T cell receptor constructs and uses thereof

Abstract

The present disclosure provides T cell receptors (TCRs) directed against peptide-MHC complexes, isolated nucleic acid molecules encoding TCRs directed against peptide-MHC complexes, T cells expressing TCRs directed against peptide-MHC complexes, and pharmaceutical compositions for treating diseases.

Description

[0001] Cross-references

[0002] This application claims the benefits of U.S. Provisional Application No. 62 / 764,817, filed August 16, 2018, and U.S. Provisional Application No. 62 / 810,112, filed February 25, 2019, both of which are incorporated herein by reference in their entirety. Background Technology

[0003] T-cell receptors (TCRs) are members of the immunoglobulin superfamily and are typically composed of two subunits, the α and β subunits. They possess an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane / transcellular membrane region, and a short cytoplasmic tail at the C-terminus. The variable domains of both the TCR α and β chains have three hypervariable regions, or complementarity-determining regions (CDRs), while the β chain's variable domain has an additional hypervariable region (HV4), which is not normally in contact with the antigen and therefore not considered a CDR.

[0004] CDR3 is the primary CDR responsible for recognizing processed antigens, although CDR1 of the α-chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the β-chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC. The constant domains of the TCR consist of short linker sequences in which cysteine ​​residues form disulfide bonds that connect the two chains. The affinity of the TCR for specific antigens makes it valuable for a variety of therapeutic approaches. For example, adoptive immunotherapy can be effective in treating cancer patients, such as melanoma patients, because the TCR is highly sensitive to its antigens and can direct the immune response to tumor cells expressing its homologous antigens. Therefore, there is a need for TCRs targeting peptide-MHC complexes for the development of new and effective therapeutic agents. Summary of the Invention

[0005] This application is based on the development of TCRs targeting peptide-MHC complexes and effective therapeutic agents containing TCRs. This document provides isolated nucleic acid molecules encoding TCRs targeting peptide-MHC complexes, T cells expressing TCRs targeting peptide-MHC complexes, and pharmaceutical compositions for treating diseases.

[0006] This document provides a recombinant nucleic acid encoding a T-cell receptor (TCR) comprising a TCRβ chain construct containing a complementarity-determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:52. In some embodiments, the T-cell receptor (TCR) construct further comprises a complementarity-determining region 1 (CDR1) and a complementarity-determining region 2 (CDR2), wherein CDR1 has the amino acid sequence shown in SEQ ID NO:50; and CDR2 has the amino acid sequence shown in SEQ ID NO:51. In some embodiments, the TCRβ chain construct contains a variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the TCRβ chain construct contains a variable region having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the TCRβ chain construct contains a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the TCRβ chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the recombinant nucleic acid encoding the T cell receptor (TCR) of any of the preceding embodiments further includes a TCRα chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 47; CDR2 has the amino acid sequence shown in SEQ ID NO: 48; and CDR3 has the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the recombinant nucleic acid of any of the preceding embodiments includes: (a) a sequence having at least 80% sequence identity with SEQ ID NO: 56 or 57, further including at least a sequence encoding SEQ ID NO: 52; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 53 or 54, further including at least a sequence encoding SEQ ID NO: 49. In some embodiments, the recombinant nucleic acid encoding the T-cell receptor (TCR) of any of the foregoing embodiments comprises a TCRα chain construct containing a variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:55. In some embodiments, the TCRα chain construct contains a variable region having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:55. In some embodiments, the TCRα chain construct contains a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:55.In some embodiments, the TCRα chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:55. In some embodiments, the recombinant nucleic acid encoding the TCR comprises: (a) a β chain having the amino acid sequence shown in SEQ ID NO:60, or an amino acid sequence at least 80% identical to SEQ ID NO:60; and (b) an α chain having the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence at least 80% identical to SEQ ID NO:59. In some embodiments, the recombinant nucleic acid encoding the TCR of any of the foregoing embodiments binds to an epitope from human RAS containing the mutant G12V.

[0007] This document provides a recombinant nucleic acid encoding a TCR comprising a TCRβ construct and a TCRα construct, wherein the TCRβ construct comprises a complementarity-determining region 3 (CDR3) having an amino acid sequence selected from SEQ ID NO: 537 and 563. In some embodiments, the TCRβ construct comprises a CDR3 having the amino acid sequence of SEQ ID NO: 537. In some embodiments, the TCRβ construct comprising a CDR3 having the amino acid sequence of SEQ ID NO: 537 comprises a variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 541. In some embodiments, the TCRβ construct has the amino acid sequence shown in SEQ ID NO: 541. In some embodiments, the TCR comprising the TCRβ chain CDR3 having the amino acid sequence of SEQ ID NO:537 further comprises a TCRα chain construct containing a variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:539 or SEQ ID NO:552. In some embodiments, the TCRα chain construct has the amino acid sequence shown in SEQ ID NO:539. In some embodiments, the TCRα chain construct has the amino acid sequence shown in SEQ ID NO:552. In some embodiments, the recombinant nucleic acid encodes a TCR comprising a β chain and an α chain, the β chain having the amino acid sequence shown in SEQ ID NO:543, or a sequence having at least 80% sequence identity with SEQ ID NO:543; the α chain having the amino acid sequence shown in SEQ ID NO:542, or a sequence having at least 80% sequence identity with SEQ ID NO:542. In some embodiments, the recombinant nucleic acid encodes a TCR comprising a β-chain and an α-chain, wherein the β-chain has the amino acid sequence shown in SEQ ID NO:543, or a sequence having at least 80% sequence identity with SEQ ID NO:543; and the α-chain has the amino acid sequence shown in SEQ ID NO:555, or a sequence having at least 80% sequence identity with SEQ ID NO:555. In some embodiments, the recombinant nucleic acid encodes a TCR comprising a β-chain variable domain and an α-chain variable domain, wherein the β-chain variable domain has the amino acid sequence shown in SEQ ID NO:541, or a sequence having at least 80% sequence identity with SEQ ID NO:541; and the α-chain variable domain has the amino acid sequence shown in SEQ ID NO:539, or a sequence having at least 80% sequence identity with SEQ ID NO:539.In some embodiments, the recombinant nucleic acid encodes a TCR comprising a β-chain variable domain and an α-chain variable domain, wherein the β-chain variable domain has the amino acid sequence shown in SEQ ID NO:541, or a sequence having at least 80% sequence identity with SEQ ID NO:541; and the α-chain variable domain has the amino acid sequence shown in SEQ ID NO:552, or a sequence having at least 80% sequence identity with SEQ ID NO:552.

[0008] In some embodiments, a recombinant nucleic acid encoding a TCR comprising a TCRβ construct and a TCRα construct, wherein the TCRβ construct comprises CDR3 having the amino acid sequence of SEQ ID NO:563. In some embodiments, the TCRβ construct comprises a variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:567. In some embodiments, the TCRα construct comprises a variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:565. In some embodiments, the recombinant nucleic acid encodes a TCR comprising a β-chain and an α-chain variable domain, wherein the β-chain has the amino acid sequence shown in SEQ ID NO:569, or a sequence having at least 80% sequence identity with SEQ ID NO:569; and the α-chain variable domain has the amino acid sequence shown in SEQ ID NO:568, or a sequence having at least 80% sequence identity with SEQ ID NO:568. This document discloses a vector comprising the recombinant nucleic acid of any of the above embodiments. This document also discloses a cell comprising the recombinant nucleic acid of any of the above embodiments or the vector described above.

[0009] This document provides a recombinant nucleic acid encoding a TCR construct comprising: (a) a TCRβ chain construct and (b) a TCRα chain construct; wherein the TCR recognizes and binds to an epitope from human RAS containing a point mutation G12V or G12C, the epitope being located in a human MHC-protein complex, wherein the human MHC-protein is an HLA antigen encoded by the HLA A03:01 allele. In some embodiments, the TCRβ chain construct comprises CDR3 having the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the TCRβ chain construct further comprises a complementarity-determining region 1 (CDR1) and a complementarity-determining region 2 (CDR2), wherein CDR1 has the amino acid sequence shown in SEQ ID NO:66; and CDR2 has the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the recombinant nucleic acid encoding the T-cell receptor (TCR) described in this paragraph further comprises a TCRα chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO:63; CDR2 has the amino acid sequence shown in SEQ ID NO:64; and CDR3 has the amino acid sequence shown in SEQ ID NO:65. In some embodiments, the TCRβ chain construct comprises a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:74. In some embodiments, the TCRβ chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:74. In some embodiments, the T-cell receptor (TCR) comprises a TCRα chain construct, wherein the TCRα chain construct contains a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:71. In some embodiments, the TCRα chain construct contains a variable region having the amino acid sequence shown in SEQ ID NO:71. In some embodiments, the TCR comprises a β chain construct and an α chain construct, the β chain construct having the amino acid sequence shown in SEQ ID NO:76, or a sequence having at least 80% sequence identity with SEQ ID NO:76; the α chain construct having the amino acid sequence shown in SEQ ID NO:75, or a sequence having at least 80% sequence identity with SEQ ID NO:75. This document provides a vector comprising a recombinant nucleic acid of any of the embodiments described in this paragraph. This document also provides a cell comprising a recombinant nucleic acid of any of the embodiments described in this paragraph or any of the vectors described in this paragraph.

[0010] In some embodiments, a TCR from human RAS containing an epitope of the point mutation G12V that recognizes and binds to an HLA antigen encoded by the HLA A03:01 allele comprises a TCRβ chain construct containing a CDR3 having the amino acid sequence shown in SEQ ID NO:84. In some embodiments, the TCRβ chain construct further comprises a complementarity-determining region 1 (CDR1) and a complementarity-determining region 2 (CDR2), wherein CDR1 has the amino acid sequence shown in SEQ ID NO:82; and CDR2 has the amino acid sequence shown in SEQ ID NO:83. In some embodiments, the TCR further comprises a TCRα construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO:79; CDR2 has the amino acid sequence shown in SEQ ID NO:80; and CDR3 has the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 90. In some embodiments, the TCRβ chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO: 90. In some embodiments, the above-described TCR further includes a TCRα chain construct, which includes a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 87. In some embodiments, the TCRα chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO: 71. In some embodiments, the TCR comprises a β-chain construct and an α-chain construct, the β-chain construct having the amino acid sequence of SEQ ID NO:92, or a sequence having at least 80% sequence identity with SEQ ID NO:92; and the α-chain construct having the amino acid sequence of SEQ ID NO:91, or a sequence having at least 80% sequence identity with SEQ ID NO:91. This document provides a vector comprising a recombinant nucleic acid of any of the embodiments described in this paragraph, and a cell comprising any of the recombinant nucleic acids of any of the embodiments described herein.

[0011] In some embodiments, the recombinant nucleic acid of the embodiments described in the preceding paragraphs recognizes and binds to a TCR from human RAS containing an epitope with a point mutation G12V or G12C that is complexed with an HLA antigen encoded by the HLA antigen encoded by the HLA03:01 allele. This TCRβ chain construct contains a CDR3 having the amino acid sequence shown in SEQ ID NO:388. In some embodiments, the TCRβ chain construct further includes a complementarity-determining region 1 (CDR1) and a complementarity-determining region 2 (CDR2), wherein CDR1 has the amino acid sequence shown in SEQ ID NO:386; and CDR2 has the amino acid sequence shown in SEQ ID NO:387. In some embodiments, the TCR further comprises a TCRα construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO:383; CDR2 has the amino acid sequence shown in SEQ ID NO:384; and CDR3 has the amino acid sequence shown in SEQ ID NO:385. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:394. In some embodiments, the TCRβ chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO:394. In some embodiments, the above-described TCR further includes a TCRα chain construct, which includes a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:391. In some embodiments, the TCRα chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO:391. In some embodiments, the TCR comprises a β-chain construct and an α-chain construct, the β-chain construct having the amino acid sequence of SEQ ID NO:396, or a sequence having at least 80% sequence identity with SEQ ID NO:396; the α-chain construct having the amino acid sequence of SEQ ID NO:395, or a sequence having at least 80% sequence identity with SEQ ID NO:395. This document provides a vector comprising a recombinant nucleic acid of any of the embodiments described in this paragraph, and a cell comprising any of the recombinant nucleic acids of any of the embodiments described herein.

[0012] In some embodiments, the recombinant nucleic acid of the embodiments described in the preceding paragraphs recognizes and binds to a TCR from human RAS containing an epitope of the point mutation G12C that is complexed with the HLA antigen encoded by the HLA antigen encoded by the HLA03:01 allele. This TCRβ chain construct contains a CDR3 having the amino acid sequence shown in SEQ ID NO:100. In some embodiments, the TCRβ chain construct further includes a complementarity-determining region 1 (CDR1) and a complementarity-determining region 2 (CDR2), wherein CDR1 has the amino acid sequence shown in SEQ ID NO:98, and CDR2 has the amino acid sequence shown in SEQ ID NO:99. In some embodiments, the TCR further comprises a TCRα construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO:95, CDR2 has the amino acid sequence shown in SEQ ID NO:96, and CDR3 has the amino acid sequence shown in SEQ ID NO:97. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106. In some embodiments, the TCRβ chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO: 106. In some embodiments, the above-described TCR further includes a TCRα chain construct, which includes a variable region having at least 80% or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCRα chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR comprises a β-chain construct and an α-chain construct, the β-chain construct having the amino acid sequence of SEQ ID NO:108, or a sequence having at least 80% sequence identity with SEQ ID NO:108; and the α-chain construct having the amino acid sequence of SEQ ID NO:107, or a sequence having at least 80% sequence identity with SEQ ID NO:107. This document provides a vector comprising a recombinant nucleic acid of any of the embodiments described in this paragraph, and a cell comprising any of the recombinant nucleic acids of any of the embodiments described herein.

[0013] In some embodiments, the epitope is 8-25 amino acids in length. In some embodiments, the epitope contains a mutation differing from the wild-type epitope by at least one amino acid. In some embodiments, the epitope binds to human MHC with a higher affinity than the corresponding wild-type epitope. In some embodiments, the epitope uses a Kc of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to human MHC. In some embodiments, the mutation is not present in the non-cancerous cells of the subject. In some embodiments, the TCR is expressed at a Kc of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to an MHC-peptide complex. In some embodiments, the nucleic acid is operatively linked to a promoter. In some embodiments, the cell is a CD4 cell. + T cells. In some embodiments, the cells are CD8 cells. + T cells. In some embodiments, the cells are isolated from subjects with RAS mutations.

[0014] This document provides a pharmaceutical composition comprising: (A) a nucleic acid of any of the above embodiments; or, a carrier of any of the above embodiments; or, a cell of any of the above embodiments; and (b) a pharmaceutically acceptable excipient or diluent. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the adjuvant is poly(I:C). In some embodiments, the pharmaceutical composition of any of the above embodiments is used to treat immune diseases or cancer.

[0015] This document provides a method for treating a subject suffering from a disease or condition, comprising administering to the subject a pharmaceutical composition of any of the above-described embodiments. In some embodiments, a method for treating a subject suffering from cancer comprises administering to the subject a pharmaceutical composition of any of the above-described embodiments.

[0016] This article also provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A03:01 allele or the HLA-A11:01 allele, wherein the therapeutic agent is a pharmaceutical composition selected from any of the above embodiments.

[0017] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC, wherein the TCRα chain construct comprises a TCRα chain construct containing an epitope from the RAS that is selected from human MHC.

[0018] The TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 84% sequence identity with an amino acid sequence selected from SEQ ID NO: 3, 18, 33, 49, 65, 81, 97, 113, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, and 433, and / or wherein the TCRβ chain construct contains a complementarity-determining region 3 (CDR3) having at least 84% sequence identity with an amino acid sequence selected from SEQ ID NO: 6, 21, 36, 52, 68, 84, 100, 116, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, and 436.

[0019] This document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC, wherein the epitope from the RAS comprises a region having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 15, 30, 45, 46, 61, 62, 77, 78, 93, 94, 109, 110, 125, 126, 219-222, 253, 254, 269, 270, 285, 286, 301, 302, 317, 318, 333, 334, 349, 350, 365, 366, 381, 382, ​​397, 398, 413, 414, 429, 430, 445, and 446.

[0020] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T-cell receptor (TCR), the T-cell receptor comprising a TCR α-chain construct and / or a TCR β-chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A03:01 allele. In some embodiments, the α-chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:71, 87, 103, 295, 311, 327, 343, 359, and 391, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A03:01 allele. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 74, 90, 106, 298, 314, 330, 346, 362, and 394, wherein the TCR specifically binds to an epitope from RAS that is encoded by the human MHC complex by the HLA-A03:01 allele.

[0021] In some embodiments, the TCRα chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 63, 79, 95, 287, 303, 319, 335, 351, and 383. In some embodiments, the TCRβ chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 66, 82, 98, 290, 306, 322, 338, 354, and 386. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 64, 80, 96, 288, 304, 320, 336, 352, and 384. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 67, 83, 99, 291, 307, 323, 339, 355, and 387. In some embodiments, the TCRα chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 65, 81, 97, 289, 305, 321, 337, 353, and 385. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 68, 84, 100, 292, 308, 324, 340, 356, and 388.

[0022] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T-cell receptor (TCR), the T-cell receptor comprising a TCR α-chain construct and / or a TCR β-chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A02:01 allele. In some embodiments, the α-chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 9 and 24, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A02:01 allele. In some embodiments, the TCR β-chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 12 and 27, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A02:01 allele.

[0023] In some embodiments, the TCRα chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:1 and 16. In some embodiments, the TCRβ chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:4 and 19. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:2 and 17. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:5 and 20. In some embodiments, the TCRα chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:3 and 18.

[0024] In some embodiments, the TCRβ chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 6 and 21.

[0025] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T-cell receptor (TCR), the T-cell receptor comprising a TCRα chain construct and / or a TCRβ chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A11:01 allele. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:39, 55, 122, 247, 263, 279, 375, 407, and 423, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A11:01 allele. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:42, 58, 125, 250, 266, 282, 378, 413, and 426, wherein the TCR specifically binds to an epitope from RAS that is encoded by the human MHC complex by the HLA-A11:01 allele.

[0026] In some embodiments, the TCRα chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 31, 47, 111, 239, 255, 271, 367, 399, and 415. In some embodiments, the TCRβ chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 34, 50, 114, 242, 258, 274, 370, 402, and 418. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 32, 48, 112, 240, 256, 272, 368, 400, and 416. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 35, 51, 115, 243, 259, 275, 371, 403, and 419. In some embodiments, the TCRα chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 33, 49, 113, 241, 257, 273, 369, 401, 417, and 433. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 36, 52, 116, 244, 260, 276, 372, 404, and 420. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:9; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:12. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:3; and the TCRβ chain construct includes CDR1 of SEQ ID NO:4, CDR2 of SEQ ID NO:5, and CDR3 of SEQ ID NO:6. In some embodiments, the TCR includes an α chain having the sequence of SEQ ID NO:13 or a sequence having at least 80% identity with SEQ ID NO:13. In some embodiments, the TCR includes a β chain having the sequence of SEQ ID NO:14 or a sequence having at least 80% identity with SEQ ID NO:14.In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:24; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:27. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:16, CDR2 of SEQ ID NO:17, and CDR3 of SEQ ID NO:18; and the TCRβ chain construct includes CDR1 of SEQ ID NO:19, CDR2 of SEQ ID NO:20, and CDR3 of SEQ ID NO:21. In some embodiments, the TCR includes an α chain having the sequence of SEQ ID NO:28 or a sequence having at least 80% identity with SEQ ID NO:28. In some embodiments, the TCR includes a β chain having the sequence of SEQ ID NO:29 or a sequence having at least 80% identity with SEQ ID NO:29. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:39; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:42. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and the TCRβ chain construct includes CDR1 of SEQ ID NO:34, CDR2 of SEQ ID NO:35, and CDR3 of SEQ ID NO:36. In some embodiments, the TCR includes an α chain having the sequence of SEQ ID NO:43 or a sequence having at least 80% identity with SEQ ID NO:43. In some embodiments, the TCR includes a β chain having the sequence of SEQ ID NO:44 or a sequence having at least 80% identity with SEQ ID NO:44. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:55; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:58. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:47, CDR2 of SEQ ID NO:48, and CDR3 of SEQ ID NO:49; and the TCRβ chain construct includes CDR1 of SEQ ID NO:50, CDR2 of SEQ ID NO:51, and CDR3 of SEQ ID NO:52.In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:71; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:74. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65; and the TCRβ chain construct includes CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. In some embodiments, the TCR includes an α chain having the sequence of SEQ ID NO:75 or a sequence having at least 80% identity with SEQ ID NO:75. In some embodiments, the TCR includes a β chain having the sequence of SEQ ID NO:76 or a sequence having at least 80% identity with SEQ ID NO:76. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:87; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:90. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:80, and CDR3 of SEQ ID NO:81; and the TCRβ chain construct includes CDR1 of SEQ ID NO:82, CDR2 of SEQ ID NO:83, and CDR3 of SEQ ID NO:84. In some embodiments, the TCR includes an α chain having the sequence of SEQ ID NO:91 or a sequence having at least 80% identity with SEQ ID NO:91. In some embodiments, the TCR includes a β chain having the sequence of SEQ ID NO:92 or a sequence having at least 80% identity with SEQ ID NO:92. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:103; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:106. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:95, CDR2 of SEQ ID NO:96, and CDR3 of SEQ ID NO:97; and the TCRβ chain construct includes CDR1 of SEQ ID NO:98, CDR2 of SEQ ID NO:99, and CDR3 of SEQ ID NO:100.In some embodiments, the TCR comprises an α-chain having the sequence of SEQ ID NO:107 or a sequence having at least 80% identity with SEQ ID NO:107. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:108 or a sequence having at least 80% identity with SEQ ID NO:108. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:119; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:122. In some embodiments, the TCR α-chain construct comprises CDR1 of SEQ ID NO:111, CDR2 of SEQ ID NO:112, and CDR3 of SEQ ID NO:113; and the TCR β-chain construct comprises CDR1 of SEQ ID NO:114, CDR2 of SEQ ID NO:115, and CDR3 of SEQ ID NO:116. In some embodiments, the TCR comprises an α-chain having the sequence of SEQ ID NO:123 or a sequence having at least 80% identity with SEQ ID NO:123. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:124 or a sequence having at least 80% identity with SEQ ID NO:124. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:247; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:250. In some embodiments, the TCR α-chain construct comprises CDR1 of SEQ ID NO:239, CDR2 of SEQ ID NO:240, and CDR3 of SEQ ID NO:241; and the TCR β-chain construct comprises CDR1 of SEQ ID NO:242, CDR2 of SEQ ID NO:243, and CDR3 of SEQ ID NO:244. In some embodiments, the TCR comprises an α-chain having the sequence of SEQ ID NO:251 or a sequence having at least 80% identity with SEQ ID NO:251. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:252 or a sequence having at least 80% identity with SEQ ID NO:252. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:263; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:266.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:255, CDR2 of SEQ ID NO:256, and CDR3 of SEQ ID NO:257; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:258, CDR2 of SEQ ID NO:259, and CDR3 of SEQ ID NO:260. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:267 or a sequence having at least 80% identity with SEQ ID NO:267. In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:268 or a sequence having at least 80% identity with SEQ ID NO:268. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:279; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:282. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:271, CDR2 of SEQ ID NO:272, and CDR3 of SEQ ID NO:273; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:274, CDR2 of SEQ ID NO:275, and CDR3 of SEQ ID NO:276. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:283 or a sequence having at least 80% identity with SEQ ID NO:283. In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:284 or a sequence having at least 80% identity with SEQ ID NO:284. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:295; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:298. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:287, CDR2 of SEQ ID NO:288, and CDR3 of SEQ ID NO:289; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:290, CDR2 of SEQ ID NO:291, and CDR3 of SEQ ID NO:292. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:299 or a sequence having at least 80% identity with SEQ ID NO:299.In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:300 or a sequence having at least 80% identity with SEQ ID NO:300. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:311; and the TCR β chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:314. In some embodiments, the TCR α chain construct comprises CDR1 of SEQ ID NO:303, CDR2 of SEQ ID NO:304, and CDR3 of SEQ ID NO:305; and the TCR β chain construct comprises CDR1 of SEQ ID NO:306, CDR2 of SEQ ID NO:307, and CDR3 of SEQ ID NO:308. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:315 or a sequence having at least 80% identity with SEQ ID NO:315. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:316 or a sequence having at least 80% identity with SEQ ID NO:316. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:327; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:330. In some embodiments, the TCR α-chain construct comprises CDR1 of SEQ ID NO:319, CDR2 of SEQ ID NO:320, and CDR3 of SEQ ID NO:321; and the TCR β-chain construct comprises CDR1 of SEQ ID NO:322, CDR2 of SEQ ID NO:323, and CDR3 of SEQ ID NO:324. In some embodiments, the TCR comprises an α-chain having the sequence of SEQ ID NO:331 or a sequence having at least 80% identity with SEQ ID NO:331. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:332 or a sequence having at least 80% identity with SEQ ID NO:332. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:343; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:346.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:335, CDR2 of SEQ ID NO:336, and CDR3 of SEQ ID NO:337; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:338, CDR2 of SEQ ID NO:339, and CDR3 of SEQ ID NO:340. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:347 or a sequence having at least 80% identity with SEQ ID NO:347. In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:348 or a sequence having at least 80% identity with SEQ ID NO:348. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:359; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:362. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:351, CDR2 of SEQ ID NO:352, and CDR3 of SEQ ID NO:353; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:354, CDR2 of SEQ ID NO:355, and CDR3 of SEQ ID NO:356. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:363 or a sequence having at least 80% identity with SEQ ID NO:363. In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:364 or a sequence having at least 80% identity with SEQ ID NO:364. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:375; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:378. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:367, CDR2 of SEQ ID NO:368, and CDR3 of SEQ ID NO:369; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:370, CDR2 of SEQ ID NO:371, and CDR3 of SEQ ID NO:372. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:379 or a sequence having at least 80% identity with SEQ ID NO:379.In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:380 or a sequence having at least 80% identity with SEQ ID NO:380. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:391; and the TCR β chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:394. In some embodiments, the TCR α chain construct comprises CDR1 of SEQ ID NO:383, CDR2 of SEQ ID NO:384, and CDR3 of SEQ ID NO:385; and the TCR β chain construct comprises CDR1 of SEQ ID NO:386, CDR2 of SEQ ID NO:387, and CDR3 of SEQ ID NO:388. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:395 or a sequence having at least 80% identity with SEQ ID NO:395. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:396 or a sequence having at least 80% identity with SEQ ID NO:396. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:407; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:410. In some embodiments, the TCR α-chain construct comprises CDR1 of SEQ ID NO:399, CDR2 of SEQ ID NO:400, and CDR3 of SEQ ID NO:401; and the TCR β-chain construct comprises CDR1 of SEQ ID NO:402, CDR2 of SEQ ID NO:403, and CDR3 of SEQ ID NO:404. In some embodiments, the TCR comprises an α-chain having the sequence of SEQ ID NO:411 or a sequence having at least 80% identity with SEQ ID NO:411. In some embodiments, the TCR comprises a β-chain having the sequence of SEQ ID NO:412 or a sequence having at least 80% identity with SEQ ID NO:412. In some embodiments, the TCR α-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:423; and the TCR β-chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:426.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:415, CDR2 of SEQ ID NO:416, and CDR3 of SEQ ID NO:417; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:418, CDR2 of SEQ ID NO:419, and CDR3 of SEQ ID NO:420. In some embodiments, the TCR comprises an α chain having the sequence of SEQ ID NO:427 or a sequence having at least 80% identity with SEQ ID NO:427. In some embodiments, the TCR comprises a β chain having the sequence of SEQ ID NO:428 or a sequence having at least 80% identity with SEQ ID NO:428. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:391; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:394.

