T cell receptor constructs and uses thereof

Abstract

The present disclosure provides T cell receptors (TCRs) for peptide-MHC complexes, isolated nucleic acid molecules encoding TCRs for peptide-MHC complexes, T cells expressing TCRs for peptide-MHC complexes, and pharmaceutical compositions for use in the treatment of disease.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 387,148, filed December 13, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] T cell receptors (TCRs) are members of the immunoglobulin superfamily and typically consist of two subunits, the α- and β-subunits, which contain an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane / transmembrane region, and a short C-terminal cytoplasmic tail. The variable domains of the α- and β-chains of the TCR contain three hypervariable regions, or complementarity-determining regions (CDRs), while the variable region of the β-chain contains an additional region of hypervariability (HV4) that does not normally contact antigen and is therefore not considered a CDR.

[0003]

[0003] While CDR3 is the primary CDR involved in recognizing processed antigens, CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is thought to recognize MHC. The constant domain of the TCR domain consists of a short connective sequence in which cysteine ​​residues form disulfide bonds, forming the link between the two chains. The affinity of TCRs for specific antigens makes them valuable for several therapeutic approaches. For example, cancer patients, such as melanoma patients, can be effectively treated using adoptive immunotherapy because TCRs are highly sensitive to those antigens and can direct the immune response to tumor cells expressing the relevant antigen. Therefore, there is a need for TCRs to target peptide-MHC complexes for the development of novel and effective therapeutic agents. Summary of the Invention

[0004] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the TCR beta chain construct further comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 1, CDR2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the recombinant nucleic acid comprises (a) a sequence having at least 80% sequence identity to SEQ ID NO: 10 or 11, and (b) a sequence having at least 80% sequence identity to SEQ ID NO: 7 or 8. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 9.In some embodiments, the TCR comprises (a) a beta chain having the amino acid sequence set forth in SEQ ID NO: 14 or 70, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or 70, and (b) an alpha chain having the amino acid sequence set forth in SEQ ID NO: 13 or 69, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13 or 69. In some embodiments, the TCR binds to an epitope derived from human RAS comprising the mutation G12V. In some embodiments, the epitope derived from human RAS comprising the mutation G12V is SEQ ID NO: 43 or 44. In some embodiments, the TCR binds to a complex comprising (i) an epitope derived from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by the HLA-A11:01 allele.

[0005] Provided herein are recombinant nucleic acids encoding a T cell receptor (TCR), comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the TCR beta chain construct further comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence set forth in SEQ ID NO:19. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 15, CDR2 has the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid comprises (a) a sequence having at least 80% sequence identity to SEQ ID NO: 24 or 25, and (b) a sequence having at least 80% sequence identity to SEQ ID NO: 21 or 22. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23.In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the TCR comprises (a) a beta chain having the amino acid sequence set forth in SEQ ID NO: 28 or 72, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 28 or 72, and (b) an alpha chain having the amino acid sequence set forth in SEQ ID NO: 27 or 71, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 27 or 71. In some embodiments, the TCR binds to an epitope derived from human RAS comprising the mutation G12V. In some embodiments, the epitope derived from human RAS comprising the mutation G12V is SEQ ID NO: 45. In some embodiments, the TCR binds to a complex comprising (i) an epitope derived from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by the HLA-C01:02 allele.

[0006] Provided herein are recombinant nucleic acids encoding a T cell receptor (TCR), comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the TCR beta chain construct further comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 29, CDR2 has the amino acid sequence set forth in SEQ ID NO: 30, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the recombinant nucleic acid comprises (a) a sequence having at least 80% sequence identity to SEQ ID NO: 38 or 39, and (b) a sequence having at least 80% sequence identity to SEQ ID NO: 35 or 36. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:37.In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the TCR comprises (a) a beta chain having the amino acid sequence set forth in SEQ ID NO: 41 or 74, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 41 or 74, and (b) an alpha chain having the amino acid sequence set forth in SEQ ID NO: 42 or 73, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 42 or 73. In some embodiments, the TCR binds to an epitope derived from human RAS comprising the mutation G12V. In some embodiments, the epitope derived from human RAS comprising the mutation G12V is SEQ ID NO: 45. In some embodiments, the TCR binds to a complex comprising (i) an epitope derived from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by the HLA-C01:02 allele.

[0007] In some embodiments, the recombinant nucleic acid is a vector. In some embodiments, the epitope has a length of 8 to 25 amino acids. In some embodiments, the epitope comprises a mutation that differs from the corresponding wild-type epitope by at least one amino acid. In some embodiments, the epitope binds to human MHC with greater affinity than the corresponding wild-type epitope. In some embodiments, the epitope binds to human MHC with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the mutation is not present in the subject's non-cancer cells. In some embodiments, the TCR binds to the MHC-peptide complex with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the recombinant nucleic acid is operably linked to a promoter.

[0008] Provided herein are cells comprising a recombinant nucleic acid described herein. In some embodiments, the cells are CD4+ T cells. In some embodiments, the cells are CD8+ T cells. In some embodiments, the cells are isolated from a subject with a RAS mutation.

[0009] Provided herein are pharmaceutical compositions comprising (a) a recombinant nucleic acid described herein or a cell described herein, and (b) a pharmaceutically acceptable excipient or diluent. In some embodiments, the pharmaceutical composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the adjuvant is poly I:C. In some embodiments, the pharmaceutical composition is for use in treating an immune disease or cancer.

[0010]

[0010] Provided herein is the use of the pharmaceutical compositions described herein for treating immune diseases or cancer.

[0011] Provided herein are methods of treating a subject having a disease or condition, comprising administering to the subject a pharmaceutical composition described herein.

[0011]

[0012] Provided herein are methods of treating a subject with cancer, comprising administering to the subject a pharmaceutical composition described herein.

[0013] Provided herein is a method for identifying a subject having cancer as a candidate for a therapeutic agent, the method comprising determining the subject as a subject that expresses a protein encoded by an HLA-A11:01 allele or an HLA C01:02 allele, wherein the therapeutic agent is a pharmaceutical composition described herein.

[0012]

[0014] Provided herein is a nucleic acid encoding a TCR, wherein the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12V and (ii) an MHC protein encoded by an HLA-A11:01 allele or an HLA C01:02 allele, and wherein complementarity-determining region 3 (CDR3) of the TCR does not comprise any of the sequences set forth in SEQ ID NOs: 46-68.

[0013]

[0015] Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs specifically bind to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-A68:01 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 82. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 80. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 77, CDR2 has the amino acid sequence set forth in SEQ ID NO: 78, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:83.In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the epitope derived from human RAS containing the mutation G12V is SEQ ID NO: 43 or 44.

[0014]

[0016] Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs specifically bind to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:03 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation. Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs specifically bind to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:04 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation. Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs specifically bind to a mutated epitope derived from human RAS in complex with human MHC encoded by an HLA-C03:03 allele and human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation. In some embodiments, the TCRs (i) bind to the mutated epitope derived from human RAS in complex with human MHC encoded by an HLA-C03:03 allele with a K of at most 500 nM, at most 250 nM, at most 50 nM, at most 25 nM, at most 10 nM, or at most 5 nM. D and (ii) a K of at most 500 nM, at most 250 nM, at most 50 nM, at most 25 nM, at most 10 nM, or at most 5 nM to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:04 allele. DIn some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 109. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 107. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 108. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 110.

[0015]

[0017] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 113. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 88. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 89. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 114.

[0016]

[0018] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 117. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 107. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 108. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 118.

[0017]

[0019] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 123. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 121. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 122. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the epitope derived from human RAS comprising the mutation G12V is SEQ ID NO: 133 or 134.

[0018]

[0020] Provided herein are recombinant nucleic acids encoding a T cell receptor (TCR), comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 127. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 128. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:29, CDR2 has the amino acid sequence set forth in SEQ ID NO:30, and CDR3 has the amino acid sequence set forth in SEQ ID NO:126. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:130. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:130. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:130. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:130. In some embodiments, the TCR binds to an epitope derived from human RAS containing the mutation G12D. In some embodiments, the epitope derived from human RAS containing the mutation G12D is SEQ ID NO:135.In some embodiments, the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12D and (ii) an MHC protein encoded by the HLA-A11:01 allele.

[0019]

[0021] Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs have a K of up to 1000 nM for a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C05:01 allele. D The mutated epitope from human RAS is characterized by a G12D mutation. Recombinant nucleic acids encoding T cell receptors (TCRs) including a TCR beta chain construct and a TCR alpha chain construct are provided herein, wherein the TCR specifically binds to the mutated epitope from human RAS in complex with human MHC encoded by the HLA-C05:01 allele with a K of 1000 nM or greater. Dand the mutated epitope derived from human RAS is characterized by a G12D mutation. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 90. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 88. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 89. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 85, CDR2 has the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 91.

[0020]

[0022] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 95. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 94. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 15, CDR2 has the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 96.

[0021]

[0023] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 101. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 94. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:98, CDR2 has the amino acid sequence set forth in SEQ ID NO:99, and CDR3 has the amino acid sequence set forth in SEQ ID NO:100. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:102. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:102. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:102. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:102. In some embodiments, the epitope derived from human RAS containing the mutation G12D is SEQ ID NO:132.

[0022]

[0024] In some embodiments, the TCR binds the mutated epitope with an EC50 It specifically binds to Incorporation by Reference

[0025] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0023]

[0026] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0024] [Figure 1]

[0027] The workflow for identifying and analyzing antigen-specific TCRs is illustrated. PBMCs from healthy donors can be stimulated with the antigen of interest, and antigen-specific T cells can then be identified using peptide-MHC multimers (left). Antigen-specific T cells can be isolated, and the TCRs can be sequenced and analyzed (middle left). TCRs can be synthesized and expressed in cell lines or PBMCs, and specificity can again be confirmed using peptide-MHC multimers (middle right). TCR-expressing cell lines or PBMCs can be co-cultured with antigen-expressing cell lines to confirm TCR functionality (right). [Figure 2]

[0028] Schematic example of antigen-specific CD8+ T cell expansion. PBMCs can be stimulated with an antigen of interest and cytokines. After expansion, antigen-specific CD8+ T cells can be identified by peptide-MHC multimers. [Figure 3A]

[0029] 1 is a schematic example of a recombinant TCR construct and vector design for expression of the TCR construct in cells. [Figure 3B]

[0030] 1 is a schematic example of a viral vector encoding a recombinant TCR for transduction or transfection into cells. [Figure 4A]

[0031] Illustrated is an example of flow cytometry analysis of RAS antigen-specific CD8+ T cell proliferation in response to stimulation with RAS peptide. [Figure 4B]

[0032] Illustrates an example of flow cytometry analysis of RAS antigen-specific Jurkat cells after sorting the top 10% multimer-positive cells expressing recombinant TCR after puromycin selection. [Figure 5]

[0033] Illustrated are examples of flow cytometry analysis of RAS peptide-HLA-A11:01 complex-specific CD8+ T cell proliferation in response to stimulation with RAS G12V peptide (left), RAS peptide-HLA-A11:01 complex-specific CD8+ T cell proliferation in response to stimulation with RAS G12C peptide (middle), and RAS peptide-HLA-A11:01 complex-specific CD8+ T cell proliferation in response to stimulation with RAS G12D peptide (right). [Figure 6]

