groups. T cell expansion is evaluated from the time of the first administration of CD19-TAC expressing T cells until no more cells are detected. Radiologic disease evaluation is performed by positron emission tomography (PET:) before treatment, about 3, 6, 9, 12, 18 and 24 months after the last administration of CD19-TAC expressing T cells, or until treatment with advanced disease or additional anticancer therapy. positron emission tomography) and/or computed tomography (CT) scan.

Example 14. Preparation of CD19-TAC-expressing T cell drug product

The manufacturing process of the CD19-TAC-expressing T cell drug product comprises the steps of selecting CD4/CD8 T cells from the leukocyte collection product, activating CD4/CD8 positive cells, and a lentiviral vector comprising a CD19-TAC construct (Example 5 As described above), expanding the transduced cells to a level suitable for the proposed dosing schedule, and collecting and cryopreserving the final product.

Leukocyte collection materials from patients with a relevant unique subject identifier (UPN) are received at the manufacturing site and given a unique specimen number (ISN). CD4/CD8 cells are selected and cryopreserved until the incubation process step begins.

T cells selected for cryopreserved CD4/CD8 positive are thawed at 37° C., resuspended in an appropriate medium and seeded into a culture bag with an activating reagent, and the cultures are incubated overnight at 37° C./5% CO 2.

Cells are transduced with a CD19-TAC lentiviral vector at an appropriate multiplicity of infection (MOI) and incubated overnight at 37° C./5% CO2. On the next day, the cultures are supplemented with complete medium to maintain the desired cell concentration and ultimately collected in transfer bags, pelleted, resuspended and seeded into larger culture bags at the desired cell density.

For the final drug product, the collected cell suspension is resuspended in excipients, cryopreserved in a controlled rate freezer and transferred to an LN2 reservoir.

The product is transported frozen to the clinical site, thawed in bed and administered intravenously.

Prior to the clinical trial, an engineering manufacturing run, including all in-process and release tests, is performed using healthy donor leukocytes. In addition to in-process and release testing, studies to support regulatory filing are conducted for the final drug product in these pilot productions. These studies include stability after thawing, onset of long-term stability, residual testing to ensure removal of growth-promoting cytokines, and early evaluation of potential function/efficacy indication assays.

Example 15. BCMA For the treatment of benign malignancies BCMA Of specific T cell-antigen coupler (TAC) therapy Preclinical Development

Fig. 27 illustrates that TAC proliferates when it encounters an antigen on a cell, but does not proliferate when the antigen is presented to artificial beads. However, CARs proliferate regardless of whether the antigen is present on the beads or cells.

[Fig. 28A] to [Fig. 28B] exemplify that TAC engineered T cells expand in vivo and provide long-term protection to show cell persistence in a myeloma model. [Fig. 28A] to [Fig. 28B] illustrate 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 engraftment, mice were treated with BCMA TAC-T cells (bearing firefly luciferase). TAC-T cells are greatly expanded after administration. It is associated with tumor regression. Treated mice were resistant to tumor re-administration showing long-term persistence of TAC-T cells.

The data show that TAC-T cells have the potential to destroy tumor cells through mechanisms that mimic the process of natural T cell activation. The TAC technique illustrates 1) potent potency in liquid, 2) proliferation in vivo, 3) persistence of T cells to protect mice from re-administration, 4) cell expansion after T cell administration.

Example 16. MSCV or EF1α Promoter hu - or muIgκ HER2 -In vivo and in vitro activity of TAC.

CD4 and CD8 T cells were engineered with various TAC expressing viruses. A set of cells was engineered with a lentivirus expressing TAC [muIgG TAC (MSCV)] specific for HER-2 using a murine IgG signal peptide using the MSCV promoter. Another set of cells was engineered with a lentivirus expressing TAC [huIgG TAC (MSCV)] specific for HER-2 using a human IgG signal peptide using the MSCV promoter. The third set of cells was engineered with a lentivirus expressing TAC [muIgG TAC(EF1)] specific for HER-2 using a murine IgG signal peptide using the EF1α promoter. As a negative control, a TAC construct without a HER2 binding domain (Δ binding domain TAC) was used. The engineered cells were then characterized in vitro for resurface expression and specific activity and in vivo for activity in an OVCAR3 HER2 positive solid tumor model.