[0027] This document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCR α-chain construct and / or a TCR β-chain construct capable of specifically binding to an epitope derived from TMPRSS2:ERG that complexes with human MHC, wherein the TCR α-chain construct and / or TCR β-chain construct comprises a complementarity-determining region 3 (CDR3) having sequences of SEQ ID NO:144 and SEQ ID NO:147, or a sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:144 and SEQ ID NO:147. In some embodiments, the TCR comprises an α-chain having a sequence of SEQ ID NO:154 or a sequence having at least 80% identity with SEQ ID NO:154. In some embodiments, the TCR comprises a β-chain having a sequence of SEQ ID NO:155 or a sequence having at least 80% identity with SEQ ID NO:155.

[0028] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the epitope from TMPRSS2:ERG comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:156.

[0029] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:150. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:153. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity determination region 1 (CDR1) having the sequence of SEQ ID NO:142 or a sequence having at least 90% sequence identity with SEQ ID NO:142, and the sequence of SEQ ID NO:145 or a sequence having at least 90% sequence identity with SEQ ID NO:145. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity determination region 2 (CDR2) having at least 90% sequence identity with SEQ ID NO:143 or SEQ ID NO:146, respectively. In some embodiments, the TCRα chain construct includes a variable region having the sequence of SEQ ID NO:150 or having at least 80% sequence identity with SEQ ID NO:150; and the TCRβ chain construct includes a sequence having SEQ ID NO:153 or having at least 80% sequence identity with SEQ ID NO:153. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:142, CDR2 of SEQ ID NO:143, and CDR3 of SEQ ID NO:144; and the TCRβ chain construct includes CDR1 of SEQ ID NO:145, CDR2 of SEQ ID NO:146, and CDR3 of SEQ ID NO:147.

[0030] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from GATA3 that complexes with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 129, 132, 191, 194, 206 and 209.

[0031] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant GATA3 peptide complexed with a protein encoded by an HLA allele from a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct.

[0032] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode T cell receptors (TCRs) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCRs specifically bind to mutant GATA3 peptides complexed with HLA-A02:01, HLA-B07:02, or HLA-B08:01 proteins; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:129, 132, 191, 194, 206, or 209; and / or specifically binding mutant GATA3 peptides comprising a region having a sequence having at least 70% sequence identity with SEQ ID NO:141, 203, or 218.

[0033] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from GATA3 that is complexed with human MHC, wherein the epitope from GATA3 comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:141, 203 or 218.

[0034] In some embodiments, the TCRα chain construct includes a variable region having the sequence of SEQ ID NO: 135, 197, or 212; or a sequence having at least 80% sequence identity with SEQ ID NO: 135, 197, or 212. In some embodiments, the TCRβ chain construct includes a variable region having the sequence of SEQ ID NO: 138, 200, or 215; or a sequence having at least 80% sequence identity with SEQ ID NO: 138, 200, or 215. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having the amino acid sequences of SEQ ID NO: 127, 130, 189, 192, 204, and 207, or having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 127, 130, 189, 192, 204, and 207. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having an amino acid sequence of SEQ ID NO:128, 131, 190, 193, 205, and 208, or having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:128, 131, 190, 193, 205, and 208. In some embodiments, the GATA3-binding TCRα chain includes a variable region having a sequence of SEQ ID NO:135, or a sequence having at least 80% identity with SEQ ID NO:135; and the β chain has a variable region having a sequence of SEQ ID NO:138, or a sequence having at least 80% identity with SEQ ID NO:138. In some embodiments, the TCR α chain construct comprises CDR1 of SEQ ID NO:127, CDR2 of SEQ ID NO:128, and CDR3 of SEQ ID NO:129; and the TCR β chain construct comprises CDR1 of SEQ ID NO:130, CDR2 of SEQ ID NO:131, and CDR3 of SEQ ID NO:132. In some embodiments, the TCR has an α chain having the sequence of SEQ ID NO:139, or a sequence having at least 80% identity with SEQ ID NO:139. In some embodiments, the TCR has a β chain having the sequence of SEQ ID NO:140, or a sequence having at least 80% identity with SEQ ID NO:140.In some embodiments, the TCRα chain construct comprises an amino acid sequence having the sequence of SEQ ID NO:197 or a variable region having at least 80% sequence identity with SEQ ID NO:197; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:200. In some embodiments, the TCRα chain comprises the sequence of SEQ ID NO:201, or a sequence having at least 80% sequence identity with SEQ ID NO:201. In some embodiments, the TCRβ chain comprises the sequence of SEQ ID NO:202, or a sequence having at least 80% sequence identity with SEQ ID NO:202. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:189, CDR2 of SEQ ID NO:190, and CDR3 of SEQ ID NO:191; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:192, CDR2 of SEQ ID NO:193, and CDR3 of SEQ ID NO:194. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:212; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:215. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:204, CDR2 of SEQ ID NO:205, and CDR3 of SEQ ID NO:206; and the TCRβ chain construct includes CDR1 of SEQ ID NO:207, CDR2 of SEQ ID NO:208, and CDR3 of SEQ ID NO:209. In some embodiments, the TCR includes an α chain having the sequence of SEQ ID NO:216, or a sequence having at least 80% identity with SEQ ID NO:216. In some embodiments, the TCR includes a β chain having the sequence of SEQ ID NO:217, or a sequence having at least 80% identity with SEQ ID NO:217.

[0035] This document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from BTK that complexes with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:161 and SEQ ID NO:176, or wherein the TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:164 and SEQ ID NO:179.

[0036] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant BTK peptide complexed with a protein encoded by an HLA allele from a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct.

[0037] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode T cell receptors (TCRs) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCRs specifically bind to a mutant BTK peptide complexed with an HLA-A02:01 protein; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:161, 164, 176 or 179; and / or specifically bind to a mutant BTK peptide comprising a region having at least 70% sequence identity with SEQ ID NO:173 or 188.

[0038] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from BTK that complexes with human MHC, wherein the epitope derived from BTK comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:173 or 188.

[0039] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 167 or 182. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 170 or 185. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 159, 162, 174, and 177. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 160, 163, 175, and 178. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:167 or 182; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:170 or 185. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:159 or 174, CDR2 of SEQ ID NO:160 or 175, and CDR3 of SEQ ID NO:161 or 176; and the TCRβ chain construct includes CDR1 of SEQ ID NO:162 or 177, CDR2 of SEQ ID NO:163 or 178, and CDR3 of SEQ ID NO:164 or 179.

[0040] This document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an EGFR-derived epitope complexed with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:449, SEQ ID NO:466, SEQ ID NO:483, SEQ ID NO:500, and SEQ ID NO:517, or wherein the TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:452, SEQ ID NO:469, SEQ ID NO:486, SEQ ID NO:503, and SEQ ID NO:520.

[0041] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant EGFR peptide complexed with a protein encoded by an HLA allele from a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct.

[0042] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode a T cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCR specifically binds a mutant EGFR peptide complexed with an HLA-A02:01 protein; comprising a complementarity-determining region 3 (CDR3) having a sequence having SEQ ID NO:449, 466, 483, 500, 517, 452, 469, 486, 503 or 520 or a sequence having at least 90% sequence identity with SEQ ID NO:449, 466, 483, 500, 517, 452, 469, 486, 503 or 520; and / or specifically binding a mutant EGFR peptide comprising a sequence having ..., wherein the mutant EGFR peptide comprises a sequence having SEQ ID NO:449, 466, 483, 500, 517, 452, 469, 486, 503 or 520; and / or specifically binding a mutant EGFR The sequence of SEQ ID NO:461, 462, 463, 478, 479, 480, 495, 496, 497, 512, 513, 514, 529, 530 or 531, or a region of a sequence having at least 70% sequence identity with SEQ ID NO:461, 462, 463, 478, 479, 480, 495, 496, 497, 512, 513, 514, 529, 530 or 531.

[0043] This document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an EGFR-derived epitope complexing with human MHC, wherein the EGFR-derived epitope comprises a region having at least 90% sequence identity with the amino acid sequences SEQ ID NO: 461, 462, 463, 478, 479, 480, 495, 496, 497, 512, 513, 514, 529, 530, or 531.

[0044] In some embodiments, the TCRα chain construct includes a variable region having a sequence of SEQ ID NO:449, 466, 483, 500, or 517, or a sequence having at least 80% sequence identity with SEQ ID NO:449, 466, 483, 500, or 517. In some embodiments, the TCRβ chain construct includes a variable region having a sequence of SEQ ID NO:452, 469, 486, 503, or 520, or a sequence having at least 80% sequence identity with SEQ ID NO:452, 469, 486, 503, or 520. In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having a sequence selected from SEQ ID NO:447, 464, 481, 498, 515, 450, 467, 484, 501, and 518, or a sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:447, 464, 481, 498, 515, 450, 467, 484, 501, and 518. In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having a sequence selected from SEQ ID NO:448, 465, 482, 499, 516, 451, 468, 485, 502, and 519, or a sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:448, 465, 482, 499, 516, 451, 468, 485, 502, and 519. In some embodiments, the TCRα chain construct includes a variable region having a sequence of SEQ ID NO:455, 472, 489, 506 or 523 or a sequence having at least 80% sequence identity with SEQ ID NO:455, 472, 489, 506 or 523; and the TCRβ chain construct includes a variable region having a sequence of SEQ ID NO:458, 475, 492, 509 or 526 or a sequence having at least 80% sequence identity with SEQ ID NO:458, 475, 492, 509 or 526.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO: 447, 464, 481, 498 or 515, CDR2 of SEQ ID NO: 448, 465, 482, 499 or 516, and CDR3 of SEQ ID NO: 449, 466, 483, 500 or 517; and the TCRβ chain construct comprises CDR1 of SEQ ID NO: 450, 467, 484, 501 or 518, CDR2 of SEQ ID NO: 451, 468, 485, 502 or 519, and CDR3 of SEQ ID NO: 452, 469, 486, 503 or 520.

[0045] In some embodiments, the epitope comprises a mutation selected from point mutations, splice site mutations, frameshift mutations, readthrough mutations, resistance mutations, gene fusion mutations, and any combination thereof. In some embodiments, the human MHC is encoded by the HLA-A02:01 allele, the HLA-A03:01 allele, or the HLA-A11:01 allele. In some embodiments, the RAS epitope comprises a point mutation. In some embodiments, the point mutation is a G12V mutation. In some embodiments, the point mutation is a G12C mutation. In some embodiments, the point mutation is a G12D mutation. In some embodiments, the human MHC is encoded by the HLA-A02:01 allele. In some embodiments, the TMPRSS2:ERG epitope comprises a gene fusion mutation. In some embodiments, the human MHC is encoded by the HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele. In some embodiments, the epitope comprises a frameshift mutation. In some embodiments, the human MHC is encoded by the HLA-A02:01 allele. In some embodiments, the BTK epitope contains a point mutation. In some embodiments, the point mutation is a C481S mutation. In some embodiments, the human MHC is encoded by the HLA-A02:01 allele. In some embodiments, the EGFR epitope contains a point mutation. In some embodiments, the point mutation is T790M.

[0046] In some embodiments, the epitope is at least 8 amino acids long. In some embodiments, the epitope is at least 16 amino acids long. In some embodiments, the epitope is 8-25 amino acids long. In some embodiments, the epitope is 8-12 amino acids long. In some embodiments, the epitope is 16-25 amino acids long. In some embodiments, the epitope is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long.

[0047] In some embodiments, the epitope binds to human MHC with a higher affinity than the corresponding wild-type epitope. In some embodiments, the epitope has a K+ concentration of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to human MHC.

[0048] In some embodiments, the mutation is not present in the subject's non-cancerous cells. In some embodiments, the epitope is encoded by a gene of the subject's cancer cells or a gene expressed thereon.

[0049] In some embodiments, the TCR is at a Kc of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to the HLA-peptide complex.

[0050] In some implementations, the nucleic acid is operatively linked to a promoter.

[0051] This document provides vectors containing the nucleic acids described herein. In some embodiments, the vector is a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, granule, virus, or virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, alphavirus, vaccinia virus, hepatitis B virus, human papillomavirus, or a pseudotype thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoparticle, cationic lipid, cationic polymer, metal nanopolymer, nanorod, liposome, micelle, microvesicle, cell-penetrating peptide, or liposphere.

[0052] This article provides proteins encoded by nucleic acids from any of the above components.

[0053] This document provides a host cell comprising the aforementioned nucleic acid, vector, or protein. In some embodiments, the host cell is a CD4 cell. + T cells. In some embodiments, the host cell is a CD8 cell. + T cells. In some embodiments, the host cell is an autologous cell. In some embodiments, the host cell is an allogeneic cell. In some embodiments, the host cell is a natural killer cell, a B cell, or a cell line that proliferates indefinitely. In some embodiments, the host cell is a human cell.

[0054] This document provides a pharmaceutical composition comprising the nucleic acid, carrier, protein, or host cell described herein; and a pharmaceutically acceptable excipient or diluent. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the immunomodulator in the pharmaceutical composition is a cytokine. In some embodiments, the adjuvant in the pharmaceutical composition is poly(I:C). In some embodiments, the pharmaceutical composition is intended for the treatment of immune diseases or cancer.

[0055] This article provides information on the use of the above-mentioned pharmaceutical compositions in the treatment of immune diseases or cancer.

[0056] This document provides the vectors, proteins, or host cells described herein for the preparation of medicaments for treating immune diseases or cancer. In some embodiments, the medicament is an adoptive T-cell therapy or a TCR gene therapy.

[0057] This article provides a method for treating a subject suffering from a disease or condition, comprising administering to the subject the pharmaceutical composition disclosed herein.

[0058] This article provides a method for treating a subject with cancer, which includes administering the pharmaceutical composition disclosed herein to the subject.

[0059] This article provides a method for treating a subject with cancer, comprising administering a pharmaceutical composition to the subject. In some embodiments, the subject expresses or is identified as expressing a protein encoded by the HLA-A02:01, HLA-B07:02, HLA-B08:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01, HLA-A33:03, HLA-A68:01, or HLA-A74:01 alleles of the subject's genome.

[0060] This article provides a method for treating a subject with cancer, comprising administering a TCR or T cells expressing the TCR to the subject, wherein the TCR specifically binds to alleles of HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01, HLA-A33:03, HLA-A68:01, or HLA-A A mutant RAS peptide encoding a protein complex of HLA-A03:01; wherein the subject expresses or is identified as expressing a protein encoded by the HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele, wherein the subject expresses that HLA allele.

[0061] This article provides a method for treating a subject with cancer, comprising administering to the subject the pharmaceutical composition described herein; wherein the TCR binds to the HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01, HLA-A33:03, HLA-A68:01, or HLA-A68:01 alleles. The LA-A74:01 complex contains a mutant RAS peptide with a mutation at G12; wherein the subject was identified as expressing a protein encoded by the HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele.

[0062] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition comprising a T-cell receptor (TCR) specific for a mutant GATA3 peptide that complexes with an HLA protein; wherein the mutant GATA3 peptide comprises at least one mutant amino acid and is a fragment of at least eight consecutive amino acids of a mutant GATA3 protein resulting from a mutation in the GATA3 gene of a cancer cell; wherein the mutant GATA3 peptide binds to a protein encoded by an allele of HLA-A02:01, HLA-B07:02, or HLA-B08:01.

[0063] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition comprising a T-cell receptor (TCR) specific for a mutant GATA3 peptide that complexes with an HLA protein; wherein the mutant GATA3 peptide comprises one or more mutant GATA3 amino acids encoded by a GATA3 neoORF sequence, wherein the mutant GATA3 peptide binds to a protein encoded by an HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele.

[0064] This article provides a method for treating a subject with cancer, comprising administering a TCR or T cells expressing the TCR to the subject; wherein the TCR specifically binds to a mutant GATA3 peptide complexed with a protein encoded by an HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele; wherein the subject expresses or is identified as expressing a protein encoded by an HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele.

[0065] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition comprising a T-cell receptor (TCR) specific to a mutant TMPRSS2:ERG peptide that complexes with an HLA protein; wherein the mutant TMPRSS2:ERG peptide comprises at least one mutant amino acid and is a fragment of a TMPRSS2:ERG gene fusion mutation; wherein the mutant TMPRSS2:ERG peptide binds to a protein encoded by the HLA-A02:01 allele.

[0066] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition comprising a T-cell receptor (TCR) that is specific to a mutant BTK peptide that complexes with an HLA protein; wherein the mutant BTK peptide comprises at least one mutant amino acid; wherein the mutant BTK peptide binds to a protein encoded by the HLA-A02:01 allele.

[0067] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition comprising a T-cell receptor (TCR) that is specific to a mutant BTK peptide that complexes with an HLA protein; wherein the mutant BTK peptide comprises a resistance mutation or a point mutation; wherein the mutant BTK peptide binds to a protein encoded by the HLA-A02:01 allele.

[0068] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition comprising a T-cell receptor (TCR) that is specific to a mutant BTK peptide that complexes with an HLA protein; wherein the mutant BTK peptide comprises a C481S mutation; wherein the mutant BTK peptide binds to a protein encoded by the HLA-A02:01 allele.

[0069] This article provides a method for treating a subject with cancer, comprising administering to the subject a composition containing a T-cell receptor (TCR) or T cells expressing the TCR, wherein the TCR specifically binds to a mutant EGFR peptide complexed with a protein encoded by HLA-A02:01; wherein the mutant EGFR peptide comprises a resistance mutation or a point mutation, and wherein the subject expresses or is identified as expressing a protein encoded by the HLA-A02:01 allele. In some embodiments, the mutant EGFR peptide comprises a T790M mutation.

[0070] This article provides a method for preventing resistance to cancer therapies, which involves administering the pharmaceutical composition described herein to a subject in need.

[0071] This article provides a method for inducing an immune response, which includes administering the pharmaceutical composition described herein to a subject in need.

[0072] This article also provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele, wherein the therapeutic agent is a pharmaceutical composition described herein.

[0073] This article also provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by an HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele, wherein the therapeutic agent comprises a T-cell receptor (TCR) specific to a mutant GATA3 peptide that complexes with an HLA protein; wherein the mutant GATA3 peptide comprises at least one mutant amino acid and is a fragment of at least eight consecutive amino acids of a mutant GATA3 protein resulting from a mutation in the GATA3 gene of a cancer cell; wherein the mutant GATA3 peptide binds to a protein encoded by an HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele.

[0074] This article provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele, wherein the therapeutic agent package The T-cell receptor (TCR) contains a mutant RAS peptide that is specific to the HLA protein complex and contains a mutation at G12; wherein the mutant RAS peptide binds to a protein encoded by the HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele.