[0034] Illustrated are examples of flow cytometry analysis of RAS peptide-HLA-A11:01 complex-specific CD8+ T cell proliferation in response to stimulation with G12V or G12C mutant RAS peptides (left), TCR clone abundance analysis after sequencing TCRs derived from pooled hits (middle), and confirmation of mutant vs. wild-type specificity of the top two recovered clones after expression of recombinant TCRs in Jurkat cells (right). [Figure 7]

[0035] 1 illustrates an example of flow cytometry analysis of Jurkat cells transduced to express RAS TCR-1. [Figure 8]

[0036] Illustrates an example of flow cytometry analysis of peripheral blood mononuclear cells transduced to express RAS TCR-1. [Figure 9]

[0037] Illustrates an example of flow cytometry analysis of electroporated Jurkat cells expressing RAS TCR-2 or RAS TCR-3. [Figure 10]

[0038] Graphs depict experimental results of a TCR functional assay assessing the specificity and affinity of RAS TCR-1. Graphs depict CD69 activation after co-culture of RAS TCR-transduced Jurkat cells with HLA-A11:01-expressing A375 cells loaded with RAS wild-type (rightmost data point in each panel), or a RAS mutant 9-mer peptide (left) or increasing amounts of a RAS mutant 10-mer peptide (right). [Figure 11]

[0039] Illustrated are experimental results of TCR functional assays assessing the specificity and affinity of RAS TCR-2 and RAS TCR-3. Graphs showing CD69 activation after co-culture of RAS TCR-transduced Jurkat cells with HLA-C01:02-expressing K562 cells loaded with RAS wild-type (right-most data point in each panel) or RAS mutant peptides. [Figure 12]

[0040] This figure illustrates the experimental results of a cytotoxicity assay in which PBMCs transduced with an irrelevant TCR or RAS TCR-1 were cocultured with SW620 (RAS G12V+) target tumor cells expressing HLA-A11:01 and GFP at increasing CD4+ cell-to-target (E:T) ratios. SW620 target tumor cells cultured alone or cocultured with PBMCs transduced with an irrelevant TCR served as negative controls. The graph illustrates target cell proliferation over 100 hours (X-axis), as measured by the GFP signal reflected by the total area of ​​the green object in the culture image (Y-axis). [Figure 13]

[0041] This figure illustrates the experimental results of a cytotoxicity assay in which CD4+ T cells isolated from PBMCs transduced with an irrelevant TCR or RAS TCR-1 were cocultured with SW620 (RAS G12V+) target tumor cells expressing HLA-A11:01 and GFP at increasing PBMC-to-target ratios. SW620 target tumor cells cultured alone or without PBMCs served as negative controls. The graph illustrates target cell proliferation over 72 hours (X-axis), as measured by the GFP signal reflected by the total area of ​​the green object in the culture image (Y-axis). [Figure 14]

[0042] 1 illustrates exemplary data showing percent cytolysis over time of the tumor cell line SNG-M after treatment with PBMCs transduced with an irrelevant TCR or RAS TCR-1. [Figure 15]

[0043]

[0023] Figure 1 illustrates exemplary data showing tumor volume over time in mice subcutaneously inoculated with the tumor cell line A375-HLA-A11:01-KRAS G12V after treatment with PBMCs transduced with an irrelevant chimeric antigen receptor (CAR) or RAS TCR-1. Mice treated with RAS TCR-1 transduced T cells exhibited tumor regression compared to PBS and irrelevant transduced T cell controls. [Figure 16]

[0044] 1 illustrates exemplary data showing the functional affinity of RAS TCR-2 and RAS TCR-3. [Figure 17A]

[0045]

[0023] Figure 1 illustrates exemplary data showing the % cytolysis after treatment of the colorectal cancer cell line SW620, engineered to express HLA C*01:02, with PBMCs transduced with RAS TCR-2 or RAS TCR-3 at the indicated effector-to-target cell ratios. The % cytolysis was calculated based on tumor cells co-cultured with untransduced T cells. [Figure 17B]

[0046] Figure 1 illustrates exemplary data showing the % cytolysis after treatment of the pancreatic tumor cell line Panc 03.27 with RAS TCR-2 or RAS TCR-3 transduced PBMCs at the indicated effector-to-target cell ratios. The % cytolysis was calculated based on tumor cells co-cultured with untransduced T cells. [Figure 18]

[0047] Illustrated are exemplary data showing the percentage of CD69+ cells in engineered cells after transduction with different concentrations of RAS TCR-5, RAS TCR-6, or RAS TCR-7 that bind to G12D RAS in complex with MHC encoded by the HLA-C05:01 allele. DETAILED DESCRIPTION OF THE INVENTION

[0025]

[0048] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within or more than one standard deviation per practice in the art. Alternatively, "about" can mean within 20%, 10%, 5%, or 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0026]

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the practice or testing of this disclosure, any methods and materials similar or equivalent to those described herein can be used, but the preferred methods and materials are described herein. The details of one or more specific embodiments are set forth in the following description.

[0027] 1.Definition

[0050] Unless otherwise stated, the term "TCR" should be understood to encompass a complete TCR, as well as antigen-binding portions or fragments thereof (also referred to as MHC-peptide-binding fragments). In some embodiments, the TCR is an intact or full-length TCR. In some embodiments, the TCR is an antigen-binding portion that is smaller than a full-length TCR but binds to a specific antigenic peptide bound to (e.g., associated with) an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR may include only a portion of the structural domains of a full-length or intact TCR, yet still be capable of binding to an epitope (e.g., an MHC-peptide complex) bound by the complete TCR. In some cases, the antigen-binding portion or fragment of a TCR includes the variable domains of the TCR, e.g., the variable α chain and variable β chain of the TCR sufficient to form a binding site for binding to a specific MHC-peptide complex, e.g., where each chain typically contains three complementarity-determining regions. Included are polypeptides or proteins having a binding domain that is, or is homologous to, an antigen-binding domain. Complementarity-determining region (CDR)-grafted TCRs and other humanized TCRs, including CDR modifications and framework region modifications, are also contemplated by these terms. It should be noted that although reference is made only to immunoglobulin chains (e.g., heavy and light chains), the disclosed invention is applicable to many 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.

[0028]

[0051] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids within a TCR variable region, which confer specificity and / or binding affinity to an MHC-peptide complex. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each alpha chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each beta chain variable region. The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the alpha and beta chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length alpha chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length beta chain variable region.

[0029]

[0052] The term "variable region" or "variable domain" refers to the domain of the alpha, beta, gamma, or delta chain of a TCR, which is involved in the binding of the TCR to an antigen-MHC complex. The variable domains of the alpha and beta chains (Vα and Vβ, respectively) and the variable domains of the gamma and delta chains (Vγ and Vδ, respectively) of natural TCRs generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. A single Vα or Vβ domain, or a Vγ or Vδ domain, may be sufficient to confer binding specificity to a peptide-MHC complex.

[0030]

[0053] Also provided herein are TCR fragments, including antigen-binding fragments. In some embodiments, the TCR is a variant of a full-length TCR that does not include its antigen-binding portion, e.g., its transmembrane and / or cytoplasmic region, which may be referred to as a fully soluble TCR. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (scTCR), e.g., an scTCR having a structure described in PCT Patent Publications WO2003 / 020763, WO2004 / 033685, or WO2011 / 044186. In certain embodiments, the TCR is a single-chain TCR fragment, e.g., an scTv, comprising an alpha chain variable region linked to a beta chain variable region. In some embodiments, an scTv is also referred to as an scFv. Single-chain Tv, or scTv, in some embodiments refers to a TCR fragment comprising the variable alpha or gamma chain (Vα or Vγ) and variable beta or delta chain (Vβ or Vδ) domains of the TCR, where these domains are present in a single polypeptide chain. Generally, the Tv polypeptide further comprises a polypeptide linker between the Vα and Vβ domains or the Vγ and Vδ domains, thereby enabling the scTv to form the desired structure for antigen binding. Diabody, in some embodiments, refers to a TCR fragment having two antigen-binding sites, where these fragments comprise a Vα connected to a Vβ (Vα-Vβ) or a Vγ connected to a Vδ (Vγ-Vδ) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Exemplary diabodies are described more fully in, for example, EP 404097 and WO 93111161. Fv, in some embodiments, refers to a TCR fragment that contains the complete peptide-MHC complex recognition and binding site. This region consists of a dimer of one TCR α and one TCR β chain or one TCR γ and one TCR δ chain in tight, non-covalent association.It is in this configuration that the three CDRs of each variable domain interact to define a peptide-MHC complex binding site on the surface of the Vα-Vβ or Vγ-Vδ dimer. Collectively, one or more combinations of CDRs from each of the Vα-Vβ or Vγ-Vδ chains confer binding specificity to the peptide-MHC complex for the TCR. For example, it will be understood that CDRα3 and CDRβ3 or CDRγ3 and CDRδ3, when transferred to the Vα and Vβ or Vγ-Vδ chains of a recipient-selected TCR or antigen-binding fragment thereof, may be sufficient to confer antigen-binding specificity to the TCR, and this CDR combination can be tested for binding, affinity, etc. Furthermore, although the two domains of a Tv fragment (Vα and Vβ or Vγ and Vδ) are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker that allows them to be produced as a single protein chain in which the Vα and Vβ or Vγ and Vδ chain regions pair to form a monovalent molecule (known as a single-chain Tv (scTv)). Such scTvs are also intended to be encompassed within the peptide-MHC complex-binding portion of a TCR.

[0031]

[0054] "Bispecific TCR" refers, in some embodiments, to a TCR that exhibits specificity for two different peptide-MHC complexes or two different types of peptide-MHC complexes. As used herein, this term specifically includes, without limitation, a TCR that exhibits binding specificity for a target peptide-MHC complex and binding specificity for another peptide-MHC complex that facilitates delivery to a specific tissue. Similarly, a multispecific TCR has two or more binding specificities. A linear TCR refers, in some embodiments, to a pair of tandem Fd fragments (e.g., Vα-Cα1-Vα-Cα1) that form a pair of antigen-binding regions. A linear TCR can be bispecific or monospecific.

[0032]

[0055] "Antigen-binding domain" refers, in some embodiments, to one or more fragments of a TCR that retain the ability to specifically bind to a peptide-MHC complex. Non-limiting examples of TCR fragments encompassed by such term include, but are not limited to, (i) Tab fragments, i.e., Vβ, Vα, C β (ii) a monovalent fragment consisting of the Vα and Cα1 domains, (ii) a T(ab')2 fragment, i.e., a bivalent fragment comprising two Tab fragments linked by a disulfide bridge at the hinge region, (iii) a Td fragment consisting of the Vα and Cα1 domains, (iv) a Tv fragment (including scTv) comprising the Vβ and Vα domains of a single arm of a TCR, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which comprises the Vα domain, and (vi) an isolated CDR. This definition includes TCRs with a single alpha chain or a single beta chain.