[Fig. 30] exemplifies T cell surface expression of the human or murine IgG leader HER2 TAC receptor under the control of the MCSV or EF1α promoter. Surface expression is a major requirement for biological activity, showing that HER2 TAC receptor expression is not affected by the species source of the IgG signal peptide.

[Fig. 31] Induces cytokine production when T cells engineered with the human or murine IgG leader HER2 TAC construct under the control of MCSV or EF1α promoter are co-cultured with HER2 positive target cells (OVCAR3) and HER2 negative cells (LOX IMVI ) And prove not. This exemplifies that HER2 TAC receptor engineered T cells can specifically bind to HER2 expressing target cells but are non-reactive to antigen negative cells.

Figure 32 demonstrates the in vivo efficacy of the human or murine IgG leader HER2 TAC construct under the control of the MCSV or EF1α promoter. After administration of divided doses of engineered T cells, HER2 TAC engineered cells have a significant effect on tumor growth, including tumor regression compared to the negative control Δbinding TAC. This in vivo experiment demonstrates that all HER2 TAC engineered cells show significant activity against in vivo solid tumor models.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will now be conceived by 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 used to practice the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and equivalents thereof are covered thereby.

SEQUENCE LISTING <110> MCMASTER UNIVERSITY TRIUMVIRA IMMUNOLOGICS USA, INC. <120> T CELL-ANTIGEN COUPLER 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 polynucleotide <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 polynucleotide <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 oligonucleotide <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 peptide <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 polynucleotide <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 oligonucleotide <400> 9 gaacagaaac tgattagcga agaagacctg 30 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <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 oligonucleotide <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 peptide <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 polynucleotide <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 oligonucleotide <400> 15 actagtggcg gaggaggatc actcgag 27 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <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 polynucleotide <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 oligonucleotide <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 peptide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 oligonucleotide <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 peptide <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 oligonucleotide <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 peptide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 oligonucleotide <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 peptide <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 oligonucleotide <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 peptide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 polynucleotide <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 peptide <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 oligonucleotide <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 polynucleotide <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 peptide <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 peptide <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 polynucleotide <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 oligonucleotide <400> 77 ggcggcggcg gaagtggagg aggaggctca ggcggaggag ggagc 45

Claims (107)