[0075] This article provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A02:01 allele, wherein the therapeutic agent comprises a T-cell receptor (TCR) specific to a mutant TMPRSS2:ERG peptide complexed with an HLA protein; wherein the mutant TMPRSS2:ERG peptide comprises at least one mutant amino acid and is a fragment of a TMPRSS2:ERG gene fusion mutation; wherein the mutant TMPRSS2:ERG peptide binds to a protein encoded by the HLA-A02:01 allele.

[0076] This article provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A02:01 allele, wherein the therapeutic agent comprises a T-cell receptor (TCR) specific to a mutant BTK peptide that complexes with an HLA protein; wherein the mutant BTK peptide comprises at least one mutant amino acid; wherein the mutant BTK peptide binds to the protein encoded by the HLA-A02:01 allele.

[0077] This article provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A02:01 allele, wherein the therapeutic agent comprises a T-cell receptor (TCR) specific to a mutant BTK peptide that complexes with an HLA protein; wherein the mutant BTK peptide comprises a resistance mutation or a point mutation; wherein the mutant BTK peptide binds to a protein encoded by the HLA-A02:01 allele.

[0078] This article provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A02:01 allele, wherein the therapeutic agent comprises a T-cell receptor (TCR) specific to a mutant BTK peptide that complexes with an HLA protein; wherein the mutant BTK peptide comprises a C481S mutation; wherein the mutant BTK peptide binds to a protein encoded by the HLA-A02:01 allele.

[0079] This article provides a method for identifying a subject with cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as expressing a protein encoded by the HLA-A02:01 allele, wherein the therapeutic agent comprises a T-cell receptor (TCR) specific to a mutant EGFR peptide that complexes with an HLA protein; wherein the mutant EGFR peptide comprises at least one mutant amino acid T790M; wherein the mutant EGFR peptide binds to the protein encoded by the HLA-A02:01 allele.

[0080] In some embodiments, the cancer is selected from breast cancer, lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, uterine cancer, melanoma, ovarian cancer, prostate cancer, endometrial cancer, chronic lymphocytic leukemia (CLL), and liver cancer. In some embodiments, the subject has breast cancer resistant to anti-estrogen therapy, MSI breast cancer, metastatic breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, ER-negative breast cancer, ER-positive breast cancer, recurrent breast cancer, metastatic breast cancer, or any combination thereof. In some embodiments, the breast cancer expresses a mutated estrogen receptor.

[0081] In some embodiments, the subject has breast cancer resistant to anti-estrogen therapy. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has CLL resistant to ibrutinib therapy. In some embodiments, the CLL expresses Bruton's tyrosine kinase (BTK) with a mutation such as C481S. In some embodiments, the subject has lung cancer resistant to tyrosine kinase inhibitors. In some embodiments, the lung cancer expresses epidermal growth factor receptor (EGFR) with a mutation such as T790M, L792F, or C797S.

[0082] In some embodiments, an immune response is induced in the subject. In some embodiments, the immune response is a humoral response. In some embodiments, the immune response is a cytotoxic T-cell response.

[0083] In some embodiments, the method further includes administering at least one additional therapeutic agent or treatment modality. In some embodiments, the at least one additional therapeutic agent or treatment modality is surgery, checkpoint inhibitors, antibodies or fragments thereof, chemotherapeutic agents, radiation, vaccines, small molecules, T cells, vectors and APCs, polynucleotides, oncolytic viruses, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, the additional therapeutic agent is administered before, simultaneously with, or after the pharmaceutical composition described herein.

[0084] In some implementations, administration includes subcutaneous or intravenous administration.

[0085] In some implementations, the subject is a subject whose disease has progressed after endocrine therapy combined with a CDK 4 / 6 inhibitor.

[0086] This article provides a method comprising: identifying neoantigen-specific T cells from a sample containing a population of T cells; identifying one or more peptides of a peptide-MHC complex presented by antigen-presenting cells (APCs); identifying a variable sequence of a T cell receptor (TCR) from the neoantigen-specific T cells; expressing a recombinant TCR containing the variable sequence of the identified TCR in the TCR cells; and performing a functional assay, wherein the functional assay comprises contacting the TCR cells with a peptide-MHC complex containing a peptide of one or more of the identified peptides.

[0087] In some embodiments, the method includes obtaining a sample comprising a cell population containing neoantigen-specific T cells. In some embodiments, obtaining the sample includes obtaining a T cell sample from a healthy subject or from a subject with cancer. In some embodiments, the T cell sample is derived from a healthy donor. In some embodiments, the T cell sample is a peripheral blood mononuclear cell (PBMC) sample.

[0088] In some embodiments, identifying neoantigen-specific T cells includes contacting a population of T cells with at least one peptide-MHC multimer complex containing a neoantigen peptide. In some embodiments, identifying neoantigen-specific T cells includes contacting a population of T cells with a peptide-MHC complex containing a neoantigen peptide. In some embodiments, identifying neoantigen-specific T cells includes contacting a population of T cells with an APC containing a peptide-MHC complex. In some embodiments, identifying neoantigen-specific T cells further includes identifying or predicting T cells with a population specific to the peptide-MHC complex based on TCR clonalness. In some embodiments, prior to identifying neoantigen-specific T cells, a variable sequence of the TCR is identified from the neoantigen-specific T cells. In some embodiments, identifying a variable sequence of the TCR from neoantigen-specific T cells includes sequencing the DNA, RNA, or amplified product thereof from one or more neoantigen-specific T cells encoding the variable sequence. In some embodiments, identifying a variable sequence of a TCR from neoantigen-specific T cells includes sequencing the DNA, RNA, or amplified product thereof from a single neoantigen-specific T cell encoding the variable sequence. In some embodiments, identifying a variable sequence of a TCR from neoantigen-specific T cells includes sequencing the barcoded DNA, barcoded RNA, or amplified product thereof from one or more neoantigen-specific T cells encoding the variable sequence. In some embodiments, identifying a variable sequence of a TCR from neoantigen-specific T cells includes pairing the TCR-α chain with the TCR-β chain.

[0089] In some embodiments, expressing a recombinant TCR includes expressing a variable sequence identified from a polynucleotide, the polynucleotide containing a sequence encoding the identified variable sequence. In some embodiments, the polynucleotide is a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, expressing a recombinant TCR includes transducing or transfecting the polynucleotide into cells. In some embodiments, the cells are T cell lines or healthy donor PMBCs.

[0090] In some embodiments, identifying one or more peptides of a peptide-MHC complex presented by an APC includes expressing said one or more peptides in cells. In some embodiments, identifying one or more peptides of a peptide-MHC complex presented by an APC includes loading said one or more peptides onto the MHC of a cell. In some embodiments, identifying one or more peptides of a peptide-MHC complex presented by an APC includes eluting or isolating peptides from said one or more peptides from the peptide-MHC complex. In some embodiments, identifying one or more peptides of a peptide-MHC complex presented by an APC includes mass spectrometry analysis of peptides from said one or more peptides isolated or eluted from the peptide-MHC complex. In some embodiments, performing a functional assay includes determining the expression of one or more cellular markers. In some embodiments, said one or more cellular markers include TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, enzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof.

[0091] Incorporation

[0092] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Attached Figure Description

[0093] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be gained by referring to the following detailed description of illustrative embodiments utilizing the principles of the invention, along with the accompanying drawings, in which:

[0094] Figure 1The workflow for identifying and analyzing antigen-specific TCRs is described. PBMCs from healthy donors can be stimulated with the target antigen, followed by identification of antigen-specific T cells using peptide-MHC multimers (left). Antigen-specific T cells can be isolated, and TCRs can be sequenced and analyzed (middle left). TCRs can be synthesized and expressed in cell lines or PBMCs, and their specificity can be reconfirmed using peptide-MHC multimers (middle right). The TCR-expressing cell lines or PBMCs can then be co-cultured with antigen-expressing cell lines to confirm TCR functionality (right).

[0095] Figure 2 It is antigen-specific CD8 + A schematic diagram illustrating T cell expansion. PBMCs can be stimulated with target antigens and cytokines. After expansion, antigen-specific CD8... + T cells can be identified using peptide-MHC multimers.

[0096] Figure 3A This is an example schematic diagram of the recombinant TCR construct and the vector design for expressing the TCR construct in cells.

[0097] Figure 3B This is an example schematic diagram of a viral vector encoding a recombinant TCR used for transduction or transfection into cells.

[0098] Figure 4A The specific CD8 antigen response to RAS peptide stimulation was described. + Example flow cytometry analysis of T cell expansion.

[0099] Figure 4B An example flow cytometry analysis of RAS antigen-specific Jurkat cells is described after sorting the top 10% of recombinant TCR-expressing multimer-positive cells following puromycin selection.

[0100] Figure 5 The specific CD8 response of the RAS-peptide-HLA-A11:01 complex to RAS G12V peptide stimulation was characterized. + T cell expansion (left), RAS-peptide-HLA-A11:01 complex-specific CD8 response to RAS G12C peptide stimulation. + T cell expansion (intermediate) and specific CD8 response of the RAS peptide-HLA-A11:01 complex to RAS G12D peptide stimulation. + Example flow cytometry analysis of T cell expansion (right).

[0101] Figure 6AThe specific CD8 response of the RAS-peptide-HLA-A11:01 complex to stimulation by G12V or G12C mutant RAS peptides was characterized. + Example flow cytometry analysis of T cell expansion (left), TCR clonal abundance analysis after sequencing of TCRs from merged hits (middle), and confirmation of mutant and wild-type specificity of the first two recovered clones after recombinant TCR expression in Jurkat cells (right).

[0102] Figure 6B The experimental results of TCR function assays are described to assess the specificity (left) and affinity (right) of three RAS TCRs (RAS TCR-3, RAS TCR-4, and RAS TCR-11). The figure shows IL-2 production after co-culturing RAS TCR-transduced Jurkat cells with HLA-A11:01-expressing A375 cells.

[0103] Figure 7A The results of TCR functional affinity assay (peptide titration) are depicted. The figure shows IL-2 production after co-culturing RAS TCR-transduced Jurkat cells with A375 cells expressing HLA-A11:01 and loaded with increased amounts of RAS wild-type or RAS mutant peptides.

[0104] Figure 7B Example diagrams are depicted showing IL-2 production after co-culturing RAS TCR-4 transduced Jurkat cells with A375 cells modified to express HLA-A11:01 only, modified to express HLA-A11:01 and KRAS G12V mutation, or modified to express HLA-A11:01 and irrelevant mutation.

[0105] Figure 8A Example figures are depicted showing IFNγ secretion from RAS TCR-4 transduced PBMCs after co-culturing with the natural RAS G12V mutant SW620 colon cancer cell line, which was unmanipulated, with increased expression of HLA-A11:01 plus RASG12V peptide, as well as expression of HLA-A11:01 only.

[0106] Figure 8B An example diagram is depicted showing caspase-3 activation after co-culturing PBMCs transduced with RAS TCR in the native RAS G12V mutant SW620 colon cancer cell line, which was unmanipulated, with increased expression of HLA-A11:01 plus RAS G12V peptide, as well as expression of HLA-A11:01 only.

[0107] Figure 8CThe results of the cytotoxicity assays are depicted, in which PBMCs transduced with indifferent TCR (left) or RAS TCR-4 (right) are co-cultured with SW620 target tumor cells expressing HLA-A11:01 and GFP within a range of gradually increasing PBMC-to-target ratios. A graph depicting target cell growth over 72 hours as measured by GFP signaling (top) and target cell death as measured by annexin V signaling (bottom) shows that RAS TCR-transduced PBMCs effectively killed the target cells.

[0108] Figure 8D The results of the cytotoxicity assays are depicted, in which PBMCs transduced with unrelated TCR (left) or RAS TCR-4 (right) were co-cultured with SNG-M target tumor cells within a range of gradually increasing PBMC-to-target ratios. A graph depicting target cell death measured over 72 hours via annexin V signaling shows that RAS TCR-transduced PBMCs killed the target cells.

[0109] Figure 8E The experimental results of the safety screening of RAS TCR-4 are depicted. The left panel shows IL-2 production after co-culturing RAS TCR-transduced Jurkat cells with A375 cells expressing HLA-A11:01 and loaded with the homologous RAS G12V epitope or a peptide in which one position of the homologous epitope is altered to alanine. The middle panel shows IL-2 production after co-culturing RAS TCR-transduced Jurkat cells with A375 cells expressing HLA-A11:01 and loaded with the RAS G12V epitope or a peptide identified from the initial safety screening. The right panel shows IL-2 production after co-culturing RAS TCR-transduced Jurkat cells with A375 cells expressing HLA-A11:01, transduced with perilipopin4 and loaded with the RAS G12V peptide or perilipopin4 peptide or without the peptide (right).

[0110] Figure 8F The workflow for identifying RAS TCR-26 and RAS TCR-27 is depicted. The upper left image illustrates the RAS-peptide-HLA-A11:01 complex-specific CD8 response to RAS G12V peptide stimulation. + Flow cytometry analysis of T cell expansion. The upper right depicts the expansion of CD8 cells. +Measurements of cell growth in target A375 cells expressing HLA-A11:01 and GFP within 54 hours of T cell co-culture. In this experiment, T cells expanded in the presence of the peptide were co-incubated with A375 cells loaded with the indicated amount of peptide. The lower left plot depicts the specific cytotoxicity calculated at 48 hours, which compares target cells loaded with progressively increasing amounts of the mutant RAS peptide relative to the control with target cells loaded with the wild-type peptide. The lower right plots the relative abundance of the TCRα and TCRβCDR3 regions after single-cell TCR sequencing of multimer-positive cells.

[0111] Figure 8G The workflow used to identify RAS TCR-28 is depicted. The upper left depicts the RAS-peptide-HLA-A11:01 complex-specific CD8 response to RAS G12V peptide stimulation. + Flow cytometry analysis of T cell expansion. The upper right depicts the expansion of CD8 cells. + Measurements of cell growth in target A375 cells expressing HLA-A11:01 and GFP and loaded with different doses of peptide were performed within 54 hours of T cell co-culture. The data showed a strong inverse correlation between cell growth and peptide dose, demonstrating the specificity of TCRs for peptides. The lower left plot depicts the specific cytotoxicity calculated at 48 hours, comparing target cells loaded with progressively increasing amounts of mutant RAS peptides (normalized relative to control) with target cells loaded with wild-type peptides. The lower right plot shows the relative abundance of the TCRα and TCRβCDR3 regions after single-cell TCR sequencing of multimer-positive cells.

[0112] Figure 9 Example flow cytometry analyses depict the specific expansion of RAS-peptide-HLA-A03:01 complex-specific CD8+ T cells in response to RAS G12V peptide stimulation (left) and the specific expansion of RAS-peptide-HLA-A03:01 complex-specific CD8+ T cells in response to RAS G12C peptide stimulation (right).

[0113] Figure 10A Depicting Figure 9 The workflow for identifying and analyzing antigen-specific TCRs in the samples shown is described.

[0114] Figure 10B Depicting Figure 9 The workflow for identifying antigen-specific TCRs in the samples shown.

[0115] Figure 10CThe results of TCR functional affinity assay (peptide titration) were depicted. The left panel shows IL-2 production after co-culturing RAS TCR-5 transduced Jurkat cells with A375 cells expressing HLA-A03:01 and loaded with increased amounts of RAS wild-type or RAS mutant peptides. The right panel shows IL-2 production after co-culturing RAS TCR-6 transduced Jurkat cells with A375 cells expressing HLA-A03:01 and loaded with increased amounts of RAS wild-type or RAS mutant peptides.

[0116] Figure 10D The results of the cytotoxicity assay are depicted, in which PBMCs transduced with irrelevant TCR (left), RAS TCR-5 (middle), or RAS TCR-6 (right) were co-cultured with A375 target tumor cells expressing HLA-A03:01 and GFP within a range of gradually increasing PBMC-to-target ratios. A graph of target cell growth over 72 hours, measured by GFP signaling, shows that RAS TCR-transduced PBMCs effectively killed the target cells.

[0117] Figure 10E The results of a cytotoxicity assay were depicted, in which PBMCs transduced with unrelated TCRs, RAS TCR-5, or RAS TCR-6 were co-cultured with NCI-H441 target tumor cells. The increase in annexin V signaling in the target cells within 48 hours indicated that the RAS TCR was able to recognize and kill the target tumor cells.

[0118] Figure 10F The experimental results of safety screening for RAS TCR-5 (top) and RAS TCR-6 (bottom) are depicted. The figure shows IL-2 production after co-culturing RAS TCR-transduced Jurkat cells with A375 cells expressing HLA-A11:01 and loaded with a homologous RAS G12V epitope or a peptide in which one position of the homologous epitope is altered to alanine.

[0119] Figure 11 An example workflow for identifying and analyzing GATA3 antigen-specific TCRs is depicted. The left side illustrates an example flow cytometry analysis of CD8+ T cell expansion specific to the GATA3-peptide-HLA-A02:01 complex in response to stimulation by the GATA3 mutant peptide.

[0120] Figure 12A The results of TCR functional affinity assay (peptide titration) are depicted. The figure shows the production of IL-2 after co-culturing GATA3 TCR-1 transduced T cells with cells loaded or transduced with gradually increasing amounts of GATA3 mutant peptide.

[0121] Figure 12BThe figure depicts IL-2 production after co-culturing Jurkat cells transduced with GATA3 TCR with cells loaded with irrelevant peptides or mutant GATA3 peptides, as well as cells transduced with irrelevant vectors or vectors encoding GATA3 neoORF mutations.

[0122] Figure 12C The plot shows the upregulation of IFNγ production in two cell samples expressing either the control peptide or the GATA3 mutant peptide.

[0123] Figure 12D The plot shows the percentage of CD8 and CD107a positive cells in two cell samples expressing either the control peptide or the GATA3 mutant peptide.

[0124] Figure 12E The plot shows the percentage of live cysteine ​​3 positive cells in two cell samples expressing either the control peptide or the GATA3 mutant peptide.

[0125] Figure 13 An example workflow for identifying and analyzing TMPRSS2:ERG antigen-specific TCRs is depicted. The left side depicts an example flow cytometry analysis of TMPRSS2:ERG-peptide-HLA-A02:01 complex-specific CD8+ T cell expansion in response to stimulation by the TMPRSS2:ERG mutant peptide.

[0126] Figure 14 The example diagram depicts IL-2 production after co-culturing Jurkat cells transduced with TMPRSS2:ERG TCR-1 with unmanipulated 293T cells loaded with irrelevant peptides or loaded with TMPRSS2:ERG peptides.

[0127] Figure 15 An example workflow for identifying and analyzing BTK antigen-specific TCRs is depicted. The left side illustrates an example flow cytometry analysis of BTK-peptide-HLA-A02:01 complex-specific CD8+ T cell expansion in response to stimulation by the BTK mutant peptide.

[0128] Figure 16A The specific CD8 response of the EGFR-peptide-HLA-A02:01 complex to EGFR T790M peptide stimulation was characterized. + Example flow cytometry analysis of T cell expansion. Figure 16A Each image above represents a representative T-cell augmentation sample (well) using the EGFR T790M peptide. The TCRs from these cells were sequenced.

[0129] Figure 16BThe specific CD8 response of the potential EGFR-peptide-HLA-A02:01 complex to EGFR T790M peptide stimulation was characterized. + Example flow cytometry analysis of the T cell population, whose TCRs are yet to be sequenced. Figure 16B Each image above represents a representative T-cell expansion sample (well) in response to the EGFR T790M peptide. Detailed Implementation

[0130] The terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by a person skilled in the art, the acceptable range of error depending in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may indicate within an order of magnitude of a value, preferably within 5 times, more preferably within 2 times. Where a particular value is described in this application and claims, unless otherwise stated, the term “about” should be presumed to mean within an acceptable range of error for that particular value.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are now described; however, any methods and materials similar to or equivalent to those described herein may also be used in practice or testing of this disclosure. The following description elucidates details of one or more specific embodiments.

[0132] I. definition

[0133] Unless otherwise stated, the term "TCR" should be understood to include the complete TCR and its antigen-binding portion or fragment (also known as an MHC-peptide-binding fragment). In some embodiments, the TCR is a complete or full-length TCR. In some embodiments, the TCR is an antigen-binding portion that is smaller than the full-length TCR but binds to a specific antigenic peptide bound to (i.e., in its environment) an MHC molecule, i.e., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR may contain only a portion of the domains of the full-length or complete TCR, but still be able to bind to the epitopes bound by the full-length TCR (e.g., MHC-peptide complexes). In some cases, the antigen-binding portion or fragment of the TCR contains variable domains of the TCR, such as variable α-chains and variable β-chains of the TCR, sufficient to form binding sites for binding to a specific MHC-peptide complex, for example, typically in cases where each chain contains three complementarity-determining regions. This includes polypeptides or proteins having binding domains that are antigen-binding domains or homologous to antigen-binding domains. These terms also refer to TCRs transplanted with complementarity-determining regions (CDRs) as well as other humanized TCRs (including CDR modifications and framework region modifications). It should be noted that while only immunoglobulin chains (e.g., heavy and light chains) may be mentioned, the disclosed invention is applicable to a variety of other different types of paired sequences, such as T cell receptor chain pairs (TCRα and TCRβ chains and TCRγ and TCRδ chains), and is not limited to immunoglobulins.

[0134] The terms “complementarity-determining region” and “CDR,” synonyms with “hypervariate region” or “HVR,” are known in the art and refer to the discontinuous amino acid sequence within the TCR variable region that confers specificity and / or binding affinity to the MHC-peptide complex. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each α-chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each β-chain variable region. The terms “frame region” and “FR,” known in the art, refer to the non-CDR portions of the variable regions of the α-chain and β-chain. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length α-chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length β-chain variable region.

[0135] The term "variable region" or "variable domain" refers to a domain of the TCR α, β, γ, or δ chain that participates in the binding of the TCR to the antigen-MHC complex. The variable domains of the α and β chains of the native TCR (Vα and Vβ, respectively) and the variable domains of the γ and δ chains (Vγ and Vδ, respectively) typically have similar structures, with each domain containing four conserved frame regions (FRs) and three core regions (CDRs). A single Vα or Vβ domain, or a Vγ or Vδ domain, may be sufficient to confer specificity for binding to the peptide-MHC complex.