[0033]

[0056] Among the TCRs provided are humanized TCRs 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 contain at least a portion of a TCR constant region derived from a human TCR. A "humanized form" of a non-human TCR typically refers to a variant of a non-human TCR that has undergone humanization to reduce immunogenicity in 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 with corresponding residues from a non-human TCR (e.g., the TCR from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the TCR. A "human TCR" is a TCR having an amino acid sequence corresponding to that of a TCR produced by a human or human cell, or a non-human source utilizing a human TCR repertoire, including a human TCR library, or other human TCR coding sequence. This term does not include humanized forms of non-human TCRs containing non-human peptide-MHC complex binding regions, such as TCRs in which all or substantially all CDRs are non-human. Human TCRs may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce an intact human TCR or an intact TCR with a human variable region in response to antigen sensitization. Such animals typically contain all or part of a human TCR locus, which replaces the endogenous TCR locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous TCR locus is generally inactivated. Human TCRs may also be derived from human TCR libraries, including phage display libraries and cell-free libraries, containing TCR coding sequences from the human repertoire.

[0034]

[0057] The term "cancer neoantigen" or "neoantigen" or "neoepitope" can refer to an antigen that is not encoded in the normal, unmutated host genome. Neoantigens can refer to antigens that contain one or more amino acid modifications compared to the parent antigen. For example, neoantigens can be tumor-associated neoantigens, and the term "tumor-associated neoantigen" can include peptides or proteins that contain amino acid modifications due to tumor-specific mutations. In some cases, neoantigens represent tumorigenic viral proteins or abnormal proteins that arise as a result of somatic mutations. For example, neoantigens can arise from disruption of cellular machinery due to the activity of viral proteins. Another example is exposure to carcinogenic compounds, which in some cases can lead to somatic mutations. This somatic mutation can ultimately lead to tumor / cancer formation. Neoantigens can be a class of tumor antigens that result from tumor-specific alterations of proteins. Neoantigens include, but are not limited to, tumor antigens resulting from protein sequence substitutions, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and expression of endogenous retroviral polypeptides. A neoepitope can be an epitope that is not present in a reference, e.g., non-diseased cell, e.g., a non-cancer cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where the corresponding epitope is found in a normal, non-diseased cell or a germline cell, but one or more mutations in the diseased cell, e.g., a cancer cell, have altered the sequence of the epitope to result in a neoantigen.

[0035]

[0058] "Epitope," in some embodiments, refers to a portion of an antigen or other macromolecule that can form a binding interaction with the binding pocket of a variable region of a TCR. In some embodiments, an epitope refers to a portion of a peptide-MHC complex that can form a binding interaction with the binding pocket of a variable region of a TCR. Such a binding interaction can be expressed as an intermolecular contact with one or more amino acid residues of one or more CDRs. Binding of a peptide-MHC complex can involve, for example, the interaction of a CDR3, a pair of CDR3s, or in some cases, up to all six CDRs of the Vα and Vβ chains or the Vγ or Vδ chains. An epitope can be a linear peptide sequence (e.g., "continuous") or can consist of a discontinuous amino acid sequence (e.g., "conformational" or "discontinuous"). A TCR can recognize one or more amino acid sequences. Thus, an epitope can define two or more different amino acid sequences. In some embodiments, a TCR can recognize one or more amino acid sequences or epitopes associated with an MHC. Epitopes recognized by TCRs can be determined by peptide mapping and sequence analysis techniques well known to those skilled in the art. Binding interactions are expressed as intermolecular contacts with one or more amino acid residues of a CDR. An epitope can refer to an antigenic determinant in a molecule, such as an antigen, e.g., a portion or fragment of a molecule that is recognized by the immune system, particularly when presented in the context of an MHC molecule, e.g., a molecule recognized by T cells. Epitopes of proteins, such as tumor antigens, can comprise continuous or non-contiguous portions of the protein and can be 5-100, 5-50, 8-30, or 10-25 amino acids in length; for example, epitopes can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0036]

[0059] The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, and ionic, and / or hydrogen-bonding interactions under physiological conditions, including interactions such as salt bridges and water bridges, as well as any other conventional means of binding.

[0037]

[0060] In some embodiments, reference to a TCR with "specific binding" refers to a situation in which the TCR does not exhibit any significant binding to molecules other than peptide-MHC complexes containing the epitope recognized by the TCR. This term is also applicable, for example, when an antigen-binding domain is specific for a particular epitope carried by several peptide-MHC complexes, in which case a selected TCR or peptide-MHC complex-binding fragment thereof carrying the peptide-MHC complex-binding domain will be able to bind to various peptide-MHC complexes carrying the epitope. The terms "preferentially bind" or "specifically bind" mean that the TCR or fragment thereof binds to the epitope with greater affinity than it binds to unrelated amino acid sequences, and, if cross-reactive with other polypeptides containing the epitope, is not toxic at levels formulated for administration to humans. In one embodiment, such affinity is at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the TCR or fragment thereof for an unrelated amino acid sequence.

[0038]

[0061] 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 and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). Affinity can be expressed as the equilibrium constant for the reversible binding of two agents, K D , K. A , K. off , or K on It can be expressed as: K Drefers to the dissociation constant between two members of a binding pair and has units of molar concentration. A K refers to the affinity constant between two members of a binding pair and is the reciprocal of the dissociation constant. Affinity may be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. off K refers to the off-rate constant of the two members of a binding pair (e.g., the off-rate constant of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR). on refers to the on-rate constant of the two members of a binding pair (e.g., the on-rate constant of an HLA-binding peptide and class I or II HLA, or of a peptide-HLA complex and a TCR). The affinity of a binding protein for a ligand, e.g., the affinity of a TCR for an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). The term "affinity" refers to the resistance of a complex of two or more agents to dissociation after dilution.

[0039]

[0062] Throughout this disclosure, the results of "combined data" are referred to as "IC 50 Affinity may be expressed in terms of inhibitory concentration (IC 50 ), i.e., the concentration at which 50% of the first member (e.g., peptide) of the binding pair is displaced. Similarly, ln(IC 50 ) is IC 50 For example, IC 50 is the concentration of test peptide at which 50% inhibition of binding of the labeled reference peptide is observed in a binding assay. Given the conditions under which the assay is performed (e.g., limiting the concentration of HLA protein and / or the concentration of labeled reference peptide), these values ​​will give the K DValues ​​can be estimated. Assays for determining binding are well known in the art and are described in detail in, for example, PCT Publications WO94 / 20127 and WO94 / 03205, as well as 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 can be expressed relative to binding by a reference standard peptide. Binding can be measured 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 cell lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), 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-flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and assays using measurements of class 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)).

[0040]

[0063] The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that contains MHC class I and MHC class II molecules and occurs in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments derived from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC regions can be divided into three subgroups: class I, class II, and class III. MHC class I proteins can contain an α chain and β2-microglobulin (not part of the MHC encoded by chromosome 15), which can present antigen fragments to cytotoxic T cells. MHC class II proteins can contain α and β chains and can present antigen fragments to T helper cells. MHC class III regions can encode other immune components, such as complement components and cytokines. The MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are many alleles of each gene).

[0041]

[0064] The term "haplotype" can refer to human leukocyte antigen (HLA) alleles found on one chromosome and the proteins encoded thereby. Haplotype can also refer to alleles present at any one locus within the MHC. Each class of MHC is represented by several loci, for example, HLA-A (human leukocyte antigen A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, and HLA-V for class I, and HLA-DRA, HLA-DRB1-9, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DPB2, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB for class II. The terms "HLA allele" and "MHC allele" are used interchangeably herein.

[0042]

[0065] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. They can refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and 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 defined by 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 polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be introduced before or after assembly of the polymer. A polynucleotide may contain non-standard nucleotides, such as nucleotide analogs or modified nucleotides. In some embodiments, the non-standard nucleotides can stabilize hybrid formation. In some embodiments, the non-standard nucleotides can destabilize hybrid formation. In some embodiments, the non-standard nucleotides can enhance the specificity of hybridization. In some embodiments, the non-standard nucleotides can reduce the specificity of hybridization.Examples of non-standard nucleotide modifications include 2'O-Me, 2'O-allyl, 2'O-propargyl, 2'O-alkyl, 2'fluoro, 2'arabino, 2'xylo, 2'fluoroarabino, phosphorothioate, phosphorodithioate, phosphoramidate, 2'amino, 5-alkyl substituted pyrimidines, 3'deoxyguanosine, 5-halo substituted pyrimidines, alkyl substituted purines, halo substituted purines, bicyclic nucleotides, 2'MOE, PNA molecules, LNA molecules, LNA-like molecules, diaminopurine, S2T, 5-fluorouracil, 5-bromo ... Uracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosyl eosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 α-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxocin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, and derivatives thereof. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0043]

[0066] "Complementarity" can refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence by conventional Watson-Crick or other non-conventional methods. The percent complementarity can refer to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" can mean that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" can refer to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or can refer to two nucleic acids that hybridize under stringent conditions. Sequence identity, for example, for purposes of assessing percent complementarity, may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (e.g., the EMBOSS Needle aligner, available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, which may be with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool, available at blast.ncbi.nlm.nih.gov / Blast.cgi, which may be with default settings), or the Smith-Waterman algorithm. Optimal alignment may be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0044]

[0067] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. For example, a polypeptide can 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 polypeptides include, but are not limited to, amino acid chains, proteins, peptides, hormones, polypeptides, saccharides, lipids, glycolipids, phospholipids, antibodies, enzymes, kinases, receptors, transcription factors, and ligands. Polypeptides, including the provided TCRs and TCR chains, and other peptides, such as linkers and connecting peptides, can contain amino acid residues, including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, a polypeptide may contain modifications relative to the native or natural sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutation of the host producing the protein or errors, for example, by PCR amplification.

[0045]

[0068] As used herein, the twenty conventional amino acids and their abbreviations known to those of skill in the art follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), unnatural 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 for the polypeptides of the present invention. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, 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 notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Percent sequence identity (%) with respect to a reference polypeptide sequence (or nucleic acid sequence) is the percentage of amino acid residues (or nucleotides) in a candidate sequence that are identical to the amino acid residues (or nucleotides, in the case of a nucleic acid sequence) in the reference polypeptide sequence (or nucleic acid sequence) after aligning the sequences without considering any conservative substitutions as part of the sequence identity and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office (Washington, DC 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the 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 do not vary. In situations where ALIGN-2 is employed for comparing amino acid sequences, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y. where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It is recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0046]

[0069] "Germline sequences" refer to gene sequences derived from haploid gametes and the diploid cells that form them. Germline DNA contains multiple gene segments that encode a single TCR alpha or beta chain, or a single TCR gamma or delta chain. These gene segments are carried in germ cells but cannot be transcribed and translated until they are arranged into functional genes. During T cell differentiation in the bone marrow, these gene segments are transcribed and translated over a period of 10 8 They are randomly recombined by a dynamic genetic system that can generate specificities exceeding 100%.