(a) a first polynucleotide encoding a ligand that selectively binds to the CD19 antigen;
(b) a second polynucleotide encoding a UCHT1 ligand that binds to CD3; And
(c) a third polynucleotide encoding a TCR signaling domain polypeptide comprising a cytosolic domain and a transmembrane domain
As a nucleic acid sequence encoding a CD19 trifunctional T cell-antigen coupler (CD19-TAC) comprising,
A nucleic acid sequence 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. The nucleic acid sequence of claim 1, wherein the ligand that selectively binds to the CD19 antigen is a single chain variable fragment (scFv). The ligand of claim 1 or 2, wherein the ligand that selectively binds to the CD19 antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 36. , A nucleic acid sequence comprising an amino acid sequence having at least 99%, or 100% sequence identity. The nucleic acid sequence according to any one of claims 1 to 3, wherein the UCHT1 ligand is a single chain antibody. The nucleic acid sequence of any one of claims 1 to 4, wherein the UCHT1 ligand comprises a Y182T mutation. The nucleic acid sequence according to any one of claims 1 to 4, wherein the UCHT1 ligand is a humanized variant of UCHT1 (huUCHT1). The nucleic acid sequence according to any one of claims 1 to 4, wherein the UCHT1 ligand is a humanized variant of UCHT1 comprising the Y177T mutation (huUCHT1 (Y177T)). The method of any one of claims 1 to 7, wherein the UCHT1 ligand is at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO: 14, SEQ ID NO: 72, SEQ ID NO: 44, or SEQ ID NO: 46. , At least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. 9. The nucleic acid sequence of any one of claims 1 to 8, wherein the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain. The method of any one of claims 1 to 9, wherein the third polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 with SEQ ID NO: 18. A nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having %, at least 99%, or 100% sequence identity. The nucleic acid sequence according to any one of claims 1 to 10, wherein the component encoded by (a) and the component encoded by (c) are fused to the component encoded by (b). The nucleic acid sequence according to any one of claims 1 to 10, wherein the component encoded by (b) and the component encoded by (c) are fused to the component encoded by (a). The nucleic acid sequence according to any one of claims 1 to 10, wherein at least one linker connects the component encoded by (a) to the component encoded by (b). The method of any one of claims 1 to 10, or 13, wherein the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helical structure, or a short helical structure. Nucleic acid sequence. The method of any one of claims 1 to 10, 13 or 14, wherein the at least one linker is at least 80%, at least with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28. A nucleic acid sequence comprising an amino acid sequence having 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The nucleic acid sequence according to any one of claims 1 to 15, wherein CD3 is of a TCR complex on a cell expressing the second polynucleotide. 17. The nucleic acid sequence of any one of claims 1 to 16, wherein binding of CD3 induces activation of a cell expressing the second polynucleotide. The method of any one of claims 1 to 17, wherein the CD19-TAC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 63. , A nucleic acid sequence comprising a nucleic acid sequence having at least 99%, or 100% sequence identity. The method of any one of claims 1 to 17, wherein the CD19-TAC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 64. , A nucleic acid sequence comprising an amino acid sequence having at least 99%, or 100% sequence identity. 20. The nucleic acid sequence according to any one of claims 1 to 19, wherein the nucleic acid sequence does not encode an auxiliary stimulatory domain. 21. The nucleic acid sequence of any one of claims 1 to 20, wherein the nucleic acid sequence does not encode an activation domain. (a) the nucleic acid sequence of any one of claims 1 to 21; And
(b) functional promoter in mammalian cells
A vector construct comprising. A T cell comprising the nucleic acid sequence of any one of claims 1 to 21. A pharmaceutical composition comprising the T cells of claim 23 and a pharmaceutically acceptable excipient. A method of treating a cancer expressing CD19 in an individual in need of treatment for a cancer expressing CD19, comprising administering the pharmaceutical composition of claim 24 to the individual. The method of claim 25, wherein the cancer is a B cell malignant tumor. The method of claim 25, wherein the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin lymphoma. 28. The method of any one of claims 25 to 27, wherein the pharmaceutical composition is administered into the carotid artery, subcutaneous, intradermal, intratumoral, intranodal, intrathecal, intramuscular, intravenous or intraperitoneal. (a) a first polynucleotide encoding a target specific ligand;