[0136] This document also provides TCR fragments, including antigen-binding fragments. In some embodiments, the TCR is its antigen-binding portion, such as a variant of a full-length TCR lacking its transmembrane and / or cytoplasmic regions, which may be referred to as a fully soluble TCR. In some embodiments, the TCR is a dimer TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (scTCR), such as a scTCR having a structure as described in PCT patent disclosures WO2003 / 020763, WO2004 / 033685, or WO2011 / 044186. In some embodiments, the TCR is a single-chain TCR fragment containing an α-chain variable region linked to a β-chain variable region, such as scTv. In some embodiments, scTv is also referred to as scFv. In some aspects, single-chain Tv or scTv refers to a TCR fragment containing variable α- or γ-chain (Vα or Vγ) and variable β- or δ-chain (Vβ or Vδ) domains of the TCR, wherein these domains are present in a single polypeptide chain. Typically, Tv peptides further include a peptide linker between the Vα and Vβ domains or the Vγ and Vδ domains, which enables scTv to form the desired structure for antigen binding. In some aspects, a biantibody refers to a TCR fragment having two antigen-binding sites, the fragment comprising a Vα linked to Vβ (Vα-Vβ) or a Vγ linked to Vδ in the same polypeptide chain (Vγ-Vδ). By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with a complementary domain of another chain, forming two antigen-binding sites. Exemplary biantibodies are described more fully in, for example, EP404097 and WO93111161. In some aspects, Fv refers to a TCR fragment containing both a complete peptide-MHC complex recognition site and a peptide-MHC complex binding site. This region consists of a dimer of a tightly, non-covalently associated TCRα chain and a TCRβ chain, or a TCRγ chain and a TCRδ chain. In this configuration, the three CDRs of each variable domain interact to define peptide-MHC complex binding sites on the surface of the Vα-Vβ dimer or Vγ-Vδ dimer. Commonly, combinations of one or more CDRs from each Vα-Vβ chain or Vγ-Vδ chain confer peptide-MHC complex binding specificity to the TCR. For example, it should be understood that, for instance, when transferred to the Vα and Vβ chains or Vγ-Vδ chains of a recipient-selected TCR or its antigen-binding fragment, CDRα3 and CDRβ3 or CDRγ3 and CDRδ3 may be sufficient to confer antigen-binding specificity to the TCR, and the binding, affinity, etc., of this CDR combination can be detected.Furthermore, although the two domains of the Tv fragment (Vα and Vβ or Vγ and Vδ) are encoded by separate genes, they can be joined together using a recombination approach via a synthetic linker that allows them to become a single protein chain, where the Vα and Vβ or Vδ and Vγ chain regions pair to form a monovalent molecule (called a single-chain Tv (scTv)). Such scTvs are also intended to be included within the peptide-MHC complex-binding moiety of the TCR.

[0137] In some respects, a “bispecific TCR” refers to a TCR that exhibits specificity for two different peptide-MHC complexes or two different types of peptide-MHC complexes. Specifically, as used herein, this term includes, but is not limited to, a TCR that exhibits binding specificity to a target peptide-MHC complex and another peptide-MHC complex that facilitates delivery to a specific tissue. Similarly, a multispecific TCR has two or more binding specificities. In some respects, a linear TCR refers to a pair of tandem Fd segments (e.g., Vα-Cα1-Vα-Cα1) that form a pair of antigen-binding regions. A linear TCR can be bispecific or monospecific.

[0138] In some respects, "antigen-binding domain" refers to one or more fragments of a TCR that retain the ability to specifically bind to the peptide-MHC complex. Non-limiting examples of TCR fragments included in this terminology include, but are not limited to: (i) Tab fragments, i.e., those composed of Vβ, Vα, C... β (ii) a monovalent fragment consisting of a Vα and a Cα1 domain; (iii) a T(ab')2 fragment, i.e., a bivalent fragment containing two Tab fragments connected by a disulfide bridge at the hinge region; (iv) a Td fragment consisting of a Vβ and a Vα1 domain; (v) a Tv fragment containing a single arm of the TCR with Vβ and Vα domains, including scTv; (v) a dAb fragment (Ward et al., (1989) Nature 341:544 546) containing a Vα domain; and (vi) a separate CDR. This definition includes TCRs with a single α chain or a single β chain.

[0139] The provided TCRs include humanized and human TCRs. A “humanized” TCR is a TCR in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized TCR may optionally include at least a portion of the constant region of a TCR derived from a human TCR. A “humanized form” of a non-human TCR refers to a variant of a non-human TCR that has undergone humanization, typically with reduced immunogenicity to humans while retaining the specificity and affinity of the parent non-human TCR. In some embodiments, some FR residues in a humanized TCR are replaced by corresponding residues from a non-human TCR (e.g., a TCR from which CDR residues are derived), for example, to restore or improve TCR specificity or affinity. A “human TCR” is a TCR whose amino acid sequence corresponds to the amino acid sequence of a TCR derived from humans or human cells, or a non-human source TCR utilizing a human TCR library or other human TCR coding sequences (including human TCR libraries). This term does not include humanized forms of nonhuman TCRs containing nonhuman peptide-MHC complex-binding regions, such as those in which all or substantially all CDRs are nonhuman CDRs. Human TCRs can be prepared by administering immunogens to transgenic animals modified to produce complete human TCRs or complete TCRs with human variable regions in response to antigen challenge. Such animals generally contain all or part of the human TCR loci, which replace endogenous TCR loci, or are present extrachromosomally or randomly integrated into the animal's chromosome. In these transgenic animals, endogenous TCR loci are usually inactivated. Human TCRs can also be derived from human TCR libraries, including phage display libraries and cell-free libraries containing TCR coding sequences derived from human genome libraries.

[0140] The terms “cancer neoantigen” or “neoantigen” or “neoepitope” can refer to an antigen not encoded in the normal, unmutated host genome. A neoantigen can involve an antigen containing one or more amino acid modifications compared to a parent antigen. For example, a neoantigen can be a tumor-associated neoantigen, where the term “tumor-associated neoantigen” can include peptides or proteins containing amino acid modifications resulting from tumor-specific mutations. In some cases, a neoantigen represents an oncogenic viral protein or aberrant protein resulting from somatic mutations. For example, a neoantigen can be generated by disrupting cellular mechanisms via viral protein activity. Another example can be exposure to an oncogenic compound that, in some cases, can cause somatic mutations. Such somatic mutations can ultimately lead to tumor / cancer formation. Neoantigens can be a class of tumor antigens resulting from tumor-specific alterations of proteins. Neoantigens include, but are not limited to, tumor antigens generated by, for example, protein sequence substitutions, frameshift mutations, fusion peptides, in-frame deletions, insertions, and the expression of endogenous retroviral peptides. A neoepitope can be an epitope that is not present in reference cells such as non-pathological cells, such as non-cancer cells or germline cells, but is found in pathological cells, such as cancer cells. This includes situations where a corresponding epitope is found in normal, non-pathological cells or germline cells, but the sequence of that epitope is altered due to one or more mutations in pathological cells, such as cancer cells, resulting in a new epitope.

[0141] In some aspects, an "epitope" refers to a portion of an antigen or other macromolecule capable of forming a binding interaction with the variable region binding pouch of a TCR. In some aspects, an epitope refers to a portion of a peptide-MHC complex capable of forming a binding interaction with the variable region binding pouch of a TCR. Such a binding interaction may manifest as an intermolecular contact with one or more amino acid residues of one or more CDRs. The peptide-MHC complex binding may involve, for example, CDR3, CDR3 pairs, or in some cases, interactions involving up to all six CDRs of the Vα and Vβ chains or the Vγ or Vδ chains. An epitope may be a linear peptide sequence (i.e., "continuous") or may consist of non-contiguous amino acid sequences (i.e., "conformal" or "discontinuous"). A TCR may recognize one or more amino acid sequences. Thus, an epitope may define more than one unique amino acid sequence. In some aspects, in the context of MHC, a TCR may recognize one or more amino acid sequences or epitopes. Epitopes recognized by a TCR can be determined by peptide mapping and sequence analysis techniques known to those skilled in the art. The binding interaction manifests as an intermolecular contact with one or more amino acid residues of a CDR. Epitopes can refer to antigenic determinants in molecules such as antigens, that is, a portion or fragment of a molecule that is recognized by the immune system, such as by T cells, especially when presented in the context of MHC molecules. Epitopes of proteins such as tumor antigens can comprise continuous or discontinuous portions of the protein, and their length can be 5 to 100, 5 to 50, 8 to 30, or 10 to 25 amino acids. For example, the length of an epitope can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0142] The term "binding" refers to the direct association between two molecules under physiological conditions due to interactions such as covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding, and includes interactions such as salt bridges and water bridges, as well as any other conventional binding mechanisms.

[0143] In some embodiments, references to a TCR having “specific binding” refer to a situation where the TCR will not exhibit any significant binding to molecules other than peptide-MHC complexes containing an epitope recognized by the TCR. The term also applies, for example, to situations where the antigen-binding domain is specific to a particular epitope carried by a large number of peptide-MHC complexes, in which case a selected TCR or peptide-MHC complex-binding fragment carrying that peptide-MHC complex-binding domain will be able to bind to various peptide-MHC complexes carrying that epitope. The terms “preferential binding” or “specific binding” mean that the TCR or a fragment thereof has a greater affinity for binding to the epitope than for binding to unrelated amino acid sequences, and that it is non-toxic at levels formulated for human drug administration if it has cross-reactivity with other peptides containing that epitope. In one respect, such affinity is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 1000 times greater than the affinity of the TCR or its fragments for unrelated amino acid sequences.

[0144] The term "affinity" refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide with class I or II HLA, or a peptide-HLA complex with a T-cell receptor (TCR)). Affinity can be expressed as the equilibrium constant for the reversible binding of two reagents and can be represented as K. D K A K off or K on K D K refers to the dissociation constant between the two members of a binding pair and has a molar concentration unit. A K refers to the affinity constant between the two members of a binding pair, and is the reciprocal of the dissociation constant. Affinity can be determined experimentally, for example, using commercially available Biacore SPR units via surface plasmon resonance (SPR). off This refers to the dissociation rate constant of the two members of a binding pair (e.g., the dissociation rate constant of an HLA-binding peptide with class I or II HLA, or the dissociation rate constant of a peptide-HLA complex with a TCR). K onThis refers to the association rate constant of the two members of a binding pair (e.g., the association rate constant of an HLA-binding peptide with class I or II HLA, or a peptide-HLA complex with a TCR). The affinity of a binding protein for a ligand, such as the affinity of a TCR for an epitope, can be, for example, from about 100 nanomolars (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). The term "affinity" refers to the resistance of a complex of two or more substances to dissociation upon dilution.

[0145] Throughout the publicly available content, the "combined data" results can be expressed as "IC". 50 Affinity can also be expressed as the inhibitory concentration (IC50). 50 ), or the concentration when 50% of the binding pair's first member (e.g., peptide) is substituted. Similarly, ln(IC) 50 ) refers to IC 50 The natural logarithm of IC. For example, IC 50 This can be the concentration of the test peptide at which 50% inhibition of binding to the labeled reference peptide is observed in the binding assay. These values ​​can be close to K, taking into account the conditions under which the assay is run (e.g., limiting HLA protein concentration and / or the concentration of the labeled reference peptide). DValues. Assays used to determine binding are well known in the art and are described in detail, for example, in PCT publications WO94 / 20127 and WO 94 / 03205 and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney et al., J. Immunol. 154:247 (1995); and Sette et al., Mol. Immunol. 31:813 (1994). Alternatively, binding may be expressed relative to the binding of a reference standard peptide. The binding can also be determined using other assay systems, including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), and immobilized purified MHC (e.g., Hill et al., J. Immunol. 152,2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)). ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-throughput soluble phase determination (Hammer et al., J. Exp. Med. 180:2353 (1994)) and measurements of type I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0146] The term "major histocompatibility complex" and the abbreviation "MHC" can include MHC class I and MHC class II molecules and refers to a complex of genes present in all vertebrates. MHC proteins or molecules may be essential for signaling between lymphocytes and antigen-presenting or diseased cells in the immune response, where MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. MHC-encoded proteins can be expressed on the cell surface and present T cells with self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms). MHC regions can be divided into three subgroups: class I, class II, and class III. MHC class I proteins may contain α chains and β2-microglobulins (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T cells. MHC class II proteins may contain α and β chains, and they can present antigen fragments to T helper cells. MHC class III regions may encode other immune components, such as complement components and cytokines. MHC can be either multigenic (with several MHC class I and MHC class II genes) or polymorphic (each gene has multiple alleles).

[0147] The term "monotype" can refer to the human leukocyte antigen (HLA) alleles and their encoded proteins found on a single chromosome. A monotype can also refer to an allele located at any locus within the MHC. Each MHC class is represented by several loci: for example, HLA-A (human leukocyte antigen-A); class I: HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, and HLA-V; and class II: HLA-DRA, HLA-DRB1-9, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DPB2, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB. The terms "HLA alleles" and "MHC alleles" are used interchangeably herein.

[0148] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably. They can refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (multiple loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be conferred before or after polymer assembly. Polynucleotides may include non-standard nucleotides, such as nucleotide analogs or modified nucleotides. In some embodiments, non-standard nucleotides can stabilize heterozygote formation. In some embodiments, non-standard nucleotides can destabilize heterozygote formation. In some embodiments, non-standard nucleotides can enhance hybridization specificity. In some embodiments, non-standard nucleotides can reduce hybridization specificity. Examples of non-standard nucleotide modifications include 2'O-Me, 2'O-allyl, 2'O-propargyl, 2'O-alkyl, 2'fluoro, 2'arabinose, 2'xylose, 2'fluoroarabinose, thiophosphate, dithiophosphate, phosphoramidide, 2'amino, 5-alkyl-substituted pyrimidine, 3'deoxyguanosine, 5-halo-substituted pyrimidine, alkyl-substituted purine, halo-substituted purine, bicyclic nucleotide, 2'MOE, PNA molecule, LNA-molecule, LNA-like molecule, diaminopurine, S2T 5-Fluorouracil, 5-Bromouracil, 5-Chlorouracil, 5-Iodouracil, Hypoxanthine, Xanthine, 4-Acetylcytosine, 5-(Carboxyhydroxymethyl)uracil, 5-Carboxymethylaminomethyl-2-thiouridine, 5-Carboxymethylaminomethyluracil, Dihydrouracil, β-D-galactosylqueosine, Inosine, N6-Isopentenyladenine, 1-Methylguanine, 1-Methylinosine, 2,2-Dimethylguanine, 2-Methyladenine, 2-Methylguanine, 3-Methylcytosine, 5-Methylcytosine, N 6-Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine and their derivatives. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example by conjugation with labeled components.

[0149] "Complementarity" can refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-conventional types. The complementarity percentage can represent the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10). "Complete complementarity" can mean that all consecutive residues in the nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. "Substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in regions having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or 100% of nucleotides, or may refer to two nucleic acids that hybridize under strict conditions. For example, to assess the percentage of complementarity, sequence identity can be measured using any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, for example, the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm. Optimal alignment can be evaluated using any suitable parameters of the chosen algorithm (including default parameters).

[0150] The terms "peptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. For example, a peptide may contain at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 peptides or amino acids. Examples of peptides include, but are not limited to, amino acid chains, proteins, peptides, hormones, polypeptide sugars, lipids, glycolipids, phospholipids, antibodies, enzymes, kinases, receptors, transcription factors, and ligands. Peptides containing a provided TCR and TCR chain, as well as other peptides such as linkers and binding peptides, may include amino acid residues, including natural and / or non-natural amino acid residues. The terminology also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some respects, peptides may contain modifications relative to the original or native sequence, as long as the protein retains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or can be accidental, such as through mutations in the host producing the protein or errors caused by PCR amplification.

[0151] As used herein, the twenty common amino acids and their abbreviations known to those skilled in the art follow conventional usage. Stereoisomers of the twenty common amino acids (e.g., D-amino acids), non-natural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide representation used herein, according to standard usage and convention, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxyl-terminal direction. The percentage of sequence identity (%) relative to a reference polypeptide (or nucleic acid) sequence is the percentage of amino acid residues (or nucleotides in the case of a nucleic acid sequence) in a candidate sequence that are identical to those in a reference polypeptide (or nucleic acid) sequence after alignment of the sequences and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments aimed at determining the percentage of amino acid sequence identity can be performed in various ways within the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the amino acid sequence identity percentage is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code has been submitted as user documentation to the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., and can also be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and / or relative to a given amino acid sequence B (or can be expressed as: a given amino acid A has or contains a certain percentage of amino acid sequence identity with or relative to a given amino acid sequence B) is calculated as follows: score X / Y multiplied by 100, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless otherwise specified, all amino acid sequence identity percentage values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0152] "German sequence" refers to the gene sequence derived from haploid gametes and the diploid cells that form them. German DNA contains multiple gene segments encoding a single TCRα or TCRβ chain or a single TCRγ or TCRδ chain. These gene segments are carried in germ cells but cannot be transcribed and translated before they are arranged into functional genes. During T cell differentiation in the bone marrow, these gene segments are able to generate more than 10 8 Random regrouping of species-specific dynamic genetic systems.

[0153] "Inhibition," "treatment," and "management" are used interchangeably and refer to, for example, the cessation of symptoms, prolongation of survival, partial or complete relief of symptoms, and partial or complete elimination of conditions, diseases, or ailments associated with excessive protein levels or protein activity. For example, cancer treatment includes, but is not limited to, the cessation, partial or complete elimination of cancer growth or tumors. Treatment or partial elimination includes, for example, a reduction in growth or tumor size and / or volume by multiples, such as a reduction of about 2, 3, 4, 5, 10, 20, or 50 times, or any multiple between. Similarly, treatment or partial elimination may include a reduction of growth or tumor size and / or volume by about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or any percentage reduction between these values. Prevention refers to the prevention or halting of the onset of symptoms, and the prevention of the progression of diseases or ailments associated with excessive protein levels or protein activity.

[0154] "Subject," "individual," "host," or "patient" refers to a living organism, such as a mammal. Examples of subjects and hosts include, but are not limited to, horses, cattle, camels, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice (e.g., humanized mice), gerbils, non-human primates (e.g., rhesus monkeys), humans, etc., and non-mammals, including, for example, non-mammalian vertebrates such as birds (e.g., chickens or ducks), fish (e.g., sharks), or frogs (e.g., clawed frogs), and non-mammalian invertebrates, as well as their transgenic species. In some respects, a subject refers to a single organism (e.g., a human). In some respects, a group of individuals is provided, comprising a small group with a common immune factor and / or disease of the study, and / or a group of individuals without the disease (e.g., negative / normal controls). Subjects from whom samples are obtained may have a disease and / or condition (e.g., one or more allergic reactions, infections, cancers, or autoimmune diseases, etc.) and may be compared with negative control subjects who do not have the disease.

[0155] "Reagent kit" refers to a delivery system for delivering materials or reagents to perform the methods disclosed herein. In some embodiments, a reagent kit includes a system that allows the storage, transport, or delivery from one location to another of reaction reagents (e.g., probes, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing the analysis, etc.). For example, a reagent kit includes one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or support materials. Such contents may be delivered together or separately to the intended recipient. For example, a first container may contain an enzyme for use in the analysis, while a second container contains multiple primers. Packaging material refers to the physical structure that contains the components of the reagent kit. Packaging material can sterilely retain the components and may be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). Labels or packaging inserts may contain appropriate written instructions. Thus, the reagent kit may also contain labels or instructions regarding the use of the reagent kit components in any of the methods of the present invention. The reagent kit may contain a compound in the package, or a dispenser, and instructions regarding the administration of the compound as described herein.

[0156] The term "resistance mutation" refers to a mutation in a gene that allows the gene or the host cell containing the gene to become resistant to drug treatment. For example, the BTK C481S mutation is a resistance mutation that can confer resistance to ibrutinib.

[0157] II. Overview

[0158] This disclosure provides a T-cell receptor (TCR) against a neoantigen, an isolated nucleic acid molecule encoding the TCR against the neoantigen, T cells expressing the TCR, and a pharmaceutical composition for treating diseases involving malignant cells expressing the neoantigen.

[0159] This document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 84% sequence identity with an amino acid sequence selected from SEQ ID NO:3, 18, 33, 49, 65, 81, 97 and 113, and / or wherein the TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 84% sequence identity with an amino acid sequence selected from SEQ ID NO:6, 21, 36, 52, 68, 84, 100 and 116.

[0160] This document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC, wherein the epitope from the RAS comprises a region having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 15, 30, 45, 46, 61, 62, 77, 78, 93, 94, 109, 110, 125, 126 and 219-222.

[0161] This article provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A03:01 allele.

[0162] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 9, 24, 39, 55, 71, 87, 103, and 119. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 12, 27, 42, 58, 74, 90, 106, and 122. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 16, 31, 47, 63, 79, 95, and 111. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 4, 19, 34, 50, 66, 82, 98, and 114. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:2, 17, 32, 48, 64, 80, 96, and 112. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:5, 20, 35, 51, 67, 83, 99, and 115. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:9; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:12. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:3; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:4, CDR2 of SEQ ID NO:5, and CDR3 of SEQ ID NO:6. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:24; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:27. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:16, CDR2 of SEQ ID NO:17, and CDR3 of SEQ ID NO:18; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:19, CDR2 of SEQ ID NO:20, and CDR3 of SEQ ID NO:21.In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:39; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:42. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and the TCRβ chain construct includes CDR1 of SEQ ID NO:34, CDR2 of SEQ ID NO:35, and CDR3 of SEQ ID NO:36. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:55; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:58. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:47, CDR2 of SEQ ID NO:48, and CDR3 of SEQ ID NO:49; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:50, CDR2 of SEQ ID NO:51, and CDR3 of SEQ ID NO:52.

[0163] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:144 and SEQ ID NO:147.

[0164] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the epitope from TMPRSS2:ERG comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:156.

[0165] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:150. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:153. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementary determination region 1 (CDR1) having at least 90% sequence identity with SEQ ID NO:142 or SEQ ID NO:145. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementary determination region 2 (CDR2) having at least 90% sequence identity with SEQ ID NO:143 or SEQ ID NO:146. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:150; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:153. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:142, CDR2 of SEQ ID NO:143, and CDR3 of SEQ ID NO:144; and the TCRβ chain construct includes CDR1 of SEQ ID NO:145, CDR2 of SEQ ID NO:146, and CDR3 of SEQ ID NO:147.

[0166] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from GATA3 that complexes with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:129 and SEQ ID NO:132.

[0167] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant GATA3 peptide complexed with a protein encoded by an HLA allele from a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct.

[0168] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode a T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCR specifically binds a mutant GATA3 peptide complexed with an HLA-A02:01 protein; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:129 or SEQ ID NO:132; and / or specifically binds a mutant GATA3 peptide comprising a region having at least 70% sequence identity with SEQ ID NO:141.

[0169] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from GATA3 that complexes with human MHC, wherein the epitope from GATA3 comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:141.

[0170] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:135. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:138. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:127 and SEQ ID NO:130. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:128 and SEQ ID NO:131. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:135; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:138. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:127, CDR2 of SEQ ID NO:128, and CDR3 of SEQ ID NO:129; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:130, CDR2 of SEQ ID NO:131, and CDR3 of SEQ ID NO:132.

[0171] In another aspect, this document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from BTK that complexes with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:161 and SEQ ID NO:176, or wherein the TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:164 and SEQ ID NO:179. In another aspect, this document provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant BTK peptide that complexes with a protein encoded by an HLA allele from a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct. In another aspect, this document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode a T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCR specifically binds a mutant BTK peptide complexed with an HLA-A02:01 protein; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:161, 164, 176, or 179; and / or specifically binds a mutant BTK peptide comprising a region having at least 70% sequence identity with SEQ ID NO:173 or 188. In another aspect, this document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from BTK that complexes with human MHC, wherein the epitope derived from BTK comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:173 or 188. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 167 or 182. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 170 or 185. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 159, 162, 174, and 177. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 160, 163, 175, and 178.In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 167 or 182; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 170 or 185. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO: 159 or 174, CDR2 of SEQ ID NO: 160 or 175, and CDR3 of SEQ ID NO: 161 or 176; and the TCRβ chain construct includes CDR1 of SEQ ID NO: 162 or 177, CDR2 of SEQ ID NO: 163 or 178, and CDR3 of SEQ ID NO: 164 or 179.