[0047]

[0070] Inhibition, "treatment," and "treating" are used interchangeably and refer to, for example, stasis of symptoms, prolongation of survival, partial or complete amelioration of symptoms, and partial or complete eradication of a condition, disease, or disorder associated with excessive levels of a protein or correlated with the activity of the protein. For example, treating cancer includes, but is not limited to, stasis, 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, such as about one-half, about one-third, about one-quarter, about one-fifth, about one-tenth, about one-twentieth, about one-fiftieth, or any percentage reduction therebetween. Similarly, treatment or partial elimination includes about a 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% reduction in growth or tumor size and / or volume, or any percentage reduction therebetween. Prevention refers to preventing, preventing the onset of symptoms, or preventing the progression of a disease or disorder associated with excessive levels of or correlated with the activity of the protein.

[0048]

[0071] "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, cows, camels, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice (e.g., humanized mice), gerbils, non-human primates (e.g., macaques), humans, and other non-mammals, including non-mammalian vertebrates, such as birds (e.g., chickens or ducks), fish (e.g., sharks), or frogs (e.g., Xenopus), and non-mammalian invertebrates, and transgenic species thereof. In certain embodiments, a subject refers to a single organism (e.g., a human). In certain embodiments, a group of individuals is provided, consisting of a small cohort of individuals with a common immune factor and / or disease to be studied and / or a cohort of individuals without the disease (e.g., negative / normal controls). The subject from which the sample is obtained may be suffering from a disease and / or disorder (e.g., one or more allergies, infections, cancer, or autoimmune disorders, etc.) and may be compared to a negative control subject who does not suffer from the disease.

[0049]

[0072] A "kit" refers to a delivery system for delivering materials or agents for carrying out the methods disclosed herein. In some embodiments, a kit includes a system that allows for the storage, transport, or delivery of reaction reagents (e.g., probes, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for conducting an assay, etc.) from one location to another. For example, a kit includes one or more containers (e.g., boxes) containing the relevant reaction reagents and / or support materials. Such contents may be delivered to an intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains multiple primers. Packaging material refers to the physical structure that houses the components of the kit. Packaging material can maintain the sterility of the components and can be made from materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). A label or package insert may include appropriate written instructions. Thus, a kit further includes a label or instructions for using the components of the kit in any of the methods of the present invention. The kit may include a dispenser in packaging with the compound or instructions for administering the compound in the manner described herein.

[0050]

[0073] The term "resistance mutation" refers to a mutation in a gene that allows the gene or a host cell containing the gene to become resistant to treatment with a drug. For example, the BTK C481S mutation is a resistance mutation that can confer resistance to ibrutinib.

[0051] II. Overview

[0074] The present disclosure provides T cell receptors (TCRs) for neoantigens, isolated nucleic acid molecules encoding TCRs for neoantigens, T cells expressing said TCRs, and pharmaceutical compositions for use in treating diseases involving malignant cells expressing said neoantigens.

[0052]

[0075] Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having at least 84% sequence identity to the amino acid sequence of SEQ ID NO: 3, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 6. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 84% sequence identity to the amino acid sequence of SEQ ID NO: 17, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 20. Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 84% sequence identity to the amino acid sequence of SEQ ID NO: 31, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 34. Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 3, 17, or 31.Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR beta chain construct comprises a CDR3 having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 6, 20, or 34.

[0053]

[0076] Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein CDR3 of the TCR does not comprise any of SEQ ID NOS: 46-68. Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOS: 43-45. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOS: 43-44. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOS: 43. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 44. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 45.

[0054]

[0077] Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 80. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 90. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), including a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:93, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO:95.Provided herein are nucleic acids encoding at least one T cell receptor (TCR), including a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 101. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 109. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), including a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 112, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 113.Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 116, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 117. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 120, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 123. Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 129.Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 79, 87, 93, 100, 106, 112, 116, 120, or 126. Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR beta chain construct comprises a CDR3 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 82, 90, 95, 101, 109, 113, 117, 123, or 129.

[0055]

[0078] Provided herein are nucleic acids encoding at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the epitope derived from RAS comprises a region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 132-135. In some embodiments, the epitope derived from RAS comprises a region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 132-135. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 132. In some embodiments, the epitope derived from RAS comprises a region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 133. In some embodiments, the epitope derived from RAS comprises a region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 134. In some embodiments, the epitope derived from RAS comprises a region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 135.

[0056]

[0079] Provided herein is an isolated nucleic acid or cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by an HLA-A11:01 allele or HLA-C01:02. In some embodiments, the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by an HLA-A11:01 allele. In some embodiments, the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by HLA-C01:02.

[0057]

[0080] Provided herein is an isolated nucleic acid or cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-A68:01 allele, HLA-C05:01, HLA-C03:04, or HLA-C03:03. In some embodiments, the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-A68:01 allele. In some embodiments, the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by HLA-C05:01. In some embodiments, the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by HLA-C03:04. In some embodiments, the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by HLA-C03:03.

[0058]

[0081] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO:9. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO:12. In some embodiments, the TCR alpha chain construct and / or the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:1. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:4. In some embodiments, the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:2. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:5. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO:9, and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO:1, a CDR2 of SEQ ID NO:2, and a CDR3 of SEQ ID NO:3, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO:4, a CDR2 of SEQ ID NO:5, and a CDR3 of SEQ ID NO:6.

[0059]

[0082] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 23. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 26. In some embodiments, the TCR alpha chain construct and / or the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 15. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 18. In some embodiments, the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 16. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 19. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 23, and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 16, and a CDR3 of SEQ ID NO: 17, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 20.

[0060]

[0083] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 37. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 40. In some embodiments, the TCR alpha chain construct and / or the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 29. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 32. In some embodiments, the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 30. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 23. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 37, and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 40. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 29, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 31, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 32, a CDR2 of SEQ ID NO: 33, and a CDR3 of SEQ ID NO: 34.

[0061]

[0084] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 83. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 84. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 77. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 80. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 78. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 81. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 77, a CDR2 of SEQ ID NO: 78, and a CDR3 of SEQ ID NO: 79, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 80, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 82.

[0062]

[0085] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 91. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 92. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 85. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 88. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 86. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 89. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 85, a CDR2 of SEQ ID NO: 86, and a CDR3 of SEQ ID NO: 87, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89, and a CDR3 of SEQ ID NO: 90.

[0063]

[0086] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 96. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 97. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 15. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 94. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 16. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 81. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 16, and a CDR3 of SEQ ID NO: 93, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 94, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 95.

[0064]

[0087] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 102. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 103. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 98. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 94. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 99. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 81. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99, and a CDR3 of SEQ ID NO: 100, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 94, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 101.

[0065]

[0088] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 110. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 111. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 104. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 107. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 108. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 106, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 107, a CDR2 of SEQ ID NO: 108, and a CDR3 of SEQ ID NO: 109.

[0066]

[0089] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 114. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 115. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 104. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 88. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 89. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 112, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89, and a CDR3 of SEQ ID NO: 113.

[0067]

[0090] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 118. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 119. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 104. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 107. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 108. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 116, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 107, a CDR2 of SEQ ID NO: 108, and a CDR3 of SEQ ID NO: 117.

[0068]

[0091] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 124. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 125. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 104. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 121. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 122. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 120, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 121, a CDR2 of SEQ ID NO: 122, and a CDR3 of SEQ ID NO: 123.

[0069]

[0092] In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 130. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 131. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 29. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 127. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 30. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 128. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 29, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 126, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 127, a CDR2 of SEQ ID NO: 128, and a CDR3 of SEQ ID NO: 129.

[0070]

[0093] In yet another aspect, the present disclosure provides a host cell comprising a nucleic acid encoding a TCR against a neo-antigen provided herein, a vector comprising the nucleic acid sequence, or a protein encoded by the nucleic acid provided herein. In some embodiments, the host cell is a CD4 +In some embodiments, the host cells are CD8 T cells. + The host cells are T cells. The host cells can be natural killer (NK) cells or B cells. The host cells can be an immortalized cell line.

[0071]

[0094] In yet another aspect, the present disclosure provides pharmaceutical compositions comprising a nucleic acid encoding a TCR against a neo-antigen provided herein, a host cell comprising a nucleic acid encoding a TCR against a neo-antigen provided herein, a vector comprising a nucleic acid sequence, or a protein encoded by a nucleic acid provided herein. In some embodiments, the present disclosure also includes methods of using the pharmaceutical compositions disclosed herein.

[0072]

[0095] Also provided herein in a further aspect is a method of treating a subject having a disease or condition, comprising administering to the subject a pharmaceutical composition disclosed herein. In some embodiments, the subject has cancer.

[0073] III. T cell receptor (TCR)

[0096] The ability of T cells to recognize antigens associated with various 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 employs unique mechanisms to generate the highly diverse TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides presented by MHC class I and class II proteins on the surface of antigen-presenting cells (APCs). Binding of the TCR to antigenic peptides on APCs is a central event in T cell activation and occurs at the immunological synapse at the point of contact between the T cell and APC.

[0074]

[0097] Each TCR comprises variable complementarity-determining regions (CDRs), as well as framework regions (FRs) and a constant region. The constant region may comprise an α chain constant region and a β chain constant region. The α chain constant region and the β chain constant region may be derived from a mouse. For example, the mouse TCR α chain constant region may be Uniprot accession number P01849, and the mouse β chain constant region may be Uniprot accession number P01852 or P01851. In some cases, the α chain constant region and the β chain constant region may be derived from a human. For example, the human TCR α chain constant region may be Uniprot accession number P01848, and the human β chain constant region may be Uniprot accession number P01850 or A0A5B9. The amino acid sequences of the third complementarity-determining region (CDR3) loops of the α and β chain variable domains primarily determine the sequence diversity of αβ T cells, resulting from recombination between variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments in the β chain locus, and recombination between similar Vα and Jα gene segments in the α chain locus. The presence of multiple such gene segments in the α and β chain loci of TCRs allows multiple different CDR3 sequences to be encoded. Independent addition and deletion of nucleotides at Vβ-Dβ, Dβ-Jβ, and Vα-Jα junctions during TCR gene rearrangement further increases the sequence diversity of CDR3. In this regard, immune competence is reflected in TCR diversity. γδ TCRs are distinguished from αβ TCRs in that they encode receptors that interact closely with the innate immune system. TCRγδ is expressed early during development, has a specialized anatomical distribution, unique specificities for pathogens and small molecules, and a wide range of innate and adaptive cellular interactions. Early in ontogeny, restricted subsets of TCRγδ cells reside in various prenatal tissues, establishing a biased pattern of TCRγ V and J segment expression.

[0075]

[0098] The TCRs provided herein may be engineered TCRs, such as chimeric antigen receptors (CARs). CARs may consist of three regions: an ectodomain, a transmembrane domain, and an endodomain.

[0076]

[0099] The ectodomain may be the region of the receptor exposed to extracellular fluid and may consist of an antigen recognition region. In some embodiments, the ectodomain further comprises a spacer. In some embodiments, the ectodomain further comprises a signal peptide. The signal peptide can direct the nascent protein to the endoplasmic reticulum. The signal protein of the CAR may be a single-chain variable fragment (scFv). A fusion protein may be formed by merging two or more genes that originally encode different proteins, which, when translated in a cell, produce one or more polypeptides with functional properties derived from each of the original genes. An scFv is a chimeric protein consisting of a light chain domain and a heavy chain variable domain connected by a short linker peptide. The linker may contain hydrophilic residues with a stretch of glycine and / or serine residues. The linker may contain a stretch of glutamate and lysine residues, which can improve solubility.