(b) a second polynucleotide encoding a ligand that binds to the protein associated with the TCR complex; And
(c) a third polynucleotide encoding a T cell receptor signal transduction domain polypeptide
As a nucleic acid sequence encoding a trifunctional T cell-antigen coupler (Tri-TAC) comprising,
The nucleic acid sequence that the ligand that binds to the protein associated with the TCR complex is selected from OKT3, F6A or L2K. The nucleic acid of claim 29, wherein the component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused directly to each other or linked by at least one linker. order. The nucleic acid sequence according to claim 29 or 30, wherein the component encoded by (a) and the component encoded by (b) are directly fused and linked by a linker to the component encoded by (c). The nucleic acid sequence according to claim 29 or 30, wherein the component encoded by (b) and the component encoded by (c) are directly fused and linked by a linker to the component encoded by (a). The nucleic acid sequence of any one of claims 30 to 32, wherein the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helical structure, or a short helical structure. The method of any one of claims 30 to 33, wherein the at least one linker is at least 80%, at least 85%, at least 90%, at least with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28. A nucleic acid sequence having an amino acid sequence having 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The nucleic acid sequence according to any one of claims 29 to 34, wherein the ligand that binds to the protein associated with the TCR complex is OKT3. The method of any one of claims 29 to 35, wherein the ligand that binds to the protein associated with the TCR complex is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least A nucleic acid sequence comprising an amino acid sequence having 97%, at least 98%, at least 99%, or 100% sequence identity. The nucleic acid sequence according to any one of claims 29 to 34, wherein the ligand that binds to the protein associated with the TCR complex is F6A. The method of any one of claims 29-34, or 37, wherein the ligand binding to the protein associated with the TCR complex is at least 80%, at least 85%, at least 90%, at least 95%, with SEQ ID NO: 24, A nucleic acid sequence comprising an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The nucleic acid sequence according to any one of claims 29 to 34, wherein the ligand that binds to the protein associated with the TCR complex is L2K. The method of any one of claims 29 to 34, or 39, wherein the ligand that binds to the protein associated with the TCR complex is at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO: 26, A nucleic acid sequence comprising an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. 41. The nucleic acid sequence according to any one of claims 29 to 40, wherein the protein associated with the TCR complex is CD3. 42. The nucleic acid sequence of any one of claims 29-41, wherein the target specific ligand selectively binds to a tumor antigen. 43. The nucleic acid sequence according to any one of claims 29 to 42, wherein the target specific ligand is a designed ankyrin repeat (DARPin) polypeptide, or a single chain variable fragment (scFv). 44. The nucleic acid sequence of any one of claims 29-43, wherein the target specific ligand selectively binds to the CD19 antigen, HER2 antigen, or BCMA antigen. The method of any one of claims 29 to 44, wherein the target-specific ligand selectively binds to the HER-2 antigen and trastuzumab, Pertuzumab, Lapatinib, neratinib Antibodies selected from (Neratinib), Ado-trastuzmab Emtansine, Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab A nucleic acid sequence comprising an antigen binding domain of. The method of any one of claims 29-44, wherein the target specific ligand selectively binds to the BCMA antigen and comprises an antigen binding domain of an antibody selected from belantamab mafodotin, and GSK2857916. Nucleic acid sequence. The method of any one of claims 29 to 44, wherein the target specific ligand is at least 80%, at least 85%, at least 90%, at least 95%, at least 96 with SEQ ID NO: 36, SEQ ID NO: 8 or SEQ ID NO: 34. A nucleic acid sequence comprising an amino acid sequence having %, at least 97%, at least 98%, at least 99%, or 100% sequence identity. 48. The nucleic acid sequence of any one of claims 29-47, wherein the T cell receptor signaling domain polypeptide comprises a cytosolic domain and a transmembrane domain. 49. The nucleic acid sequence of claim 48, wherein the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain, or the cytosolic domain is a CD8 cytosolic domain and the transmembrane domain is a CD8 transmembrane domain. 50. The nucleic acid sequence according to any one of claims 29 to 49, further comprising a leader sequence. The method of claim 50, wherein the leader sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50, A nucleic acid sequence comprising an amino acid sequence having at least 99%, or 100% sequence identity. 