[0172] In another aspect, this disclosure provides a host cell comprising a nucleic acid encoding a TCR against a neoantigen provided herein, a vector containing the sequence of that nucleic acid, or a protein encoded by the nucleic acid provided herein. In some embodiments, the host cell is a CD4 cell. + T cells. In some embodiments, the host cell is a CD8 cell. + T cells. The host cell can be a natural killer (NK) cell or a B cell. The host cell can be a cell line that proliferates indefinitely.

[0173] In another aspect, this disclosure provides pharmaceutical compositions comprising a nucleic acid encoding a TCR against a neoantigen provided herein, a host cell comprising a nucleic acid encoding a TCR against a neoantigen provided herein, a vector comprising the nucleic acid sequence, or a protein encoded by the nucleic acid provided herein. In some embodiments, this disclosure also includes methods of using the pharmaceutical compositions disclosed herein.

[0174] On the other hand, this document also provides a method for treating a subject suffering from a disease or condition, comprising administering to the subject the pharmaceutical composition disclosed herein. In some embodiments, the subject suffers from cancer.

[0175] III. T-cell receptor (TCR)

[0176] The ability of T cells to recognize antigens associated with a variety of cancers or infectious organisms is conferred by their TCRs, which consist of alpha (α) and beta (β) chains or gamma (γ) and delta (δ) chains. The proteins that make up these chains are encoded by DNA, which generates the vast diversity of TCRs through unique mechanisms. This multi-subunit immune recognition receptor is associated with the CD3 complex and binds to peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The binding of the TCR to the antigenic peptide on the APC is a central event in T cell activation, occurring at the immune synapse at the contact point between the T cell and the APC.

[0177] Each TCR contains a variable complementarity-determining region (CDR), as well as a framework region (FR) and a constant region. The amino acid sequence of the third complementarity-determining region (CDR3) loop in the variable domains of the α and β chains largely determines the sequence diversity of αβ T cells resulting from recombination between variable (Vβ), diverse (Dβ), and linking (Jβ) gene segments in the β chain locus, and between similar Vα and Jα gene segments in the α chain locus. The presence of multiple such gene segments in the TCR α and β chain loci allows for the encoding of a large number of different CDR3 sequences. During TCR gene rearrangement, independent additions and deletions of nucleotides at Vβ-Dβ, Dβ-Jβ, and Vα-Jα links further increase CDR3 sequence diversity. In this respect, immune capacity is reflected in the diversity of TCRs. The difference between γδ TCRs and αβ TCRs lies in the fact that the receptors they encode interact closely with the innate immune system. TCR γδ is expressed early in development, has a specific anatomical distribution, exhibits unique pathogen and small molecule specificity, and possesses a broad spectrum of innate and adaptive cell interactions. In early ontogenesis, a biased pattern of TCRγV and J segment expression is established because a limited subset of TCRγδ cells occupy multiple tissues in the prenatal period.

[0178] The TCR targets provided in this article can be engineered TCRs, such as chimeric antigen receptors (CARs). CARs can consist of three regions: an extracellular domain, a transmembrane domain, and an intracellular domain.

[0179] The extracellular domain can be the region of the receptor exposed to the extracellular fluid and can consist of an antigen recognition region. In some embodiments, the extracellular domain also includes a spacer region. In some embodiments, the extracellular domain also includes a signal peptide. The signal peptide can guide the nascent protein into the endoplasmic reticulum. The signal protein in the CAR can be a single-chain variable fragment (scFv). A fusion protein can be a protein formed by combining two or more genes that originally encode different proteins, but when translated in the cell, they translate to produce one or more polypeptides having functional properties derived from each original gene. scFv is a chimeric protein consisting of a light chain domain and a heavy chain variable domain linked by a short linker peptide. The linker may contain hydrophilic residues with glycine and / or serine residue segments. The linker may contain glutamic acid and lysine residue segments that improve solubility.

[0180] The transmembrane domain can be a hydrophobic transmembrane domain. In some embodiments, the transmembrane domain comprises an α-helical domain. The transmembrane domain may be functional for the stability of the receptor as a whole. In some embodiments, the transmembrane domain comprises a transmembrane domain from the component closest to the membrane from the intracellular domain. In some embodiments, the transmembrane domain comprises a CD3-zeta transmembrane domain. In some embodiments, the transmembrane domain allows for the incorporation of an artificial TCR into the natural TCR complex. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0181] The intracellular domain can be a functional intracellular portion of a receptor such as a TCR or CAR. Upon antigen recognition, the receptor clusters and can transduce signals into the cell. In some embodiments, the intracellular domain comprises a CD3-zeta intracellular domain. In some embodiments, the intracellular domain comprises at least one ITAM. In some embodiments, the intracellular domain comprises at least three or more ITAMs. In some embodiments, the intracellular domain comprises a CD28 intracellular domain. In some embodiments, the intracellular domain comprises an OX40 intracellular domain. In some embodiments, the intracellular domain comprises a chimeric intracellular domain. For example, the intracellular domain may comprise a CD28 intracellular domain, an OX40 intracellular domain, and a CD3-zeta intracellular domain.

[0182] IV. T cells

[0183] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. T cells include CD4+ cells. + T cells (helper T cells) and CD8 + T cells (cytotoxic T cells). CD4+ T cells assist other white blood cells in the immune process, which includes the maturation of B cells and the activation of cytotoxic T cells and macrophages. CD4 +T cells are activated upon presentation of MHC class II peptide antigens expressed on the surface of antigen-presenting cells (APCs). Once activated, T cells can rapidly proliferate and secrete cytokines that regulate the active immune response. CD8 + T cells can destroy virus-infected cells and tumor cells, and can also participate in transplant rejection. CD8 + T cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every type of cell in the body. Most T cells possess T cell receptors (TCRs). The ability of T cells to recognize antigens associated with a variety of cancers or infectious organisms is conferred by their TCRs, which are composed of alpha (α) and beta (β) chains or gamma (γ) and delta (δ) chains. The proteins that make up these chains are encoded by DNA, which generates the diversity of TCRs through unique mechanisms. This multi-subunit immune recognition receptor associates with the CD3 complex and binds to peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The first signal in T cell activation can be provided by the binding of the T cell receptor to a short peptide presented by MHC on another cell. This ensures that only T cells with TCRs specific to that peptide are activated. Companion cells are typically antigen-presenting cells, such as professional antigen-presenting cells, and in naive responses, dendritic cells, although B cells and macrophages may be important APCs. The binding of the TCR to the antigenic peptide on the APC can be a central event in T cell activation, occurring at the immune synapse at the contact point between the T cell and the APC.

[0184] T cells can be prepared according to methods known in the art. The T cells can be enriched T cell products, APC-depleted cell products, or substantially purified T cell products. The T cells can be a mixed population of T cells or a purified subset of T cells. The T cells can be enriched T cell products containing an increased number or percentage of T cells relative to a separated T cell population.

[0185] T cells, or subsets of T cells, can be obtained from a variety of lymphoid tissues. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, thymus, tissue biopsy, tumors, lymph node tissue, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, lymphoid tissue, and tumors. As used herein, the term "peripheral blood lymphocytes" (PBL) and its grammatical equivalents can refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes can also refer to lymphocytes that are not localized to an organ. Peripheral blood lymphocytes can include T cells, NK cells, B cells, or any combination thereof.

[0186] The method may include isolating T cells from a subject. The method may include obtaining T cells isolated from the subject. T cells may be obtained from a T cell line. T cells may be obtained from an autologous source. T cells may be obtained from an allogeneic source. T cells may also be obtained from xenogeneic sources, such as mice, rats, non-human primates, and pigs.

[0187] T cells can be APC-depleted cell products. T cells may be substantially APC-free. For example, T cells may contain T cells with more than 75% APC removed. In an exemplary embodiment, peripheral blood mononuclear cells (PBMCs) may be obtained from blood, for example, in heparinized vials. PBMCs can be separated from red blood cells by centrifugation and recovered from the interface. The recovered PBMCs may optionally be washed (e.g., with PBS).

[0188] T cell purification can be achieved, for example, through positive or negative selection, including but not limited to the use of antibodies against CD2, CD3, CD4, CD5, CD8, CD14, CD19, and / or MHC class II molecules. Specific T cell subsets, such as CD28, can be isolated using positive or negative selection techniques. + CD4 + CD8 + CD45RA + and / or CD45RO + T cells. For example, CD3 / CD28 conjugated magnetic beads can be used to positively select CD3 cells. + CD28 + T cells. In one aspect of the invention, enrichment of a T cell population by negative selection can be achieved by combining a combination of antibodies targeting cell-specific surface markers for negative selection.

[0189] For example, a T-cell sample may contain cells from the subject's circulating blood and can be obtained via apheresis or leukocyte apheresis. The T-cell sample may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets. Undesirable components can be removed from the T-cell sample, and the remaining T cells can be suspended in a culture medium. For example, cells can be washed to remove plasma fractions. This can be done, for example, by lysing erythrocytes and passing them through a PERCOLL... TM T cells were isolated from peripheral blood lymphocytes by gradient centrifugation.

[0190] In some embodiments, the T cell includes at least one T cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC. In some embodiments, the T cell includes at least one T cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the TMPRSS2:ERG that complexes with human MHC. In some embodiments, the T cell includes at least one T cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the GATA3 that complexes with human MHC. In some embodiments, the host cell includes at least one TCR disclosed herein, wherein the host cell is a CD4+ T cell. In some embodiments, the host cell is a CD8+ T cell. + T cells. In some embodiments, the host cell is an autologous cell. In some embodiments, the host cell is an allogeneic cell. In some embodiments, the host cell is a human cell. In some other cases, the host cell may be a natural killer (NK) cell, a B cell, or a cell line that proliferates indefinitely.

[0191] In some implementations, T cells can be obtained through positive and / or negative selection. In positive selection, an affinity agent (such as an antibody, antibody fragment, and aptamer) can be used to bind to a cell surface marker expressed on the cell population, such as CD3 on T cells. Using the affinity agent, T cells can be labeled. The labeled T cells can then be enriched using various methods known in the art. Non-limiting examples of these methods include fluorescence-activated cell sorting (FACS) and cell isolation based on (para)magnetic particles (e.g., the MACS Cell Isolation Kit from Miltenyi Biotec).

[0192] In negative selection, affinity agents can be used to bind to cell surface markers expressed on blood cells other than the desired population. For example, when attempting to isolate T cells, a mixture of affinity agents can be used to label B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and erythrocytes. The labeled cells can then be depleted, resulting in T cell enrichment. Exemplary methods for depleting labeled cells include FACS and cell isolation based on (para)magnetic particles.

[0193] In addition to marker-based isolation, specific growth conditions can be used to promote the growth of a particular cell population. For example, specific cytokines or growth factors can be used to obtain specific growth conditions. For another example, T cells can preferentially proliferate in media containing phytohemagglutinins (PHAs), IL-2, and / or IL-15.

[0194] In some cases, non-functional markers can be used to isolate T cells because the binding of functional markers, such as anti-CD3 antibodies (alone or conjugated with magnetic particles) to CD3, can trigger unwanted signaling events on T cells. Therefore, a mixture of antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (blood group glycoprotein A) can be used to isolate T cells.

[0195] Detailed protocols are available in publicly available literature (e.g., see Lewisor et al., J Vis Exp. 2010; (40): 2017), which is incorporated herein by reference. T-cell isolation kits are available from, for example, STEMCELL Technologies, Thermofisher, and Miltenyi Biotec.

[0196] The T cells mentioned in this article can be allogeneic T cells.

[0197] In some embodiments, the T cell may be a genetically modified cell that contains a modified human T cell receptor (TCR) α chain gene and / or a modified human TCR β chain gene in its genome, wherein the cell has reduced cell surface expression of endogenous TCR.

[0198] To disrupt the components of the TCR, gene-editing nucleases can be used. The TCRα chain (TCRα) is encoded by a single TRAC gene and pairs with the TCRβ chain (TCRβ), which is encoded by two TCRB genes. Since the TCRα / β dimer produces a fully functional TCR complex, disrupting the function of TCRα and / or TCRβ can reduce (or even eliminate) endogenous TCR expression.

[0199] Several methods can be used to disrupt endogenous TCRα or TCRβ genes. For example, there are four classes of gene-editing proteins that share a common mode of action in binding user-defined DNA sequences and mediating double-strand DNA breaks (DSBs). Zinc finger nucleases (ZFNs) are heterodimeric arrays co-localized to target DNA sites. ZFNs consist of a single zinc finger subunit that binds to DNA and is bound to a Fokl nuclease domain that cuts DNA. Transcription activator-like effector nucleases (TALENs) consist of repeating units that bind to DNA via highly variable diamino acid sequences (repeated variable two residues; RVD) that control DNA base recognition. Similar to ZFNs, TALENs act as dimer proteins fused to the Fokl endonuclease domain for DSB generation. Massive nucleases (MNs) are monomeric proteins with innate nuclease activity, derived from bacterial homing endonucleases and engineered for a single target site. Clustered, regularly spaced short palindromic repeats (CRISPR) and associated Cas9 nuclease platforms involve small guide RNA (gRNA) transcripts that contact the target DNA sequence via Watson-Crick base pairing, and Cas9 nucleases that cleave that DNA.

[0200] In some implementations, introducing a genome editing nuclease into T cells includes introducing a polynucleotide encoding the genome editing nuclease into the T cells.

[0201] In some embodiments, introducing a genome editing nuclease into T cells includes introducing a Cas9 peptide into T cells. In some embodiments, the genome editing nuclease includes a TALEN nuclease, a CRISPR / Cas9 nuclease, or a megaTAL nuclease.

[0202] In some embodiments, the CRISPR / Cas9 nuclease is derived from *Streptococcus pyogenes* or *Staphylococcus aureus*. In some of these embodiments, the CRISPR / Cas9 nuclease includes a nuclease-resistant gRNA, such as at least one 2'-OMe-phosphate-modified base, at least one 2'-O-methyl-modified base, or at least one 2'-O-methyl-3'-phosphate-modified base.

[0203] In some implementations, the TALEN nuclease or megaTAL nuclease is encoded by RNA with an exogenous polyadenylation signal.

[0204] In some implementations, the methods described herein may further include culturing T cells under conditions that effectively expand the population of genomically modified T cells.

[0205] In some embodiments, disrupting the expression of TCRα and / or TCRβ further disrupts the assembly of TCRα and TCRβ. In some embodiments, disrupting the expression of TCRα further disrupts the formation of the complex between TCR and CD3. In some embodiments, disrupting the expression of TCRα involves further disrupting the assembly of TCRα and TCRβ.

[0206] In some implementations, the genetically modified T cells contain broken TCRα and / or β chains and inactivated genes encoding immune checkpoint proteins such as PD1 and CTLA-4. This can be achieved by using a specific TALE nuclease targeting TCRα or TCRβ for gene inactivation, followed by inactivation of genes encoding immune checkpoint proteins such as PD1 and CTLA-4.

[0207] In some implementations, genetic modification depends on the inactivation of one gene or two genes selected from PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCRα, and TCRβ. In some embodiments, the genetic modification depends on the inactivation of two genes selected from PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG 3 and TCRα, LAG 3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ. In some embodiments, the genetic modification depends on the inactivation of more than two genes. The genetic modification can be performed in vitro.

[0208] V. TCR with specificity to RAS peptide-MHC complex

[0209] This document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 84% sequence identity with an amino acid sequence selected from SEQ ID NO: 3, 18, 33, 49, 65, 81, 97, 113, 241, 257, 273, 289, 305, 321, 337, 353, 369, 385, 401, 417, and 433, and / or wherein the TCRβ chain construct comprises ... The amino acid sequences of NO:6, 21, 36, 52, 68, 84, 100, 116, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, and 436 have complementarity-determining regions 3 (CDR3) with at least 84% sequence identity.

[0210] This document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from the RAS that complexes with human MHC, wherein the epitope from the RAS comprises a region having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 15, 30, 45, 46, 61, 62, 77, 78, 93, 94, 109, 110, 125, 126, 219-222, 253, 254, 269, 270, 285, 286, 301, 302, 317, 318, 333, 334, 349, 350, 365, 366, 381, 382, ​​397, 398, 413, 414, 429, 430, 445, and 446.

[0211] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T-cell receptor (TCR), the T-cell receptor comprising a TCR α-chain construct and / or a TCR β-chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A03:01 allele. In some embodiments, the α-chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:71, 87, 103, 295, 311, 327, 343, 359, and 391, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A03:01 allele. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 74, 90, 106, 298, 314, 330, 346, 362, and 394, wherein the TCR specifically binds to an epitope from RAS that is encoded by the human MHC complex by the HLA-A03:01 allele.

[0212] In some embodiments, the TCRα chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 63, 79, 95, 287, 303, 319, 335, 351, and 383. In some embodiments, the TCRβ chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 66, 82, 98, 290, 306, 322, 338, 354, and 386. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 64, 80, 96, 288, 304, 320, 336, 352, and 384. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 67, 83, 99, 291, 307, 323, 339, 355, and 387. In some embodiments, the TCRα chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 65, 81, 97, 289, 305, 321, 337, 353, and 385. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 68, 84, 100, 292, 308, 324, 340, 356, and 388.

[0213] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T-cell receptor (TCR), the T-cell receptor comprising a TCR α-chain construct and / or a TCR β-chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A02:01 allele. In some embodiments, the α-chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 9 and 24, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A02:01 allele. In some embodiments, the TCR β-chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 12 and 27, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A02:01 allele.

[0214] In some embodiments, the TCRα chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:1 and 16. In some embodiments, the TCRβ chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:4 and 19. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:2 and 17. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:5 and 20. In some embodiments, the TCRα chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with amino acid sequences selected from SEQ ID NO:3 and 18. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 6 and 21.

[0215] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode at least one T-cell receptor (TCR), the T-cell receptor comprising a TCRα chain construct and / or a TCRβ chain construct; wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A11:01 allele. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:39, 55, 122, 247, 263, 279, 375, 407, 423, and 439, wherein the TCR specifically binds to an epitope from the RAS that is complexed with human MHC encoded by the HLA-A11:01 allele. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO:42, 58, 125, 250, 266, 282, 378, 413, 426, and 442, wherein the TCR specifically binds to an epitope from RAS that is encoded by the human MHC complex by the HLA-A11:01 allele.

[0216] In some embodiments, the TCRα chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 31, 47, 111, 239, 255, 271, 367, 399, 415, and 431. In some embodiments, the TCRβ chain construct as described above includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 34, 50, 114, 242, 258, 274, 370, 402, 418, and 434. In some embodiments, the TCRα chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 32, 48, 112, 240, 256, 272, 368, 400, 416, and 432. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 35, 51, 115, 243, 259, 275, 371, 403, 419, and 435. In some embodiments, the TCRα chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 33, 49, 113, 241, 257, 273, 369, 401, 417, and 433. In some embodiments, the TCRβ chain construct includes a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 36, 52, 116, 228, 244, 260, 276, 372, 404, 420, and 434. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:9; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:12. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:3; and the TCRβ chain construct includes CDR1 of SEQ ID NO:4, CDR2 of SEQ ID NO:5, and CDR3 of SEQ ID NO:6. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:24; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:27.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:16, CDR2 of SEQ ID NO:17, and CDR3 of SEQ ID NO:18; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:19, CDR2 of SEQ ID NO:20, and CDR3 of SEQ ID NO:21. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:39; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:42. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:34, CDR2 of SEQ ID NO:35, and CDR3 of SEQ ID NO:36. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:55; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:58. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:47, CDR2 of SEQ ID NO:48, and CDR3 of SEQ ID NO:49; and the TCRβ chain construct includes CDR1 of SEQ ID NO:50, CDR2 of SEQ ID NO:51, and CDR3 of SEQ ID NO:52. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:71; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:74. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65; and the TCRβ chain construct includes CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:87; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:90.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:80, and CDR3 of SEQ ID NO:81; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:82, CDR2 of SEQ ID NO:83, and CDR3 of SEQ ID NO:84. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:103; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:106. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:95, CDR2 of SEQ ID NO:96, and CDR3 of SEQ ID NO:97; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:98, CDR2 of SEQ ID NO:99, and CDR3 of SEQ ID NO:100. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:119; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:122. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:111, CDR2 of SEQ ID NO:112, and CDR3 of SEQ ID NO:113; and the TCRβ chain construct includes CDR1 of SEQ ID NO:114, CDR2 of SEQ ID NO:115, and CDR3 of SEQ ID NO:116. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:263; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:266. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:255, CDR2 of SEQ ID NO:256, and CDR3 of SEQ ID NO:257; and the TCRβ chain construct includes CDR1 of SEQ ID NO:258, CDR2 of SEQ ID NO:259, and CDR3 of SEQ ID NO:260. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:279; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:282.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:271, CDR2 of SEQ ID NO:272, and CDR3 of SEQ ID NO:273; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:274, CDR2 of SEQ ID NO:275, and CDR3 of SEQ ID NO:276. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:295; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:298. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:287, CDR2 of SEQ ID NO:288, and CDR3 of SEQ ID NO:289; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:290, CDR2 of SEQ ID NO:291, and CDR3 of SEQ ID NO:292. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:311; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:314. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:303, CDR2 of SEQ ID NO:304, and CDR3 of SEQ ID NO:305; and the TCRβ chain construct includes CDR1 of SEQ ID NO:306, CDR2 of SEQ ID NO:307, and CDR3 of SEQ ID NO:308. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:327; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:330. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:319, CDR2 of SEQ ID NO:320, and CDR3 of SEQ ID NO:321; and the TCRβ chain construct includes CDR1 of SEQ ID NO:322, CDR2 of SEQ ID NO:323, and CDR3 of SEQ ID NO:324. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:343; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:346.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:335, CDR2 of SEQ ID NO:336, and CDR3 of SEQ ID NO:337; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:338, CDR2 of SEQ ID NO:339, and CDR3 of SEQ ID NO:340. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:343; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:346. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:351, CDR2 of SEQ ID NO:352, and CDR3 of SEQ ID NO:353; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:354, CDR2 of SEQ ID NO:355, and CDR3 of SEQ ID NO:356. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:343; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:346. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:367, CDR2 of SEQ ID NO:368, and CDR3 of SEQ ID NO:369; and the TCRβ chain construct includes CDR1 of SEQ ID NO:370, CDR2 of SEQ ID NO:371, and CDR3 of SEQ ID NO:372. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:391; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:394. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:383, CDR2 of SEQ ID NO:384, and CDR3 of SEQ ID NO:385; and the TCRβ chain construct includes CDR1 of SEQ ID NO:386, CDR2 of SEQ ID NO:387, and CDR3 of SEQ ID NO:388. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:391; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:394.In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:399, CDR2 of SEQ ID NO:400, and CDR3 of SEQ ID NO:401; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:402, CDR2 of SEQ ID NO:403, and CDR3 of SEQ ID NO:404. In some embodiments, the TCRα chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:423; and the TCRβ chain construct comprises a variable region having at least 80% sequence identity with SEQ ID NO:426. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:415, CDR2 of SEQ ID NO:416, and CDR3 of SEQ ID NO:417; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:418, CDR2 of SEQ ID NO:419, and CDR3 of SEQ ID NO:420. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:391; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:394. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:431, CDR2 of SEQ ID NO:432, and CDR3 of SEQ ID NO:433; and the TCRβ chain construct includes CDR1 of SEQ ID NO:434, CDR2 of SEQ ID NO:435, and CDR3 of SEQ ID NO:426.