[0077]

[0100] The transmembrane domain may be a membrane-spanning hydrophobic domain. In some embodiments, the transmembrane domain comprises an alpha helix 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 derived from the most membrane-proximal component of the endodomain. In some embodiments, the transmembrane domain comprises a CD3 zeta transmembrane domain. In some embodiments, the transmembrane domain allows for integration of the artificial TCR into a natural TCR complex. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0078]

[0101] The endodomain can be a functional intracellular portion of a receptor, such as a TCR or CAR. After antigen recognition, the receptors form clusters and a signal is transmitted to the cell. In some embodiments, the endodomain comprises a CD3 zeta intracellular domain. In some embodiments, the endodomain comprises at least one ITAM. In some embodiments, the endodomain comprises at least three or at least three ITAMs. In some embodiments, the endodomain comprises a CD28 intracellular domain. In some embodiments, the endodomain comprises an OX40 intracellular domain. In some embodiments, the endodomain comprises a chimeric intracellular domain. For example, the endodomain can comprise a CD28 intracellular domain, an OX40 intracellular domain, and a CD3 zeta intracellular domain.

[0079] IV.T cells

[0102] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. + T cells (helper T cells) and CD8 + Includes T cells (cytotoxic T cells). CD4 + T cells can assist other white blood cells in immune processes, including the maturation of B cells and the activation of cytotoxic T cells and macrophages. + T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, T cells can rapidly divide and secrete cytokines that regulate active immune responses. CD8 + CD8 T cells can destroy virus-infected cells and tumor cells and may also be involved in transplant rejection. +T cells can recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of almost every cell in the body. Most T cells possess a T cell receptor (TCR). The ability of T cells to recognize antigens associated with various cancers or infectious organisms is conferred by their TCR, which consists 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 employs unique mechanisms to generate diverse TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides presented by MHC class I and class II proteins on the surface of antigen-presenting cells (APCs). The initial signal for 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 a TCR specific for that peptide are activated. The partner cell is usually an antigen-presenting cell, such as a professional antigen-presenting cell, usually a dendritic cell in the case of a naive response, although B cells and macrophages can also be important APCs. Binding of the TCR to an antigenic peptide on an APC is a central event in T cell activation and occurs at the immunological synapse at the point of contact between the T cell and the APC.

[0080]

[0103] T cells can be prepared according to methods known in the art. T cells can be enriched T cell preparations, APC-depleted cell preparations, or substantially purified T cell preparations. T cells can be a mixed T cell population or a purified T cell subset. T cells can be an enriched T cell preparation containing an increased number or percentage of T cells relative to an isolated T cell population.

[0081]

[0104] T cells or subsets of T cells can be obtained from various lymphoid tissues. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, thymus, tissue biopsies, tumors, lymph node tissue, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, lymphoid tissue, and tumors. As used herein, the term "peripheral blood lymphocytes" (PBLs) and its grammatical equivalents can refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes can refer to lymphocytes that are not localized in organs. Peripheral blood lymphocytes can include T cells, NK cells, B cells, or any combination thereof.

[0082]

[0105] The method may include isolating T cells from a subject. The method may include obtaining the isolated T cells from a subject. The T cells may be obtained from a T cell line. The T cells may be obtained from an autologous source. The T cells may be obtained from an allogeneic source. The T cells may also be obtained from a xenogeneic source, such as mouse, rat, non-human primate, and pig.

[0083]

[0106] The T cells can be an APC-depleted cell preparation. The T cells can be substantially free of APCs. For example, the T cells can comprise T cells separated from more than 75% APCs. In an exemplary embodiment, peripheral blood mononuclear cells (PBMCs) can be obtained from blood, for example, in a heparinized vial. The PBMCs can be separated from red blood cells by centrifugation, and the PBMCs are recovered from the interface. The recovered PBMCs can optionally be washed (e.g., with PBS).

[0084]

[0107] Purification of T cells can be achieved by positive or negative selection, including, for example, but not limited to, the use of antibodies directed against CD2, CD3, CD4, CD5, CD8, CD14, CD19, and / or MHC class II molecules. + , CD4 + , CD8 + , CD45RA + , and / or CD45RO +T cells can be isolated by positive or negative selection techniques. For example, CD3 + , CD28 + T cells can be positively selected using CD3 / CD28 conjugated magnetic beads. In one embodiment of the invention, enrichment of a T cell population by negative selection can be achieved by a combination of antibodies directed against surface markers unique to the negatively selected cells.

[0085]

[0108] For example, a T cell sample may contain cells derived from a subject's circulating blood and may be obtained by apheresis or leukopheresis. A T cell sample may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets. Undesirable components in a T cell sample may be removed, and the remaining T cells may be suspended in culture medium. For example, the cells may be washed to remove the plasma fraction. For example, T cells may be isolated from peripheral blood lymphocytes by lysing red blood cells and centrifuging through a PERCOLL™ gradient.

[0086]

[0109] In some embodiments, the T cell comprises at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from RAS in complex with human MHC. In some embodiments, the T cell comprises at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from TMPRSS2:ERG in complex with human MHC. In some embodiments, the T cell comprises at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope derived from GATA3 in complex with human MHC. In some embodiments, the host cell comprises at least one TCR disclosed herein, and the host cell is a CD4+ T cell. In some embodiments, the host cell is a CD8+ T cell. 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 cells may be natural killer (NK) cells, B cells, or immortalized cell lines.

[0087]

[0110] In some embodiments, T cells can be obtained by positive selection and / or negative selection. Positive selection can use affinity agents (e.g., antibodies, antibody fragments, and aptamers) that bind to cell surface markers expressed on the cell population, such as CD3 for T cells. The affinity agents can be used to label T cells. The labeled T cells can then be enriched using various methods well known in the art. Non-limiting examples of these methods include fluorescence-activated cell sorting (FACS) and (para)magnetic particle-based cell separation (e.g., Miltenyi Biotec's MACS cell separation kit).

[0088]

[0111] Negative selection can use affinity agents that bind to cell surface markers expressed on blood cells other than the desired population. For example, when attempting to isolate T cells, a cocktail 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, leaving only enriched T cells. Exemplary methods for depleting labeled cells include FACS and (para)magnetic particle-based cell separation.

[0089]

[0112] In addition to label-based isolation, special growth conditions may be used to promote the proliferation of a particular cell population. For example, special growth conditions can be achieved using special cytokines or growth factors. As another example, T cells may preferentially proliferate in culture medium containing phytohemagglutinin (PHA), IL-2, and / or IL-15.

[0090]

[0113] In some situations, non-functional markers may be used to isolate T cells, as binding of functional markers, such as binding of CD3 by anti-CD3 antibodies (alone or conjugated to magnetic particles), may induce unwanted signaling events in T cells. Thus, a cocktail of antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (glycophorin A) can be used to isolate T cells.

[0091]

[0114] Detailed protocols can be found in published literature (see, e.g., Lefort et al., J Vis Exp. 2010;(40):2017), which is incorporated herein by reference. T cell isolation kits can be obtained, for example, from STEMCELL Technologies, Thermofisher, and Miltenyi Biotec.

[0092]

[0115] The T cells described herein can be allogeneic T cells.

[0116] In some embodiments, the T cells may be genetically modified cells that comprise an altered human T cell receptor (TCR) alpha chain gene and / or an altered human TCR beta chain gene in their genome, and the cells have reduced cell surface expression of an endogenous TCR.

[0093]

[0117] Gene-editing nucleases may be employed to disrupt components of the TCR. The TCR alpha chain (TCRα) is encoded by a single TRAC gene and pairs with the TCR beta chain (TCRβ), which is encoded by two TCRB genes. Because the TCRα / β dimer can form a fully functional TCR complex, disrupting the function of TCRα and / or TCRβ may reduce (even eliminate) endogenous TCR expression.

[0094]

[0118] Various 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: binding user-defined DNA sequences and mediating double-stranded DNA breaks (DSBs). Zinc finger nucleases (ZFNs) are heterodimeric arrays that colocalize to target DNA sites. ZFNs contain individual finger subunits that bind to DNA and are linked to a Fokl nuclease domain that cleaves the DNA. Transcription activator-like effector nucleases (TALENs) contain repeating units that bind to DNA via a hypervariable two-amino acid sequence (repeated variable dinucleotides, RVDs) that governs DNA base recognition. Similar to ZFNs, TALENs function as dimeric proteins fused to a Fokl endonuclease domain for DSB generation. Meganucleases (MNs) are derived from bacterial homing endonucleases and are monomeric proteins with intrinsic nuclease activity engineered for specific target sites. The clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying Cas9 nuclease platform involves a small guide RNA (gRNA) transcript that contacts the target DNA sequence via Watson-Crick base pairing and a Cas9 nuclease that cleaves the DNA.

[0095]

[0119] In some embodiments, introducing a genome editing nuclease into a T cell comprises introducing a polynucleotide encoding the genome editing nuclease into the T cell.

[0120] In some embodiments, introducing a genome-editing nuclease into the T cell comprises introducing a Cas9 polypeptide into the T cell. In some embodiments, the genome-editing nuclease comprises a TALEN nuclease, a CRISPR / Cas9 nuclease, or a megaTAL nuclease.

[0096]

[0121] In some embodiments, the CRISPR / Cas9 nuclease is derived from Streptococcus pyogenes or Staphylococcus aureus. In some of these embodiments, the CRISPR / Cas9 nuclease comprises a nuclease-resistant gRNA, such as at least one 2'-OMe-phosphorothioate modified base, at least one 2'-O-methyl modified base, or at least one 2'-O-methyl 3'thioPACE modified base.

[0097]

[0122] In some embodiments, the TALEN nuclease or megaTAL nuclease is encoded by an RNA with an exogenous polyadenylation signal.

[0123] In some embodiments, the methods described herein may further comprise culturing the T cells under conditions effective to expand the population of genomically modified T cells.

[0098]

[0124] In some embodiments, disruption of TCRα and / or TCRβ expression further disrupts assembly of TCRα and TCRβ. In some embodiments, disruption of TCRα expression further disrupts formation of a TCR and CD3 complex. In some embodiments, disruption of TCRα expression comprises further disruption of assembly of TCRα and TCRβ.

[0099]

[0125] In some embodiments, the genetically modified T cells comprise disrupted TCR alpha and / or beta chains and inactivated genes encoding immune checkpoint proteins such as PD1 and CTLA-4. This can be made possible by gene inactivation using specific TALE-nucleases directed against TCR alpha or TCR beta coupled with inactivation of genes encoding immune checkpoint proteins such as PD1 and CTLA-4.

[0100]

[0126] In some embodiments, the genetic modification relies on inactivation of one or two genes selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha, and TCR beta. In some embodiments, the genetic modification relies on the inactivation of two genes selected from the group consisting of PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, and 2B4 and TCR beta. In some embodiments, the genetic modification relies on the inactivation of three or more genes. The genetic modification may be performed ex vivo.