52. The nucleic acid sequence of any one of claims 29-51, wherein CD3 is of a TCR complex on a cell expressing the second polynucleotide. 53. The nucleic acid sequence of any one of claims 29 to 52, wherein binding of CD3 induces activation of a cell expressing the second polynucleotide. The method of any one of claims 29 to 53, wherein the Tri-TAC is 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: A nucleic acid sequence 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 with 61. . The method of any one of claims 29 to 53, wherein the Tri-TAC is 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: A nucleic acid sequence comprising 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 with 62 . 56. The nucleic acid sequence of any one of claims 29-55, wherein the nucleic acid sequence does not encode an auxiliary stimulatory domain. 57. The nucleic acid sequence of any one of claims 29-56, wherein the nucleic acid sequence does not encode an activation domain. (a) a first polynucleotide encoding a target specific ligand;
(b) a second polynucleotide encoding a ligand that binds to the protein associated with the TCR complex; And
(c) a third polynucleotide encoding a T cell receptor signal transduction domain polypeptide
As a nucleic acid sequence encoding a trifunctional T cell-antigen coupler (Tri-TAC) comprising,
The nucleic acid sequence further comprises a leader sequence, and the component encoded by (a), the component encoded by (b), and the component encoded by (c) are fused directly to each other or by at least one linker. The nucleic acid sequence to be linked. 59. The nucleic acid sequence of claim 58, wherein the target specific ligand selectively binds to a tumor antigen. 60. The nucleic acid sequence of claim 58 or 59, wherein the target specific ligand is a designed ankyrin repeat (DARPin) polypeptide, or a single chain variable fragment (scFv). 61. The nucleic acid sequence of any one of claims 58-60, wherein the target specific ligand selectively binds to the CD19 antigen, HER2 antigen, or BCMA antigen. The method according to any one of claims 58 to 61, wherein the target specific ligand selectively binds to the HER-2 antigen and trastuzumab, pertuzumab, lapatinib, neratinib, ado-trastuzumab emtansine, A nucleic acid sequence comprising the antigen binding domain of an antibody selected from gankotamab, margetuximab, thymigutuzumab, and ertumaksomab. The nucleic acid sequence of any one of claims 58-61, wherein the target specific ligand selectively binds to the BCMA antigen and comprises an antigen binding domain of an antibody selected from belantamab mapodotin, and GSK2857916. . The method of any one of claims 58 to 61, wherein the target specific ligand is at least 80%, at least 85%, at least with SEQ ID NO: 36, SEQ ID NO: 8, SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO: 54. A nucleic acid sequence comprising an amino acid sequence having 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The method according to any one of claims 58 to 64, wherein the ligand that binds to the protein associated with the TCR complex is selected from UCHT1, UCHT1 (Y182T), huUCHT1, huUCHT1 (Y177T), OKT3, F6A, or L2K. Nucleic acid sequence. The method of any one of claims 58 to 65, wherein the ligand binding to the protein associated with the TCR complex is 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 A nucleic acid having 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 with SEQ ID NO: 26. order. 67. The nucleic acid sequence of any one of claims 58 to 66, wherein the protein associated with the TCR complex is CD3. 68. The nucleic acid sequence of any one of claims 58-67, wherein the T cell receptor signaling domain polypeptide comprises a cytosolic domain and a transmembrane domain. 69. The nucleic acid sequence of claim 68, wherein the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain, or the cytosolic domain is a CD8 cytosolic domain and the transmembrane domain is a CD8 transmembrane domain. The method of any one of claims 58 to 69, wherein the leader sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96% with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50. , A nucleic acid sequence comprising an amino acid sequence having at least 97%, at least 98%, at least 99%, or 100% sequence identity. The method of any one of claims 58 to 70, wherein the component encoded by (a) and the component encoded by (b) are directly fused and linked by a linker to the component encoded by (c). Nucleic acid sequence. The method of any one of claims 58 to 70, wherein the component encoded by (b) and the component encoded by (c) are directly fused and linked by a linker to the component encoded by (a). Nucleic acid sequence. 73. The nucleic acid sequence of any one of claims 58-72, wherein the at least one linker is a G ₄ S flexible linker (SEQ ID NO: 73), a large protein domain, a long helical structure, or a short helical structure. The method of any one of claims 58-73, wherein the at least one linker is at least 80%, at least 85%, at least 90%, at least with SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 30, or SEQ ID NO: 28. A nucleic acid sequence having an amino acid sequence having 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. 