[0217] In several implementation schemes, the nucleic acid sequence encoding the TCR is codon-optimized.

[0218] Mutations in any of the three ras genes—H-ras, K-ras, and N-ras—are among the most common events in human tumorigenesis. Approximately 30% of all human tumors are found to carry at least one mutation in any typical ras gene. Ras mutations are prominent in cancers such as: biliary adenocarcinoma, bladder transitional cell carcinoma, breast cancer, cervical adenocarcinoma, colonic adenocarcinoma, colonic adenoma, neuroblastoma (autonomic ganglion), acute myeloid leukemia, chronic myeloid leukemia, chronic myeloid monocytic leukemia, juvenile myeloid monocytic leukemia, acute lymphoblastic leukemia, Burkitt lymphoma, Hodgkin lymphoma, plasma cell myeloma, hepatocellular carcinoma, large cell carcinoma, non-small cell carcinoma, ductal carcinoma, endocrine tumors, prostate adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, angiosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, myxoma, malignant fibrous histiocytoma, pleomorphic sarcoma, germ cell tumor, seminoma, anaplastic carcinoma, follicular carcinoma, papillary carcinoma, and Hurthle cell carcinoma. Ras mutations can be found in cancers that affect many tissues and organs in the body, such as lung cancer, liver cancer, breast cancer, bladder cancer, colon cancer, cervical cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, testicular cancer, soft tissue cancer, skin cancer, and blood cancer.

[0219] In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the RAS peptide contains a G12V mutation. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the RAS peptide contains a G12C mutation. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the RAS peptide contains a G12D mutation. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the RAS peptide contains a mutation at Q61. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the RAS peptide contains the sequence VVGAVGVGK, VVVGAVGVGK, VVGADGVGK, VVVGADGVGK, VVGACGVGK, VVVGACGVGK, KLVVVGACGV, LVVVGACGV, KLVVVGADGV, LVVVGADGV, KLVVVGAVGV, or LVVVGAVGV.

[0220] In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein human MHC is encoded by the HLA-A02:01 allele. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein human MHC is encoded by the HLA-A03:01 allele. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein human MHC is encoded by the HLA-A11:01 allele.

[0221] In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele, and wherein the RAS peptide comprises the sequence VVGAVGVGK, VVVGAVGVGK, VVGADGVGK, VVVGADGVGK, VVGACGVGK, VVVGACGVGK, KLVVVGACGV, LVVVGACGV, KLVVVGADGV, LVVVGADGV, KLVVVGAVGV, or LVVVGAVGV. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the human MHC is encoded by the HLA-A03:01 allele, and wherein the RAS peptide comprises the sequence VVGAVGVGK, VVVGAVGVGK, VVGADGVGK, VVVGADGVGK, VVGACGVGK, VVVGACGVGK, KLVVVGACGV, LVVVGACGV, KLVVVGADGV, LVVVGADGV, KLVVVGAVGV, or LVVVGAVGV. In several embodiments, the TCR binds to the MHC:RAS peptide complex, wherein the human MHC is encoded by the HLA-A11:01 allele, and wherein the RAS peptide comprises the sequence VVGAVGVGK, VVVGAVGVGK, VVGADGVGK, VVVGADGVGK, VVGACGVGK, VVVGACGVGK, KLVVVGACGV, LVVVGACGV, KLVVVGADGV, LVVVGADGV, KLVVVGAVGV, or LVVVGAVGV.

[0222] In some embodiments, when the epitope peptide is complexed with MHC encoded by a specific allele, a TCR disclosed herein exhibits specific binding affinity for epitope peptides containing point mutations found in cancer; additionally, when the epitope peptide is complexed with MHC encoded by a specific allele, the TCR may exhibit different binding affinity for another epitope peptide of the same cancer protein containing different point mutations, but does not exhibit binding affinity for wild-type peptides without any mutations.

[0223] VI. TCRs with specificity to the TMPRSS2:ERG peptide-MHC complex

[0224] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:144 and SEQ ID NO:147.

[0225] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the epitope from TMPRSS2:ERG comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:156.

[0226] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:150. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:153. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementary determination region 1 (CDR1) having at least 90% sequence identity with SEQ ID NO:142 or SEQ ID NO:145. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementary determination region 2 (CDR2) having at least 90% sequence identity with SEQ ID NO:143 or SEQ ID NO:146. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:150; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:153. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:142, CDR2 of SEQ ID NO:143, and CDR3 of SEQ ID NO:144; and the TCRβ chain construct includes CDR1 of SEQ ID NO:145, CDR2 of SEQ ID NO:146, and CDR3 of SEQ ID NO:147.

[0227] In some embodiments, the TCR comprises a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO:144 or 147.

[0228] In some embodiments, the TCR comprises a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from TMPRSS2:ERG that is complexed with human MHC, wherein the epitope from TMPRSS2:ERG comprises a region having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO:156.

[0229] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a variable region having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO:150 or 153.

[0230] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementary determination region 1 (CDR1) having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO:142 or 145.

[0231] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementary determination region 2 (CDR2) having at least 90% sequence identity with SEQ ID NO:143 or 146.

[0232] In some embodiments, the nucleic acid encoding the TCRα strand construct and / or the TCRβ strand construct comprises a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any one of SEQ ID NO: 148, 149, 151, and 152. In several embodiments, the nucleic acid sequence encoding the TCR is codon-optimized.

[0233] In several embodiments, the TCR binds to the MHC:TMPRSS2::ERG peptide complex, wherein the TMPRSS2::ERG peptide comprises a gene fusion mutation. In several embodiments, the TCR binds to the MHC:TMPRSS2::ERG peptide complex, wherein the TMPRSS2::ERG peptide comprises a gene fusion mutation containing at least two consecutive amino acids, wherein said at least two consecutive amino acids comprise at least one amino acid of TMPRSS2 and at least one amino acid of ERG. In several embodiments, the TCR binds to the MHC:TMPRSS2::ERG peptide complex, wherein the TMPRSS2::ERG peptide comprises the sequence ALNSEALSV.

[0234] In several embodiments, the human MHC is encoded by the HLA-A02:01 allele. In several embodiments, the TCR binds to the MHC:TMPRSS2::ERG peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele.

[0235] In several embodiments, the human MHC is encoded by the HLA-A02:01 allele. In several embodiments, the TCR binds to the MHC:TMPRSS2::ERG peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele, and wherein the TMPRSS2::ERG peptide contains the sequence ALNSEALSV.

[0236] VII. TCR with specificity to GATA3 peptide-MHC complex

[0237] In some other embodiments, the TCR comprises a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from GATA3 that is complexed with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 129, 132, 191, 194, 206, or 209.

[0238] In some embodiments, the TCR comprises a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from GATA3 that is complexed with human MHC, wherein the epitope from GATA3 comprises a region having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 141, 203, or 218.

[0239] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a variable region having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 135, 138, 197, 200, 212, or 215.

[0240] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementary determination region 1 (CDR1) having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 127, 130, 189, 192, 204, or 207.

[0241] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementary determination region 2 (CDR2) having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 128, 131, 190, 193, 205, or 208.

[0242] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from GATA3 that complexes with human MHC, wherein the TCRα chain construct and / or TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 129, 132, 191, 194, 206 or 209.

[0243] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant GATA3 peptide that complexes with a protein encoded by the HLA-A02:01, HLA-B07:02, or HLA-B08:01 alleles of a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct.

[0244] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode T cell receptors (TCRs) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCRs specifically bind to mutant GATA3 peptides complexed with HLA-A02:01, HLA-B07:02, or HLA-B08:01 proteins; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:129, 132, 191, 194, 206, or 209; and / or specifically bind to mutant GATA3 peptides comprising a region having at least 70% sequence identity with SEQ ID NO:141, 203, or 218.

[0245] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope from GATA3 that is complexed with human MHC, wherein the epitope from GATA3 comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:141, 203 or 218.

[0246] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 135, 197, or 212. In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO: 138, 200, or 215. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 127, 130, 189, 192, 204, and 207. In some embodiments, the TCRα chain construct and / or the TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 128, 131, 190, 193, 205, and 208.

[0247] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:135; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:138. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:197; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:200. In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:212; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:215.

[0248] In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:127, CDR2 of SEQ ID NO:128, and CDR3 of SEQ ID NO:129; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:130, CDR2 of SEQ ID NO:131, and CDR3 of SEQ ID NO:132. In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO:189, CDR2 of SEQ ID NO:190, and CDR3 of SEQ ID NO:191; and the TCRβ chain construct comprises CDR1 of SEQ ID NO:192, CDR2 of SEQ ID NO:193, and CDR3 of SEQ ID NO:194. In some embodiments, the TCRα chain construct includes CDR1 of SEQ ID NO:204, CDR2 of SEQ ID NO:205, and CDR3 of SEQ ID NO:206; and the TCRβ chain construct includes CDR1 of SEQ ID NO:207, CDR2 of SEQ ID NO:208, and CDR3 of SEQ ID NO:209.

[0249] In some embodiments, the nucleic acids encoding the TCRα chain construct and / or the TCRβ chain construct comprise nucleic acid sequences having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any one of SEQ ID NO: 133, 134, 136, or 137. In some embodiments, the nucleic acids encoding the TCRα chain construct and / or the TCRβ chain construct comprise nucleic acid sequences having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any one of SEQ ID NO: 195, 196, 198, or 199. In some embodiments, the nucleic acid encoding the TCRα chain construct and / or the TCRβ chain construct comprises a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any one of SEQ ID NO: 210, 211, 213, or 214.

[0250] In several implementation schemes, the nucleic acid sequence encoding the TCR is codon-optimized.

[0251] In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least one amino acid encoded by a GATA3neoORF sequence. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids encoded by a GATA3 neoORF sequence. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least one amino acid encoded by a GATA3 neoORF sequence and at least one amino acid encoded by a GATA3 wild-type sequence. In several embodiments, the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids encoded by a GATA3 neoORF sequence and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids encoded by a GATA3 wild-type sequence. In several embodiments, the TCR binds to an MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least one amino acid encoded by a GATA3 neoORF sequence and at least one amino acid not encoded by a GATA3 neoORF sequence. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the GATA peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids encoded by the GATA3 neoORF sequence and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids not encoded by the GATA3 neoORF sequence. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein each amino acid of the GATA peptide is an amino acid encoded by the GATA3neoORF sequence. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the GATA3 peptide contains the sequence MLTGPPARV, KPKRDGYMF, or ESKIMFATL.

[0252] In several embodiments, the human MHC is encoded by the HLA-A02:01, HLA-B07:02, or HLA-B08:01 allele. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the human MHC is encoded by the HLA-B07:02 allele. In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein the human MHC is encoded by the HLA-B08:01 allele.

[0253] In several embodiments, the TCR binds to the MHC:GATA3 peptide complex, wherein human MHC is encoded by the HLA-B08:01 allele, and wherein the GATA3 peptide contains the sequence MLTGPPARV, KPKRDGYMF, or ESKIMFATL.

[0254] VIII. TCR with specificity to BTK peptide-MHC complex

[0255] The BTK gene encodes the Bruton's tyrosine kinase (BTK) protein, which may be involved in the development and maturation of B cells. The BTK protein can transmit chemical signals that instruct B cells to mature and produce antibodies.

[0256] This document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from BTK that complexes with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:161 and SEQ ID NO:176, or wherein the TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:164 and SEQ ID NO:179.

[0257] This document also provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode a T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCR specifically binds a mutant BTK peptide complexed with an HLA-A02:01 protein; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:161, 164, 176 or 179; and / or specifically binds a mutant BTK peptide comprising a region having at least 70% sequence identity with SEQ ID NO:173 or 188.

[0258] This article also provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an epitope derived from BTK that complexes with human MHC, wherein the epitope derived from BTK comprises a region having at least 90% sequence identity with the amino acid sequence SEQ ID NO:173 or 188.

[0259] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:167 or 182.

[0260] In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:170 or 185.

[0261] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 159, 162, 174 and 177.

[0262] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 160, 163, 175 and 178.

[0263] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:167 or 182; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:170 or 185.

[0264] In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO: 159 or 174, CDR2 of SEQ ID NO: 160 or 175, and CDR3 of SEQ ID NO: 161 or 176; and the TCRβ chain construct comprises CDR1 of SEQ ID NO: 162 or 177, CDR2 of SEQ ID NO: 163 or 178, and CDR3 of SEQ ID NO: 164 or 179.

[0265] In some embodiments, the epitope comprises a point mutation. In some embodiments, the point mutation is a C481S mutation.

[0266] In several implementation schemes, the nucleic acid sequence encoding the TCR is codon-optimized.

[0267] In several embodiments, the TCR binds to the MHC:BTK peptide complex, wherein the BTK peptide contains a point mutation. In several embodiments, the TCR binds to the MHC:BTK peptide complex, wherein the BTK peptide contains a C481S point mutation. In several embodiments, the TCR binds to the MHC:BTK peptide complex, wherein the BTK peptide contains the sequence SLLNYLREM.

[0268] In some embodiments, the human MHC is encoded by the HLA-A02:01 allele. In multiple embodiments, the human MHC is encoded by the HLA-A02:01 allele. In multiple embodiments, the TCR binds to the MHC:BTK peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele.

[0269] In several embodiments, the TCR binds to the MHC:BTK peptide complex, wherein human MHC is encoded by the HLA-A02:01 allele, and wherein the BTK peptide contains the sequence of SLLNYLREM.

[0270] IX. TCR with specificity to EGFR peptide-MHC complex

[0271] This document provides nucleic acids encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an EGFR-derived epitope complexed with human MHC, wherein the TCRα chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:449, SEQ ID NO:466, SEQ ID NO:483, SEQ ID NO:500, and SEQ ID NO:517, or wherein the TCRβ chain construct comprises a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:452, SEQ ID NO:469, SEQ ID NO:486, SEQ ID NO:503, and SEQ ID NO:520.

[0272] This article provides a nucleic acid encoding at least one T-cell receptor (TCR) capable of specifically binding to a mutant EGFR peptide complexed with a protein encoded by an HLA allele from a cancer subject, wherein the TCR comprises a TCRα chain construct and / or a TCRβ chain construct.

[0273] This document provides isolated nucleic acids or cells containing recombinant nucleic acids, wherein the nucleic acids encode a T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct, wherein the TCR specifically binds a mutant EGFR peptide complexed with an HLA-A02:01 protein; comprising a complementarity-determining region 3 (CDR3) having at least 90% sequence identity with SEQ ID NO:449, 466, 483, 500, 517, 452, 469, 486, 503 or 520; and / or specifically binds a mutant EGFR peptide comprising a region having at least 70% sequence identity with SEQ ID NO:461, 462, 463, 478, 479, 480, 495, 496, 497, 512, 513, 514, 529, 530 or 531.

[0274] This document provides a nucleic acid encoding at least one T-cell receptor (TCR) comprising a TCRα chain construct and / or a TCRβ chain construct capable of specifically binding to an EGFR-derived epitope complexing with human MHC, wherein the EGFR-derived epitope comprises a region having at least 90% sequence identity with the amino acid sequences SEQ ID NO: 461, 462, 463, 478, 479, 480, 495, 496, 497, 512, 513, 514, 529, 530, or 531.

[0275] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:449, 466, 483, 500 or 517.

[0276] In some embodiments, the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:452, 469, 486, 503 or 520.

[0277] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementarity-determining region 1 (CDR1) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 447, 464, 481, 498, 515, 450, 467, 484, 501 and 518.

[0278] In some embodiments, the TCRα chain construct and / or TCRβ chain construct includes a complementarity-determining region 2 (CDR2) having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 448, 465, 482, 499, 516, 451, 468, 485, 502 and 519.

[0279] In some embodiments, the TCRα chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:455, 472, 489, 506 or 523; and the TCRβ chain construct includes a variable region having at least 80% sequence identity with SEQ ID NO:458, 475, 492, 509 or 526.

[0280] In some embodiments, the TCRα chain construct comprises CDR1 of SEQ ID NO: 447, 464, 481, 498 or 515, CDR2 of SEQ ID NO: 448, 465, 482, 499 or 516, and CDR3 of SEQ ID NO: 449, 466, 483, 500 or 517; and the TCRβ chain construct comprises CDR1 of SEQ ID NO: 450, 467, 484, 501 or 518, CDR2 of SEQ ID NO: 451, 468, 485, 502 or 519, and CDR3 of SEQ ID NO: 452, 469, 486, 503 or 520.

[0281] In some embodiments, the epitope comprises a point mutation. In some embodiments, the point mutation is the T790M mutation. In some embodiments, the human MHC is encoded by the HLA-A02:01 allele.

[0282] In several implementation schemes, the nucleic acid sequence encoding the TCR is codon-optimized.

[0283] In several embodiments, the TCR binds to the MHC:EGFR peptide complex, wherein the EGFR peptide contains a point mutation. In several embodiments, the TCR binds to the MHC:EGFR peptide complex, wherein the EGFR peptide contains a T790M point mutation. In several embodiments, the TCR binds to the MHC:EGFR peptide complex, wherein the EGFR peptide contains the sequence QLIMQLMPF, LIMQLMPFGC, or MQLMPFGCLL.

[0284] In several embodiments, the human MHC is encoded by the HLA-A02:01 allele. In several embodiments, the TCR binds to the MHC:EGFR peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele.

[0285] In several embodiments, the TCR binds to the MHC:EGFR peptide complex, wherein the human MHC is encoded by the HLA-A02:01 allele, and wherein the EGFR peptide contains the sequence QLIMQLMPF, LIMQLMPFGC, or MQLMPFGCLL.

[0286] Delivery of nucleic acids or vectors

[0287] Nucleic acids encoding TCRs or vectors containing such nucleic acids can be delivered to host cells for expression and processing.

[0288] Terms such as “transfer,” “introduction,” or “transfection” are used interchangeably in this article and refer to the introduction of nucleic acids, particularly exogenous or heterologous nucleic acids, into cells.

[0289] Cells can be transfected using any carrier that can be associated with nucleic acids, for example, by forming a complex with the nucleic acid or forming vesicles that encapsulate or encapsulate the nucleic acid, resulting in increased stability of the nucleic acid compared to naked nucleic acids. Carriers useful according to this disclosure include, for example, lipid-containing carriers such as cationic lipids, liposomes, particularly cationic liposomes, and micelles, as well as nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. Any cationic lipid can be used according to this disclosure.

[0290] In several embodiments, the nucleic acid encoding the TCR disclosed herein is operatively linked to a promoter. Furthermore, this disclosure provides a vector, such as a plasmid, shuttle vector, phage particle, granule, expression vector, retroviral vector, adenoviral vector, or particle, and / or a vector intended for gene therapy, comprising one or more nucleic acids as disclosed above. A “vector” is a nucleic acid molecule capable of transporting another nucleic acid. A vector contains a nucleic acid insert that encodes a desired polypeptide or protein expressed in a cell such as a host cell. For the purposes of this disclosure, the insert may be a nucleic acid encoding a TCR; the α-chain or β-chain of the TCR, or both. The term “incorporated” in a vector may mean preparing a suitable expression vector using an insert containing said nucleic acid sequence. An “expression vector” is a vector that, when present in a suitable environment, can direct the expression of a protein encoded by one or more genes carried by that vector. A “retrovirus” is a virus having an RNA genome. A “gamma retrovirus” refers to a genus within the family Retroviridae. Exemplary gamma retroviruses include, but are not limited to, mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendothelial proliferative disease virus. "Lentinvirus" refers to a genus of retroviruses capable of infecting both dividing and non-dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV types 1 and 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). Vectors encoding the core virus are also referred to as "viral vectors." A large number of viral vectors suitable for use in this invention are available, including those identified for human gene therapy applications, such as those described in Pfeifer and Verma (Pfeifer, A. and I.M. Verma. 2001. Ann. Rev. Genomics Hum. Genet. 2:177-211). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors, like those derived from Moloney murine leukemia virus (MLV), and more complex retrovirus-derived vectors, such as lentivirus-derived vectors. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and maedi / visna viruses.Methods for transducing mammalian target cells with viral particles containing TCR transgenes using retroviral and lentiviral vectors and packaging cells are well known in the art and have been previously described, for example, in U.S. Patent 8,119,772; Walchli et al., 2011, PLoS One 6:327930; Zhao et al., J. Immunol., 2005, 174:4415-4423; Engels et al., 2003, Hum. Gene Ther. 14:1155-68; Frecha et al., 2010, Mol. Ther. 18:1748-57; Verhoeyen et al., 2009, Methods Mol. Biol. 506:97-114. Retroviral and lentiviral vector constructs and expression systems are also commercially available. In some embodiments, a viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a TCRα chain specific to a peptide antigen into hematopoietic progenitor cells. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a transduction marker. Transduction markers of the viral vector are known in the art and include selectable markers that can confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. When the viral vector genome contains more than one nucleic acid sequence to be expressed as a separate transcript in the host cell, the viral vector may also include additional sequences between two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, and viral 2A peptides. Other vectors may also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; and vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., 1998, Gene Ther. 5:1517-30). Other vectors include those derived from baculoviruses and alphaviruses. (Jolly D J. 1999. Emerging viral vectors. pp 209-40, in Friedmann T. ed. 1999. The development of human gene therapy. New York: ColdSpring Harbor Lab).

[0291] The vector may contain nucleic acid sequences, such as origins of replication, that allow the nucleic acid to replicate in a host cell. The vector may also contain one or more selective marker genes and other genetic elements known to those skilled in the art. The vector is preferably an expression vector containing a nucleic acid according to the invention operatively linked to a sequence that allows the expression of the nucleic acid.

[0292] In some embodiments, this document provides a vector comprising nucleic acid encoding the TCR disclosed herein. In some embodiments, the vector is a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, granulosome, virus, or virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, alphavirus, vaccinia virus, hepatitis B virus, human papillomavirus, or a pseudotype thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoparticle, cationic lipid, cationic polymer, metal nanopolymer, nanorod, liposome, micelle, microvesicle, cell-penetrating peptide, or liposphere.

[0293] This document provides constructs, such as nucleic acid constructs encoding the α and β chains of a TCR, for expression in cells. In some embodiments, the construct comprises one or more polynucleotides encoding the TCR α and TCR β chains. In some embodiments, the polynucleotides are incorporated into a suitable vector. In some embodiments, the polynucleotides encoding the α and β chains are incorporated into the same vector. In some embodiments, the polynucleotides encoding the α and β chains are incorporated into different vectors, and both vectors are delivered for expression in a single cell.