[0101] V. TCR specific for RAS peptide-MHC complexes

[0127] Provided herein are nucleic acids encoding at least one T cell receptor (TCR), including a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 3, and / or the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 6. Provided herein are nucleic acids encoding at least one TCR, including a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 17, and / or the TCR beta chain construct comprises a CDR3 having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 20. Provided herein are nucleic acids encoding at least one TCR, including a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in a complex with human MHC, wherein the TCR alpha chain construct comprises a CDR3 having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 31, and / or the TCR beta chain construct comprises a CDR3 having at least 84% sequence identity to an amino acid sequence selected from SEQ ID NO: 34.

[0102]

[0128] Provided herein are nucleic acids encoding at least one T cell receptor (TCR), comprising a TCR alpha chain construct and / or a TCR beta chain construct, capable of specifically binding to an epitope derived from RAS in a complex with human MHC, wherein the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 43-45. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 43-44. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 43. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 44. In some embodiments, the epitope derived from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 45.

[0103]

[0129] Provided herein is an isolated nucleic acid or cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-A11:01 allele. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO:9, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-A11:01 allele. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO:12, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-A11:01 allele. Provided herein is an isolated nucleic acid or cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one TCR, including a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-C01:02 allele. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 23, and the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-C01:02 allele. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 26, and the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-C01:02 allele. Provided herein is an isolated nucleic acid or cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-C01:02 allele.In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 37, and the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-C01:02 allele. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 40, and the TCR specifically binds to an epitope derived from RAS in complex with human MHC encoded by the HLA-C01:02 allele.

[0104]

[0130] In some embodiments, the TCR alpha chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 1. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4. In some embodiments, the TCR alpha chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 2. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 5. In some embodiments, the TCR alpha chain construct comprises a complementarity determining region 3 (CDR3) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 6. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO:9, and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO:12. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO:1, a CDR2 of SEQ ID NO:2, and a CDR3 of SEQ ID NO:3, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO:4, a CDR2 of SEQ ID NO:5, and a CDR3 of SEQ ID NO:6. In some embodiments, the TCR alpha chain construct comprises a CDR1 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:15. In some embodiments, the TCR beta chain construct comprises a CDR1 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:18. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:16. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:19.In some embodiments, the TCR alpha chain construct comprises a CDR3 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 17. In some embodiments, the TCR beta chain construct comprises a CDR3 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 20. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 23, and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, the TCR alpha chain construct comprises a CDR1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 16, and a CDR3 of SEQ ID NO: 17, and the TCR beta chain construct comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 20. In some embodiments, the above TCR alpha chain constructs comprise a CDR1 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 29. In some embodiments, the TCR beta chain constructs described above comprise a CDR1 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 32. In some embodiments, the TCR alpha chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 30. In some embodiments, the TCR beta chain construct comprises a CDR2 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 33. In some embodiments, the TCR alpha chain construct comprises a CDR3 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 31. In some embodiments, the TCR beta chain construct comprises a CDR3 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 34. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 37, and the TCR beta chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 40. In some embodiments, the TCR alpha chain construct comprises CDR1 of SEQ ID NO: 29, CDR2 of SEQ ID NO: 30, and CDR3 of SEQ ID NO: 31, and the TCR beta chain construct comprises CDR1 of SEQ ID NO: 32, CDR2 of SEQ ID NO: 33, and CDR3 of SEQ ID NO: 34.

[0105]

[0131] Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs are capable of specifically binding to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-A68:01 allele. The mutated epitope derived from human RAS can be characterized by a G12V mutation. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 82. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 80. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 77, CDR2 has the amino acid sequence set forth in SEQ ID NO: 78, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:83.In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the epitope derived from human RAS comprising the mutation G12V has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43 or 44.

[0106]

[0132] Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs are capable of specifically binding to a mutated epitope derived from human RAS in a complex with human MHC encoded by the HLA-C03:03 allele. The mutated epitope derived from human RAS can be characterized by a G12V mutation. Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs are capable of specifically binding to a mutated epitope derived from human RAS in a complex with human MHC encoded by the HLA-C03:04 allele. The mutated epitope derived from human RAS can be characterized by a G12V mutation. Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs are capable of specifically binding to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:03 allele and human MHC encoded by the HLA-C03:04 allele. The mutated epitope derived from human RAS can be characterized by a G12V mutation. In some embodiments, the TCRs (i) have a K of at most 500 nM, at most 250 nM, at most 50 nM, at most 25 nM, at most 10 nM, or at most 5 nM for the mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:03 allele.D and (ii) a K of at most 500 nM, at most 250 nM, at most 50 nM, at most 25 nM, at most 10 nM, or at most 5 nM to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:04 allele. DIn some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 109. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 107. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 108. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 110.

[0107]

[0133] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 113. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 88. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 89. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:114.

[0108]

[0134] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 117. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 107. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 108. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:118.

[0109]

[0135] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 123. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 121. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 122. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:124.In some embodiments, the epitope derived from human RAS comprising the mutation G12V has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 133 or 134.

[0110]

[0136] Provided herein are recombinant nucleic acids encoding a T cell receptor (TCR), comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 127. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 128. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 29, CDR2 has the amino acid sequence set forth in SEQ ID NO: 30, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 126. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the TCR binds to an epitope derived from human RAS containing the mutation G12D.In some embodiments, the epitope derived from human RAS comprising the mutation G12D has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 135. In some embodiments, the TCR binds to a complex comprising (i) an epitope derived from human RAS comprising the mutation G12D, and (ii) an MHC protein encoded by the HLA-A11:01 allele.

[0111]

[0137] Provided herein are recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, wherein the TCRs have a K of at most 0.001 nM, at most 0.01 nM, at most 0.1 nM, at most 1 nM, at most 10 nM, at most 100 nM, at most 1000 nM, at most 10000 nM, or at most 100000 nM, or at most 100000 nM, to a mutated epitope derived from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele. D The mutated epitope from human RAS can be characterized by a G12D mutation. Recombinant nucleic acids encoding T cell receptors (TCRs), including TCR beta chain constructs and TCR alpha chain constructs, are provided herein, wherein the TCR binds to the mutated epitope from human RAS in complex with a human MHC encoded by the HLA-C05:01 allele with a K of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM, 100000 nM, or greater. DThe TCR beta chain construct can specifically bind to the amino acid sequence set forth in SEQ ID NO: 92. The mutated epitope derived from human RAS can be characterized by a G12D mutation. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 90. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 88. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 89. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 85, CDR2 has the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 91.

[0112]

[0138] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 95. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 94. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 15, CDR2 has the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:96.

[0113]

[0139] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 101. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 94. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR further comprises a TCR alpha chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence set forth in SEQ ID NO: 98, CDR2 has the amino acid sequence set forth in SEQ ID NO: 99, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:102.In some embodiments, the epitope derived from human RAS containing the mutation G12D has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 132.

[0114]

[0140] In some embodiments, the TCR binds the mutated epitope with an EC 50 It specifically binds to

[0141] In various embodiments, the nucleic acid sequence encoding the TCR is codon optimized.

[0115]

[0142] Mutations in any one 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 have been found to carry at least one mutation in one of the canonical ras genes. Ras mutations are evident in, for example, adenocarcinoma of the bile duct, transitional cell carcinoma of the bladder, breast cancer, cervical adenocarcinoma, colon adenocarcinoma, colon adenoma, neuroblastoma (autonomic ganglion), acute myeloid leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's 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, germinoma, seminoma, anaplastic carcinoma, follicular adenocarcinoma, papillary carcinoma, and Hürthle cell carcinoma. Ras mutations have been found in cancers affecting many tissues and organs of the body, including lung, liver, breast, bladder, colon, cervix, pancreas, prostate, stomach, thyroid, testes, soft tissue, skin, and blood.

[0116]

[0143] In various embodiments, the TCR binds to an MHC:RAS peptide complex and the RAS peptide comprises a G12V mutation. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the RAS peptide comprises a G12C mutation. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the RAS peptide comprises a G12D mutation. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the RAS peptide comprises a mutation at Q61. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the RAS peptide comprises the sequence VVGAVGVGK (SEQ ID NO: 43), VVVGAVGVGK (SEQ ID NO: 44), or AVGVGKSAL (SEQ ID NO: 45). In various embodiments, the TCR binds to an MHC:RAS peptide complex and the RAS peptide comprises the sequence VVGAVGVGK (SEQ ID NO: 43) or VVVGAVGVGK (SEQ ID NO: 44). In various embodiments, the TCR binds to an MHC:RAS peptide complex, and the RAS peptide comprises the sequence AVGVGKSAL (SEQ ID NO: 45).

[0117]

[0144] In various embodiments, the TCR binds to an MHC:RAS peptide complex and the human MHC is encoded by an HLA-A02:01 allele. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the human MHC is encoded by an HLA-A03:01 allele. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the human MHC is encoded by an HLA-A11:01 allele. In various embodiments, the TCR binds to an MHC:RAS peptide complex and the human MHC is encoded by an HLA-C01:02 allele.

[0118]

[0145] In various embodiments, the TCR binds to an MHC:RAS peptide complex, the human MHC is encoded by an HLA-A11:01 allele, and the RAS peptide comprises the sequence VVGAVGVGK (SEQ ID NO: 43) or VVVGAVGVGK (SEQ ID NO: 44). In various embodiments, the TCR binds to an MHC:RAS peptide complex, the human MHC is encoded by an HLA-C01:02 allele, and the RAS peptide comprises the sequence AVGVGKSAL (SEQ ID NO: 45).

[0119]

[0146] In some embodiments, one TCR disclosed herein exhibits specific binding affinity for an epitope peptide containing a point mutation found in cancer when the epitope peptide is present in a complex with MHC encoded by a particular allele, and the TCR exhibits a different binding affinity for another epitope peptide containing a different point mutation of the same cancer protein when the epitope peptide is present in a complex with MHC encoded by a particular allele, but does not exhibit binding affinity for a WT peptide that does not contain any mutations.

[0120] VI. Delivery of Nucleic Acids or Vectors

[0147] A nucleic acid encoding a TCR or a vector containing such a nucleic acid can be delivered to a host cell for expression and processing.

[0121]

[0148] The terms "transfer," "introduce," or "transfect" are used interchangeably herein and relate to the introduction of nucleic acid, particularly exogenous or heterologous nucleic acid, into a cell.

[0122]

[0149] Cells can be transfected with any carrier that can accompany nucleic acid, for example, by forming complex with nucleic acid or by forming vesicles in which nucleic acid is trapped or encapsulated, thereby resulting in nucleic acid with increased stability compared with naked nucleic acid.Useful carriers according to the present disclosure include, for example, lipid-containing carriers, such as cationic lipids, liposomes, particularly cationic liposomes, and micelles, and nanoparticles.Cationic lipids can form complexes with negatively charged nucleic acid.Any cationic lipid can be used according to the present disclosure.