75. The nucleic acid sequence of any one of claims 58-74, wherein the protein associated with the TCR complex is CD3 and CD3 is of the TCR complex on a cell expressing the second polynucleotide. 76. The nucleic acid sequence of claim 75, wherein binding of CD3 induces activation of a cell expressing the second polynucleotide. The method of any one of claims 58-76, wherein the Tri-TAC is 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: A nucleic acid sequence 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 with 61. . The method of any one of claims 58-76, wherein the Tri-TAC is 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: A nucleic acid sequence comprising 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 with 62 . 79. The nucleic acid sequence of any one of claims 58-78, wherein the nucleic acid sequence does not encode an auxiliary stimulatory domain. 80. The nucleic acid sequence of any one of claims 58-79, wherein the nucleic acid sequence does not encode an activation domain. A polypeptide encoded by the nucleic acid sequence of any one of claims 1 to 21 or 29 to 80. (a) the nucleic acid sequence of any one of claims 29 to 80; And
(b) functional promoter in mammalian cells
A vector construct comprising. A T cell comprising the nucleic acid sequence of any one of claims 29 to 80. A pharmaceutical composition comprising the T cells of claim 83, and a pharmaceutically possible excipient. A method of treating cancer in an individual in need thereof, comprising administering the pharmaceutical composition of claim 84 to the individual. 86. The method of claim 85, wherein the subject is a mammal. 87. The method of claim 85 or 86, wherein the cancer is a solid cancer or a liquid cancer. The method of any one of claims 85-87, wherein the cancer is lung cancer, breast cancer, multiple myeloma, glioma, gastrointestinal cancer, ovarian cancer, gastric cancer, colorectal cancer, urinary tract cancer, endometrial cancer, or colon cancer. 87. The method of claim 85 or 86, wherein the cancer comprises CD19 expressing cancer cells. 89. The method of any one of claims 85, 86, or 89, wherein the cancer is a B cell malignant tumor. The method of any one of claims 85, 86, or 89, wherein the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma. 87. The method of claim 85 or 86, wherein the cancer comprises HER-2 expressing cancer cells. The method of any one of claims 85, 86, or 92, wherein the cancer is breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, or gastric cancer. The method of claim 85 or 86, wherein the cancer comprises BCMA expressing cancer cells. The method of any one of claims 85, 86, or 94, wherein the cancer is leukemia, lymphoma, or multiple myeloma. The method of any one of claims 85-95, wherein the pharmaceutical composition is administered to the subject by carotid artery, subcutaneous, intradermal, intratumoral, intranodal, intrathecal, intramuscular, intravenous or intraperitoneal. The method of any one of claims 85-96, wherein the pharmaceutical composition is in unit dosage form. 98. The method of any one of claims 85-97, wherein the pharmaceutical composition comprises about 0.5-2 x 10 ⁹ T cells. The method of any one of claims 85-98, wherein the pharmaceutical composition is administered daily, weekly, biweekly, monthly, bimonthly or annually. 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 with SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 75 A nucleic acid sequence encoding a HER2 trifunctional T cell-antigen coupler (HER2-TAC) comprising a nucleic acid sequence having. The method of claim 100, wherein the HER2-TAC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 76. , A nucleic acid sequence comprising an amino acid sequence having at least 99%, or 100% sequence identity. The nucleic acid sequence of claim 100 or 101, wherein the nucleic acid sequence does not encode an auxiliary stimulatory domain. 103. The nucleic acid sequence of any one of claims 100-102, wherein the nucleic acid sequence does not encode an activation domain. SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61 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 A nucleic acid sequence encoding a BCMA trifunctional T cell-antigen coupler (BCMA-TAC) comprising a nucleic acid sequence with 100% sequence identity. The method of claim 104, wherein the BCMA-TAC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% with SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62. , A nucleic acid sequence comprising an amino acid sequence having at least 98%, at least 99%, or 100% sequence identity. 106. The nucleic acid sequence of claim 104 or 105, wherein the nucleic acid sequence does not encode an auxiliary stimulatory domain. The nucleic acid sequence of any one of claims 104-106, wherein the nucleic acid sequence does not encode an activation domain.

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