[0294] In some embodiments, cells can be transduced or transfected with nucleic acids encoding TCRs, wherein the cells are capable of expressing TCRs, and the cells are used as a therapeutic agent. In some embodiments, the cells are derived from a subject or host, wherein the subject or host is human. In some embodiments, the subject or host comprises cells with mutations in epitopes, and the TCR expressed in these cells is capable of specifically binding to the mutated epitopes. In some embodiments, the cells are lymphocytes. In some embodiments, they are T lymphocytes. In some embodiments, the cells are lymphocyte precursor cells. In some embodiments, the cells are T lymphocyte precursor cells. In some embodiments, the cells are T lymphocyte progenitor cells. In some embodiments, the cells are thymocytes.

[0295] In some embodiments, the T cells are immature T cells. In some embodiments, the T cells are antigen-naïve T cells. The host cells can be cultured in vitro for 1, 2, 3, 4, 5 days or more for monitoring and recovered after transfection or transduction with polynucleotides encoding TCRs.

[0296] X. neoantigen

[0297] The TCRs disclosed herein are specific to immunogenic neoantigens. In some embodiments, the neoantigen peptide is derived from RAS. In some embodiments, the neoantigen peptide is derived from GATA3. In some embodiments, the neoantigen peptide is derived from BTK. In some embodiments, the neoantigen peptide is derived from TMPRSS2:ERG. In some embodiments, one or more neoantigen peptides are loaded onto an APC, wherein T cells are then stimulated with the peptide-loaded APC to generate antigen-specific T cells. In some embodiments, the APC used for peptide loading is a dendritic cell. Immunogenic neoantigen sequences can be identified by any suitable method known in the art.

[0298] In several embodiments, the neoantigen comprises an epitope. In animals and humans, mutated epitopes can be effective in inducing immune responses or activating T cells. In some embodiments, the epitope comprises a mutation. In some embodiments, the mutation is selected from point mutations, splice site mutations, frameshift mutations, readthrough mutations, gene fusion mutations, and any combination thereof.

[0299] In some embodiments, the epitope is at least 8 amino acids long. In some embodiments, the epitope is at least 16 amino acids long. In some embodiments, the epitope is 8-25 amino acids long. In some embodiments, the epitope is 8-12 amino acids long. In some embodiments, the epitope is 16-25 amino acids long. In some embodiments, the epitope is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long.

[0300] In some embodiments, the neoantigen or its epitope may comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues and any range thereof. In specific implementation schemes, the neoantigen or its epitope has 100 or fewer amino acids.

[0301] In some embodiments, the length of a neoantigen of MHC class I or its epitope is 13 or fewer residues, and typically consists of about 8 to about 11 residues, especially 9 or 10 residues. In some embodiments, the length of a neoantigen of MHC class II or its epitope is 9-24 residues.

[0302] In some embodiments, the neoantigen binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the neoantigen binds to the HLA protein with a higher affinity than the corresponding wild-type peptide. In specific embodiments, the IC50 of the neoantigen peptide or polypeptide is at least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM, or lower.

[0303] In some embodiments, the epitope binds to human MHC with a higher affinity than the corresponding wild-type epitope. In some embodiments, the epitope has a K+ concentration of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to human MHC. In some embodiments, the epitope contains a mutation that is not present in the subject's non-cancerous cells. In some embodiments, the epitope is encoded by a gene of the subject's cancer cells or a gene expressed thereon. In some embodiments, the TCR uses a Kc of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to the HLA-peptide complex.

[0304] In some embodiments, the length of the neoantigenic peptide may be about 8 to about 50 amino acid residues, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues. In some embodiments, the length of the neoantigenic peptide may be about 8 to about 500 amino acid residues, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues.

[0305] In some embodiments, the length of the neoantigen peptide may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues. In some embodiments, the length of the neoantigen peptide may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues. In some implementations, the length of the neoantigen peptide can be up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues. In some implementations, the length of the neoantigen peptide can be up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or fewer amino acid residues.

[0306] In some embodiments, the total length of the neoantigen peptide is at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0307] In some implementations, the total length of the neoantigen peptide is up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids.

[0308] In some embodiments, the neoantigen peptide may have a pI value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the neoantigen peptide may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or higher. In some embodiments, the neoantigen peptide may have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or lower.

[0309] In some embodiments, the neoantigen peptide may have an HLA binding affinity of about 1 pM to about 1 mM, about 100 pM to about 500 μM, about 500 pM to about 10 μM, about 1 nM to about 1 μM, or about 10 nM to about 1 μM. In some embodiments, the neoantigen peptide may have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM or higher. In some implementations, the neoantigen peptide may have an HLA binding affinity of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, or 900 μM.

[0310] In some embodiments, the neoantigen peptides described herein may include carriers such as those known in the art, such as thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acid residues (such as poly-L-lysine, poly-L-glutamic acid), influenza virus proteins, hepatitis B virus core proteins, etc.

[0311] In some embodiments, the neoantigen peptides described herein may be modified by terminal -NH2 acylation (e.g., via alkyl acyl (C1-C20) or thiohydroxyacetylation) or terminal carboxyl amidation (e.g., with ammonia, methylamine, etc.). In some embodiments, these modifications may provide sites for attachment to a support or other molecules.

[0312] In some embodiments, the neoantigen peptides described herein may include modifications, such as, but not limited to, glycosylation, side-chain oxidation, biotinylation, phosphorylation, addition of surfactants (e.g., lipids), or may be chemically modified, such as acetylation. Furthermore, the bonds in the peptide can be bonds other than peptide bonds, such as covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0313] In some embodiments, the neoantigen peptides described herein may include substitutions to alter the physical properties (e.g., stability or solubility) of the resulting peptide. For example, a neoantigen peptide can be modified by replacing cysteine ​​(C) with α-aminobutyric acid (“B”). Due to its chemical properties, cysteine ​​has a tendency to form disulfide bonds and structurally alters the peptide to reduce its binding capacity. Replacing C with α-aminobutyric acid not only alleviates this problem but, in some cases, actually improves binding and cross-binding capacity. Replacing cysteine ​​with α-aminobutyric acid can occur at any residue of the neoantigen peptide, such as at anchored or unanchored sites on an epitope or analogue, or at other sites on the peptide.

[0314] In some embodiments, the neoantigen peptides described herein may comprise amino acid mimics or non-natural amino acid residues, such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, 2, 3, or 4-pyrenealanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine. Acids; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- or L-ρ-methoxybiphenylalanine; D- or L-2-indole(allyl)alanine; and D- or L-alkylalanine, wherein the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or non-acidic amino acid residues. Aromatic rings of non-natural amino acids include, for example, thiazolyl, thiophene, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrroleyl, and pyridyl aromatic rings. Modified peptides with various amino acid mimics or non-natural amino acid residues are particularly useful because they tend to exhibit increased in vivo stability. Such peptides may also have improved shelf life or preparative properties.

[0315] Peptide stability can be determined in various ways. For example, peptidases and various biological media such as human plasma and serum have been used to determine stability. See, for example, Verhoef et al., Eur. J. Drug Metab. Pharmacokinetics 11:291 (1986). The half-life of the peptides described herein is conveniently determined using 25% human serum (v / v). The protocol is as follows: The pooled human serum (type AB, non-heat-inactivated) is destroyed by centrifugation before use. The serum is then diluted to 25% using RPMI-1640 or another suitable tissue medium. At predetermined time intervals, small amounts of the reaction solution are taken out and added to 6% trichloroacetic acid (TCA) aqueous solution or ethanol. The turbid reaction sample is cooled (4°C) for 15 minutes and then rotated to clump the precipitated serum proteins. The presence of the peptide is then determined by reversed-phase HPLC using stability-specific chromatographic conditions.

[0316] In some embodiments, the neoantigen peptides described herein can be prepared synthetically via recombinant DNA technology or chemical synthesis, or isolated from natural sources such as the original tumor or pathogenic organism. Epitopes can be synthesized alone or directly or indirectly linked to the peptide. Although the neoantigen peptides described herein are substantially free of other naturally occurring host cell proteins and their fragments, in some embodiments, the peptides can be synthetically conjugated to link with natural fragments or particles.

[0317] In some embodiments, the neoantigen peptides described herein can be prepared in a variety of ways. In some embodiments, the peptides can be synthesized in solution or on a solid support using conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols (see, for example, Stewart & Young, SOLID PHASE PEPTIDESYNTHESIS, 2nd edition, Pierce Chemical Co., 1984). Furthermore, individual peptides can be linked using chemical linkages to produce larger peptides that are still within the scope of this invention.

[0318] Alternatively, recombinant DNA techniques can be used, in which the nucleotide sequence encoding a peptide inserted into an expression vector is transformed or transfected into a suitable host cell and cultured under conditions suitable for expression. These methods are generally known in the art, as outlined in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Therefore, recombinant peptides containing one or more of the epitopes described herein or composed thereof can be used to present suitable T-cell epitopes.

[0319] XI. Pharmaceutical Composition

[0320] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, said carriers comprising excipients and adjuvants that facilitate the processing of the active agent into a pharmaceutically acceptable article. Suitable formulations may depend on the chosen route of administration. Any known techniques, carriers, and excipients may be used as suitable techniques, carriers, and excipients as understood in the art.

[0321] In some cases, the pharmaceutical composition is formulated as a cell-based therapeutic agent, such as a T-cell therapeutic agent. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic agent or a nucleic acid-based therapeutic agent, wherein the nucleic acid encodes a polypeptide. The composition may comprise T cells specific to two or more immunogenic antigens or neoantigen peptides. In some embodiments, one or more additional therapies may be used to supplement the T-cell-specific therapy.

[0322] In some embodiments, the pharmaceutical composition comprises: a nucleic acid encoding a TCR targeting a neoantigen disclosed herein, a vector containing the nucleic acid, a protein encoded by the nucleic acid, or a host cell containing the nucleic acid, protein, or vector; and a pharmaceutically acceptable excipient or diluent. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the immunomodulator is a cytokine. In some embodiments, the adjuvant is poly(I:C).

[0323] This article also provides information on the use of the pharmaceutical composition in the treatment of immune diseases or cancer.

[0324] In addition to the active ingredient, the pharmaceutical composition may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration. Acceptable carriers, excipients, or stabilizers are those that are non-toxic to the recipient at the dose and concentration used, and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as... Or polyethylene glycol (PEG).

[0325] An acceptable carrier is physiologically acceptable to the patient to whom it is administered and maintains the therapeutic properties of the administered compound. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences (18th edition, A. Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting the subject compound from one site of administration to another, or in an in vitro assay system. An acceptable carrier is compatible with the other components of the formulation and is harmless to the subject to which it is administered. An acceptable carrier should also not alter the specific activity of other components.

[0326] In one respect, this article provides pharmaceutically acceptable or physiologically acceptable compositions comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating materials, isotonic agents, and absorption enhancers or delay agents compatible with drug administration. Therefore, a pharmaceutical composition or pharmaceutical formulation refers to a composition suitable for use as a drug in a subject. Compositions may be formulated to be compatible with a specific route of administration (i.e., systemic or local). Therefore, compositions comprise carriers, diluents, or excipients suitable for administration via various routes.

[0327] In some embodiments, the pharmaceutical composition may further comprise acceptable additives to improve the stability of the composition. Acceptable additives may not alter the specific activity of the active agent, such as immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbitol, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. Acceptable additives may be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants used to increase the stability of the peptide and reduce gelation of the solution, such as polysorbate 20 or polysorbate 80. Surfactants may be added to the composition in amounts from 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0328] In some embodiments, the pharmaceutical composition comprises a therapeutic agent, which is a T cell expressing one or more polynucleotides encoding a T cell receptor. In some embodiments, the pharmaceutical composition comprises a physiologically acceptable carrier suitable for cell suspension.

[0329] Pharmaceutical compositions can be administered, for example, by injection. Pharmaceutical compositions for injection include aqueous solutions (which are water-soluble) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof. For example, in the case of dispersions, flowability can be maintained by using a coating such as lecithin, by maintaining the desired particle size, and by using surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyols like mannitol, sorbitol, and sodium chloride, may be included in the composition. The resulting solution can be packaged for use as is or lyophilized; the lyophilized product can then be combined with a sterile solution prior to administration. For intravenous or site-specific injection, the active ingredient will be in the form of a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. A sterile injectable solution can be prepared by incorporating the desired amount of the active ingredient, along with one or a combination of the ingredients listed above (if desired), into a suitable solvent, followed by sterile filtration. Typically, a dispersion is prepared by incorporating the active ingredient into a sterile medium containing an alkaline dispersion medium and any other desired ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterilized filtered solution.

[0330] For example, the pharmaceutical composition can be routinely administered intravenously, such as by injecting a unit dose. For injection, the active ingredient can be in the form of a parenteral-acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity, and stability. Suitable solutions can be prepared using, for example, isotonic media such as sodium chloride injection, Ringer's solution, lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Alternatively, the composition can be administered via nebulization.

[0331] When considering the use of a pharmaceutical composition in a drug product or any of the methods described herein, it is anticipated that the composition will be substantially pyrogen-free, thereby preventing inflammatory or unsafe allergic reactions when administered to human patients. Testing for pyrogens in a composition and preparing substantially pyrogen-free compositions are well known to those skilled in the art and can be accomplished using commercially available kits.

[0332] Acceptable carriers may contain compounds that stabilize, increase, or delay absorption, or that increase or delay clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions, including liposome carriers. To prevent digestion, compounds may be conjugated to the composition to make it resistant to acid and enzymatic hydrolysis, or compounds may be conjugated in a suitable resistant carrier such as liposomes. Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and U.S. Patent 5,391,377).

[0333] Pharmaceutical compositions can be administered in a manner compatible with dosage forms and at a therapeutically effective amount. The amount to be administered depends on the subject being treated, the subject's immune system's ability to utilize the active ingredient, and the desired degree of binding capacity. The precise amount of active ingredient to be administered depends on the practitioner's judgment and is specific to each individual. Appropriate protocols for initial administration and booster injections also differ, but typically involve an initial administration followed by repeated administration at intervals of one or more hours via subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations can be considered.

[0334] In some embodiments, this disclosure relates to an immunogenic composition, such as a pharmaceutical composition capable of eliciting a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent described herein (e.g., peptide, polynucleotide, TCR, CAR, TCR- or CAR-containing cells, peptide-containing dendritic cells, polynucleotide-containing dendritic cells, antibody, etc.) corresponding to a tumor-specific antigen or neoantigen.

[0335] In some embodiments, the pharmaceutical compositions described herein can induce specific cytotoxic T cell responses, specific helper T cell responses, or B cell responses.

[0336] In some embodiments, the antigenic peptide or polynucleotide may be provided as an antigen-presenting cell (e.g., dendritic cells) containing such a peptide or polynucleotide. In other embodiments, such antigen-presenting cells are used to stimulate T cells for a patient. In some embodiments, the antigen-presenting cell is a dendritic cell. In relevant embodiments, the dendritic cell is an autologous dendritic cell pulsed with a neoantigen peptide or nucleic acid. The neoantigen peptide may be any suitable peptide that produces an appropriate T cell response. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is an HTL. Thus, one embodiment of this disclosure is an immunogenic composition containing at least one antigen-presenting cell (e.g., dendritic cells) pulsed or loaded with one or more of the neoantigen peptides or polynucleotides described herein. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient may be loaded with neoantigen peptides or polynucleotides ex vivo. In relevant embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide can be any suitable polynucleotide capable of transducing dendritic cells, thereby leading to the presentation of neoantigen peptides and the induction of immunity. In some embodiments, such antigen-presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells or allogeneic T cells). In relevant embodiments, the T cell is a CTL. In other relevant embodiments, the T cell is an HTL. In some embodiments, the T cell is a CD8+ TTL. + T cells. In some implementations, these T cells are CD4+ cells. + T cells. These T cells are then injected into the patient. In some embodiments, CTLs are injected into the patient. In some embodiments, HTLs are injected into the patient. In some embodiments, both CTLs and HTLs are injected into the patient. The administration of any therapeutic agent can be simultaneous or sequential and in any order.

[0337] In some embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment may be formulated for parenteral, topical, nasal, oral, or external administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the composition may be administered intratumorally. The composition may be administered at the surgical resection site to induce a local immune response against the tumor. In some embodiments, compositions for parenteral administration are described herein comprising a solution of a neoantigenic peptide, and the immunogenic composition is dissolved or suspended in an acceptable carrier, such as an aqueous carrier. Various aqueous carriers may be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions may be sterilized using conventional, known sterilization techniques or by filtration. The resulting aqueous solution may be packaged for use as is or lyophilized, with the lyophilized product combined with the sterile solution prior to administration. The composition may contain pharmaceutically acceptable adjuvants required to approximate physiological conditions, such as pH adjusters and buffers, tension regulators, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0338] The ability of adjuvants to enhance the immune response to antigens typically manifests as a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity can be reflected in a significant increase in antibody titers against antigens, and an increase in T cell activity can be reflected in increased cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also alter immune responses, for example, by changing a primarily humoral or type 2 T helper cell response to a primarily cellular or type 1 T helper cell response.

[0339] Suitable adjuvants are known in the art (see WO2015 / 095811) and include, but are not limited to, poly(I:C), poly-ICLC, STING agonists, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCMATRIX, JuvImmune, LipoVac, MF59, monophospholipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, Vector systems, PLG microparticles, remiquimod, SRL172, viromas and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, saponin-derived Aquila QS21 stimulant (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several dendritic cell-specific immune adjuvants have been described (Dupuis M et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev BiolStand. 1998; 92:3-11)(Mosca et al., Frontiers in Bioscience, 2007; 12:4050-4060)(Gamvrellis et al., Immunol & Cell Biol. 2004; 82:506-516). Cytokines may also be used. Several cytokines are directly associated with influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into effective antigen-presenting cells (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1β, IL-4, IL-6, and CD40L) (US Patent 5,849,589, which is incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0340] The effects of CpG immunostimulatory oligonucleotides on adjuvants in a therapeutic setting have also been reported. Unbound by theory, CpG oligonucleotides function by activating the innate (adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic drug compositions, autologous cell immunogenic drug compositions, and polysaccharide conjugates of both prophylactic and therapeutic immunogenic drug compositions. Importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and the generation of highly cytotoxic T lymphocytes (CTLs), even in the absence of CD4+ T cell assistance. Even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IFA), which typically promote TH2 bias, the TLR9-induced TH1 bias is maintained. When formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, CpG oligonucleotides exhibit even greater adjuvant activity, which is particularly useful for inducing strong responses when the antigen is relatively weak. They can also accelerate immune responses and allow for reduced antigen doses, showing comparable antibody responses in some studies to full-dose immunogenic drug compositions without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). U.S. Patent 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. One commercially available CpG TLR9 antagonist is dSLIM (a dual-stem-ring immunomodulator) from Mologen (Berlin, DE), which is a component of the drug compositions described herein. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[0341] Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), poly(I:C) (e.g., polyI:CI2U), polyIC:LC, non-CpG bacterial DNA or RNA, ssRNA40 targeting TLR8, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, trimemumab, and SC58175, which can act therapeutically and / or as adjuvants. The amount and concentration of adjuvants and additives that can be used in the context of this invention can be readily determined by those skilled in the art without requiring extensive experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, saxaglastine).

[0342] In some embodiments, the immunogenic composition according to this disclosure may contain more than one different adjuvant. Furthermore, the invention includes pharmaceutical compositions containing any adjuvant substance, including any of the adjuvants described above and combinations thereof. In some embodiments, the immunogenic composition contains a neoantigen therapeutic agent (e.g., peptide, polynucleotide, TCR, CAR, TCR- or CAR-containing cells, polypeptide-containing dendritic cells, polynucleotide-containing dendritic cells, antibodies, etc.), and the adjuvants may be administered alone in any suitable order.

[0343] Lipidification can be categorized into several different types, such as N-myristoylation, palmitoylation, GPI-anchoring, isopentenylation, and several other types of modification. N-myristoylation is the covalent attachment of a myristate ester (C14 saturated acid) to a glycine residue. Palmitoylation is the thioester linkage of a long-chain fatty acid (C16) to a cysteine ​​residue. GPI-anchoring is the linkage via an amide bond to glycosyl-phosphatidylinositol (GPI). Isopentenylation is the thioether linkage of an isoprene-like lipid (e.g., farnesyl (C-15), geraniol (C-20)) to a cysteine ​​residue. Other types of modification may include S-diacylglycerol attachment via the sulfur atom of cysteine, O-octanoyl conjugation via serine or threonine residues, S-archaeol conjugation to cysteine ​​residues, and cholesterol attachment.

[0344] Fatty acids used to generate lipotropic peptides may include C2 to C30 saturated, monounsaturated, or polyunsaturated fatty acyl groups. Exemplary fatty acids may include palmitoyl, myristoyl, stearoyl, and decanoyl. In some cases, a lipid moiety with adjuvant properties is attached to the target peptide to initiate or enhance immunogenicity in the absence of an exogenous adjuvant. Lipotropic peptides or lipopeptides may be referred to as self-adjuvanted lipopeptides. Any fatty acid described above and elsewhere herein may initiate or enhance the immunogenicity of the target peptide. Fatty acids that may initiate or enhance immunogenicity may include palmitoyl, myristoyl, stearoyl, lauroyl, capryloyl, and decanoyl.

[0345] Peptides such as naked peptides or liposomes can be incorporated into liposomes. Sometimes, liposomes can be incorporated into liposomes. For example, the lipid portion of a liposome can spontaneously integrate into the lipid bilayer of a liposome. Therefore, lipopeptides can be presented on the "surface" of the liposome.

[0346] Liposomes can also be used to deliver nucleic acids into cells. The target nucleic acid contains one or more sequences encoding T cell receptors. Liposomes can be used to deliver DNA or RNA. Liposomes can be used to deliver nucleic acids incorporated into a vector. The nucleic acid can be 50-200,000 nucleotides long, or 100-500,000 nucleotides long, or 20-500,000 nucleotides long. Exemplary liposomes suitable for incorporation into formulations include, but are not limited to, multilayer vesicles (MLV), oligolayer vesicles (OLV), monolayer vesicles (UV), small monolayer vesicles (SUV), medium-sized monolayer vesicles (MUV), large monolayer vesicles (LUV), giant monolayer vesicles (GUV), multivesicles (MVV), monolayer or oligolayer vesicles prepared by reverse-phase evaporation (REV), multilayer vesicles prepared by reverse-phase evaporation (MLV-REV), stable multilayer vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion (VET), vesicles prepared by Freund's crusher (FPV), vesicles prepared by fusion (FUV), dehydrated-rehydrated vesicles (DRV), and foam bodies (BSV).

[0347] Depending on the preparation method, liposomes can be monolayered or multilayered and can vary in size, ranging from about 0.02 μm to greater than about 10 μm in diameter. Liposomes can adsorb onto many types of cells and then release incorporated reagents (e.g., peptides described herein). In some cases, liposomes fuse with target cells, thereby subsequently emptying the contents of the liposome into the target cell. Liposomes can be endocytosed by phagocytes. Endocytosis can be followed by lysosomal degradation of liposomal lipids and release of the encapsulating agent.