[0123]

[0150] In various embodiments, the nucleic acid encoding the TCR disclosed herein is operably linked to a promoter. The present disclosure further provides vectors, such as plasmids, shuttle vectors, phagemids, cosmids, expression vectors, retroviral vectors, adenoviral vectors, or particles, and / or vectors for use in gene therapy, which contain one or more of the nucleic acids disclosed above. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector contains a nucleic acid insert, which encodes a polypeptide or protein desired for expression in a cell, e.g., a host cell. For purposes of this disclosure, the insert may be a nucleic acid encoding a TCR, the alpha or beta chain of a TCR, or both. The term "incorporating" a nucleic acid sequence into a vector may refer to preparing a suitable expression vector with an insert containing the nucleic acid sequence. An "expression vector" is a vector that, when present in an appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector. A "retrovirus" is a virus with an RNA genome. A "gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include, but are not limited to, murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "Lentivirus" refers to a genus of retroviruses that can infect dividing and non-dividing cells. Some examples of lentiviruses include HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Vectors encoding core viruses are also known as "viral vectors." There are numerous viral vectors available that are suitable for use with the present invention, including those identified for human gene therapy applications, such as those described by Pfeifer and Verma (Pfeifer, A. and I.M. Verma. 2001. Ann. Rev. Genomics Hum. Genet. 2:177-211).Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, as well as more complex retrovirus-derived vectors, e.g., 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 packaging mammalian target cells with viral particles containing TCR transgenes using retroviral and lentiviral viral vectors for transduction are well known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., 2011, PLoS One 6:327-930; 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 TCR α chain specific for a peptide antigen into hematopoietic progenitor cells. The viral vector may be a retroviral or lentiviral vector. The viral vector may contain a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selection markers. The selection marker may confer drug resistance or a detectable marker, such as a fluorescent marker or a cell surface protein that can be detected by methods such as flow cytometry. When the viral vector genome contains two or more nucleic acid sequences that are expressed in a host cell as separate transcripts, the viral vector may contain additional sequences between the two (or more) transcripts, thereby enabling 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 can also be used for polynucleotide delivery, including DNA viral vectors, including adenovirus-based and adeno-associated virus (AAV)-based vectors, amplicon vectors, and vectors derived from herpes simplex virus (HSV), including replication-deficient 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 (Friedmann T., ed., 1999. The development of human gene therapy. New York: Cold Spring Harbor Lab)).

[0124]

[0151] A vector may contain a nucleic acid sequence that allows the nucleic acid to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known to those skilled in the art. The vector is preferably an expression vector that contains the nucleic acid of the present invention operably linked to a sequence that allows the expression of the nucleic acid.

[0125]

[0152] In some embodiments, provided herein is a vector comprising a nucleic acid encoding a TCR disclosed herein. In some embodiments, the vector is a self-amplifying RNA replicon, a plasmid, a phage, a transposon, a cosmid, a virus, or a virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpesvirus, a poxvirus, an alphavirus, a vaccinia virus, a hepatitis B virus, a 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, a cationic lipid, a cationic polymer, a metal nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere.

[0126]

[0153] Constructs, for example, nucleic acid constructs encoding TCR alpha and beta chains for expression in cells are provided herein. In some embodiments, the constructs comprise one or more polynucleotides encoding the TCR alpha and beta chains. In some embodiments, the polynucleotides are incorporated into a suitable vector. In some embodiments, the polynucleotides encoding the alpha and beta chains are incorporated into the same vector. In some embodiments, the polynucleotides encoding the alpha and beta chains are incorporated into different vectors, and both vectors are delivered for expression in a single cell.

[0127]

[0154] In some embodiments, a nucleic acid encoding a TCR may be transduced or transfected into a cell, which can express the TCR and be used as a therapeutic. In some embodiments, the cell is derived from a subject or host, and the subject or host is human. In some embodiments, the subject or host comprises a cell with a mutation in an epitope, and the TCR expressed in the cell is capable of specifically binding to the epitope with the mutation. In some embodiments, the cell is a lymphocyte cell. In some embodiments, it is a T lymphocyte. In some embodiments, the cell is a lymphocyte progenitor cell. In some embodiments, the cell is a T lymphocyte precursor cell. In some embodiments, the cell is a T lymphocyte progenitor cell. In some embodiments, the cell is a thymocyte.

[0128]

[0155] In some embodiments, the T cells are immature T cells. In some embodiments, the T cells are antigen-naive T cells. After transfection or transduction with a polynucleotide encoding a TCR, the host cells may be cultured ex vivo for 1, 2, 3, 4, 5 or more days for monitoring and recovery.

[0129] VII. Neoantigens

[0156] The TCRs disclosed herein are specific for immunogenic neo-antigens. In some embodiments, the neo-antigen peptide is derived from RAS. In some embodiments, the neo-antigen peptide is derived from GATA3. In some embodiments, the neo-antigen peptide is derived from BTK. In some embodiments, the neo-antigen peptide is derived from TMPRSS2:ERG. In some embodiments, one or more neo-antigen peptides are loaded onto APCs, and the peptide-loaded APCs are then used to stimulate T cells to produce antigen-specific T cells. In some embodiments, the APCs used for peptide loading are dendritic cells. The sequence of the immunogenic neo-antigen can be identified by any suitable method known in the art.

[0130]

[0157] In various embodiments, the neoantigen comprises an epitope. Mutated epitopes are more likely to be effective in eliciting an immune response or activating T cells in both animals and humans. In some embodiments, the epitope comprises a mutation. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a read-through mutation, a gene fusion mutation, and any combination thereof.

[0131]

[0158] In some embodiments, the epitope is at least 8 amino acids in length. In some embodiments, the epitope is at least 16 amino acids in length. In some embodiments, the epitope is 8-25 amino acids in length. In some embodiments, the epitope is 8-12 amino acids in length. In some embodiments, the epitope is 16-25 amino acids in length. 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 in length.

[0132]

[0159] In certain embodiments, the neoantigen or epitope thereof 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 derivable therein. In certain embodiments, the neoantigen or epitope thereof is 100 amino acids or less.

[0133]

[0160] In some embodiments, MHC class I neoantigens or epitopes thereof are 13 residues or less in length, typically about 8 to about 11 residues, particularly 9 or 10. In some embodiments, MHC class II neoantigens or epitopes thereof are 9 to 24 residues in length.

[0134]

[0161] In some embodiments, the neoantigen binds to an HLA protein (e.g., HLA class I or HLA class II). In certain embodiments, the neoantigen binds to an HLA protein with greater affinity than the corresponding wild-type peptide. In certain embodiments, the neoantigenic peptide or polypeptide has an IC of at least 5000 nM or less, at least 500 nM or less, at least 100 nM or less, at least 50 nM or less, or less. 50 It has.

[0135]

[0162] In some embodiments, the epitope binds to human MHC with greater affinity than the corresponding wild-type epitope, ie, the epitope binds to human MHC with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. D or IC 50 In some embodiments, the epitope binds to human MHC with a K of at most 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. D or IC 50 In some embodiments, the epitope binds to human MHC with an EC of less than 100000 nM, 10000 nM, 1000 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. 50 In some embodiments, the epitope binds to human MHC with an EC of at least about 100,000 nM, 10,000 nM, 1000 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. 50In some embodiments, the epitope comprises a mutation, and the mutation is not present in the subject's non-cancer cells. In some embodiments, the epitope is encoded by a gene or expressed gene in the subject's cancer cells. In some embodiments, the TCR binds to the HLA-peptide complex with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. D or IC 50 In some embodiments, the TCR binds to an HLA-peptide complex with a K of at most 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. D or IC 50 In some embodiments, the TCR binds to an HLA-peptide complex with an EC of less than 100000 nM, 10000 nM, 1000 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. 50 In some embodiments, the TCR binds to an HLA-peptide complex with an EC50 of at least about 100,000 nM, 10,000 nM, 1000 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM. 50 Or bind by affinity.

[0136]

[0163] In some embodiments, the neo-antigenic peptide can be about 8 to about 50 amino acid residues in length, 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 length. In some embodiments, the neo-antigenic peptide can be about 8 to about 500 amino acid residues in length, 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 in length.

[0137]

[0164] In some embodiments, the neo-antigenic peptide can 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 length. In some embodiments, the neo-antigenic peptide can 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 length. In some embodiments, the neo-antigenic 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 length. In some embodiments, the neo-antigenic 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 in length.

[0138]

[0165] In some embodiments, the neo-antigenic peptide has an overall length of 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.

[0139]

[0166] In some embodiments, the neo-antigenic peptide has an overall length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.

[0140]

[0167] In some embodiments, the neo-antigenic peptides 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 neo-antigenic peptides may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the neo-antigenic peptides may have a pI value of up to 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0141]

[0168] In some embodiments, the neo-antigenic peptides 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 neo-antigenic peptides 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 more. In some embodiments, the neo-antigenic 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, 900 μM.

[0142]

[0169] In some embodiments, the neo-antigenic peptides described herein may comprise carriers known in the art, such as thyroglobulin, albumins 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 protein, and other carriers.

[0143]

[0170] In some embodiments, the neoantigenic peptides described herein can be modified by terminal NH2 acylation, e.g., alkanoyl (C1-C20) or thioglycolyl acetylation, terminal carboxylamidation, e.g., ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linkage to a support or other molecule.

[0144]

[0171] In some embodiments, the neo-antigenic peptides described herein may include modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surfactant materials, e.g., lipids, or can be chemically modified, e.g., by acetylation, etc. Furthermore, the bonds of the peptides can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0145]

[0172] In some embodiments, the neo-antigenic peptides described herein can include substitutions to alter the physical properties (e.g., stability or solubility) of the resulting peptide. For example, neo-antigenic peptides can be modified by replacing cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine ​​tends to form disulfide bridges, significantly altering the peptide's structure and thereby reducing its binding capacity. Replacing C with α-aminobutyric acid not only alleviates this problem, but in some cases actually improves binding and cross-linking capabilities. Substitution of cysteine ​​with α-aminobutyric acid can occur at any residue in the neo-antigenic peptide, for example, at anchor or non-anchor positions of an epitope or analog within the peptide, or at other positions in the peptide.

[0146]

[0173] In some embodiments, the neo-antigenic peptides described herein contain amino acid mimetics or non-natural amino acid residues, such as D- or L-naphylalanine, D- or L-phenylglycine, D- or L-2-thienylalanine, D- or L-1, -2, 3-, or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)alanine, D- or L-(3-pyridinyl)alanine, D- or L-(2-pyrazinyl)alanine, D- or L-(4-isopropyl)phenylglycine, D-( The amino acid residues may include (trifluoromethyl)phenylglycine, D-(trifluoromethyl)phenylalanine, D-rho-fluorophenylalanine, D- or L-rho-biphenylphenylalanine, D- or L-rho-methoxybiphenylphenylalanine, D- or L-2-indole(allyl)alanine, and D- or L-alkylalanine, where the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or a non-acidic amino acid residue. Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides containing various amino acid mimetics or unnatural amino acid residues are particularly useful, as they tend to exhibit increased stability in vivo. Such peptides may also have improved product shelf life or manufacturing characteristics.

[0147]

[0174] Peptide stability can be assayed in several ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al., Eur. J. Drug Metab. Pharmacokinetics 11:291 (1986). The half-life of the peptides described herein is conveniently determined using a 25% human serum (v / v) assay. The protocol is as follows: Pooled human serum (type AB, not heat-inactivated) is dilapidated by centrifugation before use. The serum is then diluted to 25% with RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, aliquots of the reaction solution are removed and added to 6% aqueous trichloroacetic acid (TCA) or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and centrifuged to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatographic conditions.