[0348] The liposomes provided herein may also include carrier lipids. In some embodiments, the carrier lipid is a phospholipid. Carrier lipids capable of forming liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Other suitable phospholipids include distearylphosphatidylcholine (DSPC), myristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), myristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPA); myristoylphosphatidylglycerol (DMPA), distearylphosphatidylphosphatidylglycerol (DSPA), dipalmitoylphosphatidylserine (DPPS), myristoylphosphatidylserine (DMPS), distearylphosphatidylserine (DSPS), dipalmitoylphosphatidylethanolamine (DPPE), myristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and combinations thereof. In some embodiments, the liposomes also contain sterols (e.g., cholesterol) that regulate liposome formation. The carrier lipid can be any known non-phosphorylated polar lipid.

[0349] Known techniques can be used to encapsulate pharmaceutical compositions within liposomes. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of this invention.

[0350] The pharmaceutical composition can be administered in liposomes or microspheres (or particles). Methods for preparing liposomes and microspheres for administration to patients are known to those skilled in the art. Essentially, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids and surfactants (if desired) are added, and the material is dialyzed or sonicated as needed.

[0351] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be adapted for direct entry into the bloodstream via the gastrointestinal tract. Alternatively, compounds can be incorporated into and implanted into microspheres or microsphere complexes for slow release over days to months.

[0352] Cell-based immunogenic pharmaceutical compositions can also be administered to subjects. For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any known techniques, carriers, and excipients as understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, APC-based immunogenic pharmaceutical compositions can be dendritic cell-based immunogenic pharmaceutical compositions.

[0353] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared by in vitro or in vivo methods. In vitro methods may include using autologous dendritic cells (DCs) pulsed with the peptide described herein in vitro to activate or load DCs prior to administration to a patient. In vivo methods may include using an antibody conjugated to the peptide described herein to target a specific DC receptor. DC-based immunogenic pharmaceutical compositions may further comprise DC activators, such as TLR3, TLR-7-8, and CD40 agonists. DC-based immunogenic pharmaceutical compositions may further comprise adjuvants and pharmaceutically acceptable carriers.

[0354] Adjuvants can be used to enhance the immune response (humoral and / or cellular response) induced in patients receiving immunogenic drug compositions. Sometimes, adjuvants induce a Th1 response. At other times, they induce a Th2 response. A Th1 response is characterized by the production of cytokines such as IFN-γ, while a Th2 response is characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0355] In some respects, lipid-based adjuvants, such as MPLA and MDP, can be used with the immunogenic pharmaceutical compositions disclosed herein. For example, monophospholipid A (MPLA) is an adjuvant that induces increased presentation of liposomal antigens to specific T lymphocytes. Additionally, muramyl dipeptide (MDP) can also be used as a suitable adjuvant with the immunogenic pharmaceutical formulations described herein.

[0356] Adjuvants may also contain stimulating molecules, such as cytokines. Non-limiting examples of cytokines include: CCL20, α-interferon (IFNα), β-interferon (IFNβ), γ-interferon (IFNγ), platelet-derived growth factor (PDGF), TNFα, GM-CSF, epidermal growth factor (EGF), skin T-cell attraction chemokine (CTACK), epidermal thymus expression chemokine (TECK), mucosa-associated epithelial cell chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA. -1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, cysteine ​​ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-I, JNK, interferon response gene, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

[0357] Other adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor, and fibroblast growth factor. IL-7, IL-22, Nerve Growth Factor, Vascular Endothelial Growth Factor, Fas, TNF Receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, Killer, Trail-R2, Trick2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, Inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0358] In some respects, adjuvants can be modulators of Toll-like receptors. Examples of Toll-like receptor modulators include TLR9 agonists, and are not limited to small molecule modulators of Toll-like receptors, such as imiquimod. Sometimes, adjuvants are selected from bacterial toxoids, polyoxypropylene-polyethylene glycol block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Sometimes, the adjuvant is an oil-in-water emulsion. An oil-in-water emulsion may contain at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion may have a diameter of less than 5 μm, or even submicron diameters; these small sizes are achieved via microfluidics to provide a stable emulsion. Droplets smaller than 220 nm can be used for filtration sterilization.

[0359] In some cases, immunogenic pharmaceutical compositions may include carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and / or preservatives), water, oils (including petroleum, animal, plant or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), salt solutions, aqueous solutions of dextran and glycerol, flavoring agents, coloring agents and other acceptable additives, adjuvants or binders, and other pharmaceutically acceptable excipients required to achieve approximate physiological conditions, such as pH buffers, tension regulators, emulsifiers, wetting agents, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. In another case, the pharmaceutical product is substantially preservative-free. In other cases, the pharmaceutical product may contain at least one preservative. It should be recognized that while any suitable carrier known to those skilled in the art may be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the mode of administration.

[0360] Immunogenic pharmaceutical compositions may include preservatives such as thimerosal or 2-phenoxyethanol. In some cases, immunogenic pharmaceutical compositions are substantially free of mercury (e.g., <10 μg / mL), for example, free of thimerosal. α-Tocopherol succinate can be used as a substitute for mercury compounds.

[0361] To control tension, immunogenic drug compositions may contain physiological salts, such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride.

[0362] The immunogenic pharmaceutical composition may have a weight molar osmolality of 200 mOsm / kg to 400 mOsm / kg, 240-360 mOsm / kg or 290-310 mOsm / kg.

[0363] Immunogenic drug compositions may contain one or more buffer solutions, such as Tris buffer; borate buffer; succinate buffer; histidine buffer (especially containing aluminum hydroxide adjuvant); or citrate buffer. In some cases, the buffer solution is contained in 5-20 or 10-50 mM.

[0364] The pH of the immunogenic pharmaceutical composition may be from about 5.0 to about 8.5, from about 6.0 to about 8.0, from about 6.5 to about 7.5, or from about 7.0 to about 7.8.

[0365] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be pyrogen-free, for example, containing <1 EU (endotoxin unit, standard measure) per dose, and may be <0.1 EU per dose. The composition may be gluten-free.

[0366] Immunogenic pharmaceutical compositions may contain detergents, such as polyoxyethylene sorbitan ester surfactants (known as "Tween") or poly(oxyethylene styrene) alcohols (such as poly(oxyethylene styrene) alcohol-9 (Triton X-100) or tert-octylphenoxypolyethoxyethanol). The detergent may be present only in trace amounts. Immunogenic pharmaceutical compositions may contain less than 1 mg / mL of each of poly(oxyethylene styrene) alcohol-10 and polysorbate 80. Other trace residual components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0367] Immunogenic pharmaceutical compositions can be formulated into sterile solutions or suspensions in suitable media known in the art. The pharmaceutical compositions can be sterilized using conventional, known sterilization techniques, or by filtration. The resulting aqueous solution can be packaged for use as is, or lyophilized, with the lyophilized product combined with the sterile solution prior to administration.

[0368] For example, a pharmaceutical composition comprising an active agent (such as the immune cells disclosed herein) can be formulated with one or more adjuvants to have a specific molar ratio. For instance, a molar ratio of approximately 99:1 to approximately 1:99 may be used between the active agent, the immune cells as described herein, and one or more adjuvants. In some cases, the range of the molar ratio of the active agent, the immune cells as described herein, and one or more adjuvants may be selected from approximately 80:20 to approximately 20:80; approximately 75:25 to approximately 25:75; approximately 70:30 to approximately 30:70; approximately 66:33 to approximately 33:66; approximately 60:40 to approximately 40:60; approximately 50:50; and approximately 90:10 to approximately 10:90. The molar ratio of the active agent, the immune cells as described herein, to one or more adjuvants may be approximately 1:9, and in some cases, it may be approximately 1:1. Active agents, such as immune cells as described herein, can be combined with one or more adjuvants and formulated together in the same dosage unit, such as a vial, suppository, tablet, capsule, or aerosol spray; or each agent, form, and / or compound can be formulated in a separate unit, such as two vials, two suppositories, two tablets, two capsules, one tablet and one vial, or aerosol spray.

[0369] In some cases, immunogenic pharmaceutical compositions may be administered in conjunction with adjuvant agents. The choice of adjuvant agents may depend at least in part on the condition being treated. Adjuvant agents may include, for example, checkpoint inhibitors such as anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agents (e.g., anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibodies); or any agent that is effective against pathogen infection (e.g., viral infection), including, for example, drugs used to treat inflammatory conditions such as NSAIDs, such as ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, checkpoint inhibitors can be PD-1 / PD-L1 agonists selected from: nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulation may additionally contain one or more supplements, such as vitamin C, E, or other antioxidants.

[0370] Pharmaceutical compositions comprising an active agent, such as immune cells as described herein, combined with one or more adjuvants, can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients, diluents, and / or adjuvants, such as carriers that facilitate the processing of the active agent into an administerable formulation. Suitable formulations may depend at least in part on the chosen route of administration. The pharmaceutical agents described herein can be delivered to patients using a variety of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular administration, as well as inhalation.

[0371] The active agent can be formulated for parenteral administration (e.g., by injection, such as bolus or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small-volume infusions, or in multi-dose containers with added preservatives. The composition can be in the form of suspensions, solutions, or emulsions in oily or aqueous media, such as solutions in aqueous polyethylene glycol.

[0372] For injectable formulations, the carrier can be selected from those suitable in the art, including aqueous solutions or oily suspensions or emulsions (containing sesame oil, corn oil, cottonseed oil, or peanut oil), as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical carriers. The formulation may also contain biocompatible, biodegradable polymer compositions, such as poly(lactic acid-co-glycolic acid). These materials can be formed into microspheres or nanospheres, loaded with the drug, and further encapsulated or derivatized to provide excellent sustained release properties. Carriers suitable for periocular or intraocular injection include, for example, suspensions of the therapeutic agent in injectable water, liposomes, and carriers suitable for lipophilic substances. Other carriers for periocular or intraocular injection are well known in the art.

[0373] In some cases, pharmaceutical compositions are formulated according to standard procedures to be suitable for intravenous administration to the human body. Generally, compositions for intravenous administration are solutions in sterile isotonic buffer solutions. When necessary, the composition may also contain solubilizers and local anesthetics such as lidocaine to reduce pain at the injection site. Typically, the components are supplied separately or mixed together in unit dosage forms, for example, as lyophilized powders or anhydrous concentrates in sealed containers such as ampoules or sachets indicating the amount of active agent. When administered by infusion, the composition can be dispensed using infusion bottles containing sterile pharmaceutical-grade water or saline. When administered by injection, ampoules containing sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0374] When administered by injection, the active agent can be formulated in an aqueous solution, particularly in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or saline buffer. This solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. In another embodiment, the pharmaceutical composition does not contain adjuvants or any other substances added to enhance the immune response.

[0375] In addition to the formulations previously described, active agents can also be formulated as depot products. Such long-acting formulations can be administered via implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or the use of transdermal patches. Thus, for example, the agent can be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or formulated as a slightly soluble derivative, such as a slightly soluble salt.

[0376] In some cases, pharmaceutical compositions containing one or more agents exert local and regional effects when applied topically or injected at or near a specific site of infection. For example, direct topical application of viscous liquids, solutions, suspensions, dimethyl sulfoxide (DMSO)-based solutions, liposomes, gels, jelly, creams, lotions, ointments, suppositories, foams, or aerosols can be used to produce, for example, local and / or regional effects. Pharmaceutically suitable mediators for such formulations include, for example, lower aliphatic alcohols, polyethylene glycols (e.g., glycerin or polyethylene glycol), fatty acid esters, oils, fats, siloxanes, etc. Such articles may also contain preservatives (e.g., parabens) and / or antioxidants (e.g., ascorbic acid and tocopherol). See also Dermatological Formulations: Percutaneous absorption, Barry (Ed.), Marcel Dekker Incl, 1983. In another embodiment, a topical / external formulation containing a transporter, carrier, or ion channel inhibitor is used to treat epidermal or mucosal viral infections.

[0377] Pharmaceutical compositions may contain adjuvants, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, sunscreens, odor absorbers, and dyes. The amounts of these various adjuvants are those conventionally used in the field under consideration, and are, for example, from about 0.01% to about 20% of the total weight of the composition. Depending on their properties, these adjuvants may be incorporated into the fatty phase, aqueous phase, and / or lipid vesicles.

[0378] XII. Treatment

[0379] This article provides methods for preparing drugs for treating immune diseases or cancer using any of the nucleic acids disclosed above, vectors containing any of the nucleic acid sequences disclosed above, proteins encoded by any of the nucleic acids disclosed above, or the host cells disclosed above.

[0380] This document also provides methods for treating a subject suffering from a disease, condition, or illness. Treatment methods may include administering the disclosed pharmaceutical composition to a subject suffering from a disease, condition, or illness. This disclosure provides treatment methods including immunogenic therapy. Methods for treating diseases (such as cancer or viral infections) are provided. Methods may include administering to a subject an effective amount of a pharmaceutical composition comprising immunogenic antigen-specific T cells. In some embodiments, the antigen includes a tumor antigen.

[0381] In some embodiments, a method of treating a subject with a disease or condition includes administering the pharmaceutical composition disclosed herein to the subject. In some embodiments, the method is a method of preventing resistance to cancer therapies, wherein the method includes administering the pharmaceutical composition disclosed herein to a subject in need. In some embodiments, the method is a method of inducing an immune response, wherein the method includes administering the pharmaceutical composition disclosed herein to a subject in need. In some embodiments, the immune response is a humoral response. In some embodiments, the immune response is a cytotoxic T-cell response.

[0382] In some implementations, the subject has cancer, selected from melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL).

[0383] In some embodiments, the subject has breast cancer resistant to anti-estrogen therapy. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has CLL resistant to ibrutinib therapy. In some embodiments, the CLL expresses Bruton's tyrosine kinase with a mutation such as C481S. In some embodiments, the subject has lung cancer resistant to tyrosine kinase inhibitors. In some embodiments, the lung cancer expresses epidermal growth factor receptor (EGFR) with a mutation such as T790M, L792F, or C797S.

[0384] In some embodiments, the method further includes administering at least one additional therapeutic agent or treatment modality. In some embodiments, the at least one additional therapeutic agent or treatment modality is surgery, checkpoint inhibitors, antibodies or fragments thereof, chemotherapeutic agents, radiation, vaccines, small molecules, T cells, vectors and APCs, polynucleotides, oncolytic viruses, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, the additional therapeutic agent is administered before, simultaneously with, or after the administration of the pharmaceutical composition disclosed herein.

[0385] In some other aspects, this document provides for the use of pharmaceutical compositions in the preparation of medicaments for treatment. In some embodiments, the treatment method includes administering to a subject an effective amount of T cells that specifically recognize an immunogenic neoantigen peptide. In some embodiments, the treatment method includes administering to a subject an effective amount of a TCR that specifically recognizes an immunogenic neoantigen peptide, such as a TCR expressed in T cells.

[0386] In some implementations, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, colorectal cancer, rectal cancer, soft tissue sarcoma, Kaposi's sarcoma, B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma) NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunogenic NHL, high-grade lymphocytic NHL, high-grade small non-lytic cell NHL, large mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with nevus hamartomatosis, edema, Meigs syndrome, and combinations thereof.

[0387] The methods disclosed herein can be used to treat any type of cancer known in the art. Non-limiting examples of cancers treated by the methods disclosed herein may include melanoma (e.g., metastatic malignant melanoma), renal cell carcinoma (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

[0388] Additionally, the diseases or conditions described herein include refractory or recurrent malignant tumors whose growth can be suppressed using the treatment methods of the present invention. In some embodiments, the cancers treated by the treatment methods of the present disclosure are selected from carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the genital region, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematologic malignancies, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, cancers treated by the methods of this disclosure include, for example, carcinomas, squamous cell carcinomas (e.g., cancers of the cervix, eyelids, conjunctiva, vagina, lungs, oral cavity, skin, bladder, tongue, larynx, and esophagus) and adenocarcinomas (e.g., cancers of the prostate, small intestine, endometrium, cervix, large intestine, lungs, pancreas, esophagus, rectum, uterus, stomach, breast, and ovaries). In some embodiments, cancers to be treated by the methods of this disclosure further include sarcomas (e.g., myogenic sarcoma), leukemia, neuroma, melanoma, and lymphoma. In some embodiments, the cancer to be treated by the methods of this disclosure is breast cancer. In some embodiments, the cancer to be treated by the methods of this disclosure is triple-negative breast cancer (TNBC). In some embodiments, the cancer to be treated by the methods of this disclosure is ovarian cancer. In some embodiments, the cancer to be treated by the methods of this disclosure is colorectal cancer.

[0389] In some embodiments, the patient or patient group to be treated with the pharmaceutical composition of this disclosure has a solid tumor. In some embodiments, the solid tumor is melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer, thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma. In some embodiments, the patient or patient group to be treated with the pharmaceutical composition of this disclosure has a hematologic malignancy. In some embodiments, the patient has a hematologic malignancy such as diffuse large B-cell lymphoma (“DLBCL”), Hodgkin lymphoma (“HL”), non-Hodgkin lymphoma (“NHL”), follicular lymphoma (“FL”), acute myeloid leukemia (“AML”), or multiple myeloma (“MM”). In some embodiments, the cancer of the patient or patient group to be treated is selected from ovarian cancer, lung cancer, and melanoma.

[0390] Specific examples of cancers that can be prevented and / or treated according to this disclosure include, but are not limited to, the following: renal cell carcinoma, kidney cancer, glioblastoma multiforme, metastatic breast cancer; breast cancer; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; epidermal cancer; leukemia, such as but not limited to acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia, and myelodysplastic syndromes; chronic leukemia, such as but not limited to chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphoma, such as but not limited to Hodgkin's lymphoma. Diseases, non-Hodgkin's disease; multiple myeloma, such as but not limited to accumulating multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenström macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcoma, such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced osteosarcoma, Paget's bone disease, osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, nerve sheath Tumors, rhabdomyosarcomas, and synovial sarcomas; brain tumors, such as but not limited to gliomas, astrocytomas, brainstem gliomas, ependymomas, oligodendrogliomas, non-glial tumors, acoustic neuromas, craniopharyngiomas, medulloblastomas, meningiomas, pineal cell tumors, pinealoblastomas, and primary brain lymphomas; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, ductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease), and inflammatory breast cancer; adrenal cancer, such as but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as but not limited to papillary or follicular thyroid carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma; pancreatic cancer, Examples include, but are not limited to, insulinomas, gastrinomas, glucagonomas, VIP tumors, tumors that secrete somatostatin, and carcinoid or islet cell tumors; pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers, such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; and ovarian cancers, such as, but not limited to, ovarian epithelial carcinoma, junctional tumor, germ cell tumor, and stromal tumor.Esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, including but not limited to adenocarcinoma, mycosis fungoides, ulcerative, superficial, diffuse, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; colorectal cancer; KRAS-mutant colorectal cancer; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, such as adenocarcinoma; bile duct cancer, including but not limited to papillary, nodular, and diffuse; lung cancer, including KRAS-mutant non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; lung cancer; testicular cancer, including but not limited to germ cell tumors, seminoma, anaplastic, classic (typical), and seminoma. Non-seminomatous tumors, embryonal carcinomas, teratomas, choriocarcinomas (yolk sac tumors), prostate cancer, including but not limited to androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; pineal gland cancer; oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial diffuse melanoma, nodular melanoma, nevus-like malignant melanoma, acral lentiginous melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); kidney cancer; Wilms' tumor; bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. In addition, the cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangiosarcoma, mesothelioma, synovoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma.

[0391] Cancers include, but are not limited to, B-cell cancers, such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases (e.g., alpha chain disease, gamma chain disease, and μ chain disease), benign monoclonal gammopathy and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-resistant prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, cancers of the brain or central nervous system, cancers of the peripheral nervous system, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancers of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and cancers of blood tissues. Other non-limiting examples of cancer types suitable for the methods covered in this disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, and Ewing's tumor. Tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland. Somatic tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas; leukemias, such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's and non-Hodgkin's), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the methods of this disclosure is epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In other embodiments, the epithelial carcinoma is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial carcinoma can be characterized in various other ways, including but not limited to serou...

Claims

1. A recombinant nucleic acid encoding a T cell receptor (TCR), the T cell receptor comprising a TCR β chain construct and a TCR α chain construct, the TCR β chain construct comprising a β chain CDR1, a β chain CDR2 and a β chain CDR3, the TCR α chain construct comprising an α chain CDR1, an α chain CDR2 and an α chain CDR3, wherein the β chain CDR1 consists of the amino acid sequence as shown in SEQ ID NO: 50, the β chain CDR2 consists of the amino acid sequence as shown in SEQ ID NO: 51, and the β chain CDR3 consists of the amino acid sequence as shown in SEQ ID NO: 52; the α chain CDR1 consists of the amino acid sequence as shown in SEQ ID NO: 47, the α chain CDR2 consists of the amino acid sequence as shown in SEQ ID NO: 48, and the α chain CDR3 consists of the amino acid sequence as shown in SEQ ID NO: 49, wherein the TCR specifically binds to a mutated RAS epitope complexed with a human MHC encoded by an HLA-A11:01 allele, wherein the mutated RAS epitope is VVGAVGVGK or VVVGAVGVGK.

2. The recombinant nucleic acid according to claim 1, wherein the TCR β chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

58.

3. The recombinant nucleic acid according to claim 2, wherein the TCR β chain construct comprises a variable region having an amino acid sequence shown in SEQ ID NO:

58.

4. The recombinant nucleic acid of claim 1, wherein the TCR α chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

55.

5. The recombinant nucleic acid according to claim 4, wherein the TCR α chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

55.

6. The recombinant nucleic acid according to claim 1, wherein the TCRβ chain construct comprises: an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:60; and the TCRα chain construct comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

59.

7. The recombinant nucleic acid according to claim 1, wherein the TCR comprises a TCRβ chain construct having the amino acid sequence shown in SEQ ID NO: 60, and a TCRα chain construct having the amino acid sequence shown in SEQ ID NO:

59. The recombinant nucleic acid of claim 1 , wherein the recombinant nucleic acid is operably linked to a promoter.

9. A vector comprising the recombinant nucleic acid according to any one of claims 1 to 8.

10. A cell comprising the recombinant nucleic acid of any one of claims 1 to 8 or the vector of claim 9.

11. The cell of claim 10, wherein the cell is a CD4+ T cell.

12. A pharmaceutical composition comprising: (a) the cell of claim 10; and (b) pharmaceutically acceptable excipients.

13. Use of the recombinant nucleic acid according to any one of claims 1 to 8, the vector according to claim 9, the cell according to any one of claims 10 to 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating an immune disease or cancer.

14. The method of claim 13, wherein the cancer is selected from the group consisting of biliary adenocarcinoma, bladder transitional cell carcinoma, cervical adenocarcinoma, colon adenocarcinoma, colon adenoma, neuroblastoma, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, plasma cell myeloma, hepatocellular carcinoma, large cell carcinoma, ductal carcinoma, prostate adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, angiosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, myxoma, malignant fibrous histiocytoma, pleomorphic sarcoma, germ cell tumor, and Hurthle cell carcinoma.

15. The use according to claim 13, wherein the cancer is seminoma.

16. The use according to claim 13, wherein the cancer is an endocrine tumor.

Images