[0148]

[0175] In some embodiments, the neo-antigenic peptides described herein can be synthetically prepared by recombinant DNA technology or chemical synthesis, or isolated from natural sources, such as naturally occurring tumors or pathogenic organisms. The epitopes can be synthesized separately or can be directly or indirectly linked to the peptide. The neo-antigenic peptides described herein are substantially free of other naturally occurring host cell proteins and fragments thereof, although in some embodiments, the peptides can be synthetically conjugated to bind to naturally occurring fragments or particles.

[0149]

[0176] In some embodiments, the neo-antigenic peptides described herein can be prepared in a wide variety of ways. In some embodiments, peptides can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols (see, e.g., Stewart and Young, SOLID PHASE PEPTIDE SYNTHESIS, 2nd ed., Pierce Chemical Co., 1984). Additionally, chemical ligation can be used to join individual peptides to produce larger peptides that are still within the scope of the present invention.

[0150]

[0177] Alternatively, recombinant DNA technology can be employed in which a nucleotide sequence encoding the peptide is inserted into an expression vector, transformed, or transfected into a suitable host cell, and cultured under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Thus, recombinant peptides comprising or consisting of one or more epitopes described herein can be used to present appropriate T cell epitopes.

[0151] VIII. Pharmaceutical Compositions

[0178] Pharmaceutical compositions can be formulated with one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate the processing of active agents into pharmaceutically acceptable preparations.Appropriate formulation can depend on the selected route of administration.Any well-known method, carrier and excipient can be used as appropriate and understood in the art.

[0152]

[0179] In some cases, the pharmaceutical composition is formulated as a cell-based therapeutic, e.g., a T cell therapeutic. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic, a nucleic acid-based therapeutic, an antibody-based therapeutic, and a cell-based therapeutic. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic or a nucleic acid-based therapeutic, wherein the nucleic acid encodes a polypeptide. The composition may comprise T cells specific for two or more immunogenic antigens or neo-antigen peptides. In some embodiments, the T cell-specific therapeutic may be supplemented with one or more additional therapeutic agents.

[0153]

[0180] In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding a TCR that targets a neo-antigen disclosed herein, a vector comprising the nucleic acid, a protein encoded by the nucleic acid, or a host cell comprising the nucleic acid, protein, or vector, and a pharmaceutically acceptable excipient or diluent. In some embodiments, the pharmaceutical composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the adjuvant is poly I:C.

[0154]

[0181] Also provided herein is the use of the pharmaceutical composition in the treatment of immune diseases or cancer.

[0182] In addition to the active ingredient, a pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum alcohols; The composition may comprise a protein such as riboflavin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, disaccharide, and other carbohydrate, including glucose, mannose, or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0155]

[0183] An acceptable carrier is physiologically acceptable to the patient to which it is administered and retains the therapeutic properties of the compound with which it is administered. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences (18th ed., A. Gennaro, Mack Publishing Co., Easton, PA 1990). One example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in the transport or transfer of the target compound from the administration site in one organ or part of the body to another organ or part of the body, or in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the formulation and is not harmful to the subject to which it is administered. An acceptable carrier should not alter the specific activity of the other ingredients.

[0156]

[0184] In one aspect, provided herein is a pharmaceutically acceptable or physiologically acceptable composition, including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonicity, and absorption-promoting or absorption-delaying agents that are suitable for pharmaceutical administration. Thus, a pharmaceutical composition or pharmaceutical preparation refers to a composition that is suitable for pharmaceutical use in a subject. The composition may be formulated to suit a specific administration route (e.g., systemic or local). That is, the composition includes carriers, diluents, or excipients that are suitable for administration by various routes.

[0157]

[0185] In some embodiments, the pharmaceutical composition may further contain an acceptable additive to improve the stability of the composition. Acceptable additives should not alter the specific activity of the active agent, e.g., immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. Acceptable additives can be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80, which increase peptide stability and reduce solution gelation. Surfactants can be added to the composition in amounts of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0158]

[0186] In some embodiments, the pharmaceutical composition comprises a therapeutic agent that is a T cell that expresses one or more polynucleotides encoding a T cell receptor. In some embodiments, the pharmaceutical composition comprises a physiologically acceptable carrier suitable for cell suspension.

[0159]

[0187] Pharmaceutical compositions can be administered, for example, by injection. Pharmaceutical compositions for injection include aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic 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, etc.), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride can be included in the composition. The resulting solution can be packaged for use as is or lyophilized. The lyophilized formulation can then be combined with a sterile solution prior to administration. For intravenous injection or injection at the affected site, the active ingredient is in the form of a pyrogen-free, parenterally acceptable aqueous solution with suitable pH, isotonicity, and stability.Those skilled in the art can prepare suitable solutions using isotonic vehicles such as sodium chloride for injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.Sterile injectable solutions can be prepared by incorporating the required amount of active ingredient in a suitable solvent, optionally with one of the combinations of ingredients listed above, followed by sterile filtration.Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method can be vacuum drying and freeze-drying, which can produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0160]

[0188] The pharmaceutical composition can be conventionally administered intravenously, for example, by injection of a unit dose.For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity, and stability.Suitable solutions can be prepared using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.Furthermore, the composition can be administered via aerosolization.

[0161]

[0189] When a pharmaceutical composition is contemplated for use in any of the methods provided herein, it is envisioned that the composition is substantially pyrogen-free, and therefore will not provoke an inflammatory or unsafe allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing substantially pyrogen-free compositions is well understood by those skilled in the art and can be accomplished using commercially available kits.

[0162]

[0190] Acceptable carriers may contain compounds that stabilize, enhance, or delay absorption, or enhance or delay elimination. Such compounds include carbohydrates such as glucose, sucrose, or dextran, compositions that reduce the elimination or hydrolysis of low-molecular-weight proteins and peptides, or excipients, or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents, including liposomal carriers, can also be used to stabilize, enhance, or reduce the absorption of pharmaceutical compositions. To protect against digestion, the compound can be complexed with the composition to provide resistance to acidic and enzymatic hydrolysis, or the compound can be complexed with an appropriately resistant carrier such as a liposome. Means for 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. Pat. No. 5,391,377).

[0163]

[0191] Pharmaceutical compositions can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.The amount to be administered depends on the subject to be treated, the capacity of the subject's immune system to utilize the active ingredient, and the desired degree of binding capacity.The exact amount of active ingredient required to be administered is specific to each individual, depending on the judgment of the clinician.Suitable regimes for initial administration and booster injections also vary, but are typified by an initial administration followed by multiple doses at intervals of one hour or more by subsequent injections or other administrations.Alternatively, continuous intravenous infusion sufficient to maintain blood levels is also contemplated.

[0164]

[0192] In some embodiments, the present disclosure is directed to immunogenic compositions, e.g., pharmaceutical compositions capable of eliciting a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic compositions comprise a neoantigen therapeutic agent described herein (e.g., a peptide, polynucleotide, TCR, CAR, cell comprising a TCR or CAR, dendritic cells comprising a polypeptide, dendritic cells comprising a polynucleotide, antibody, etc.) that corresponds to a tumor-specific antigen or neoantigen.

[0165]

[0193] In some embodiments, the pharmaceutical compositions described herein are capable of eliciting a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0194] In some embodiments, antigenic polypeptides or polynucleotides can be provided into antigen-presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient. In some embodiments, the antigen-presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells pulsed with a neo-antigenic peptide or nucleic acid. The neo-antigenic peptide can be any suitable peptide that generates an appropriate T cell response. In some embodiments, the T cells are CTLs. In some embodiments, the T cells are HTLs. Thus, one embodiment of the present disclosure is an immunogenic composition comprising at least one antigen-presenting cell (e.g., a dendritic cell) pulsed or loaded with one or more neo-antigenic polypeptides 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 can be loaded with the neo-antigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide may be any suitable polynucleotide capable of transducing dendritic cells, thereby resulting in presentation of neo-antigenic peptides and eliciting 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 related embodiments, the T cells are CTLs. In other related embodiments, the T cells are HTLs. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + In some embodiments, the therapeutic agent is a T cell. Such 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. Administration of either therapeutic agent may be performed simultaneously or sequentially in any order.

[0166]

[0195] In some embodiments, pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment can be formulated for parenteral, topical, nasal, oral, or local administration. In some embodiments, pharmaceutical compositions described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the compositions can be administered intratumorally. The compositions can be administered at a surgical resection site to elicit a localized immune response against the tumor. In some embodiments, compositions for parenteral administration are described herein, comprising a solution of neo-antigenic peptides, wherein the immunogenic composition is dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is or lyophilized, and the lyophilized formulation can be combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0167]

[0196] The ability of an adjuvant to enhance the immune response to an antigen is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity can be manifested by a significant increase in the titer of antibodies elicited against the antigen, and an increase in T cell activity can be manifested by increased cell proliferation or cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a predominantly humoral, i.e., T helper 2, response to a predominantly cellular, i.e., T helper 1, response.

[0168]

[0197] Suitable adjuvants are known in the art (see WO2015 / 095811) and include, but are not limited to, poly(I:C), polyICLC, STING agonists, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide These include ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® Vector System, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), which is derived from saponin, mycobacterium extracts, and synthetic bacterial wall mimics, as well as other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparation have been described (Dupuis M et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 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 can also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), promoting the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1β, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, 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).

[0169]

[0198] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in therapeutic settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide range of antigens, including peptide or protein antigens, live or killed viruses, immunogenic pharmaceutical compositions of dendritic cells, immunogenic pharmaceutical compositions of autologous cells, and polysaccharide conjugates in prophylactic and therapeutic immunogenic pharmaceutical compositions. Importantly, this enhances dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and the generation of potent cytotoxic T lymphocytes (CTLs) even in the absence of CD4+ T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit significant adjuvant activity when formulated or co-administered with other adjuvants, or even in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which is particularly useful for eliciting strong responses when the antigen is relatively weak. They can also accelerate immune responses, and in some experiments have been shown to reduce antigen doses while achieving comparable antibody responses to full-dose immunogenic pharmaceutical compositions without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, June 5, 2006, pp. 471-484). U.S. Patent No. 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to elicit antigen-specific immune responses. Commercially available CpG TLR9 antagonists include dSLIM (double stem-loop immunomodulator) by Mologen (Berlin, DE), which is a component of the pharmaceutical compositions described herein. Other TLR binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, can also be used.

[0170]

[0199] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., polyI:CI2U), polyIC:LC, non-CpG bacterial DNA or RNA, ssRNA40 for TLR8, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in connection with the present invention can be readily determined by one of ordinary skill in the art without undue experimentation. Further adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0171]

[0200] In some embodiments, immunogenic compositions according to the present disclosure may include two or more different adjuvants. Additionally, the present invention encompasses pharmaceutical compositions comprising any adjuvant agent, including any of the above or combinations thereof. In some embodiments, immunogenic compositions include neo-antigen therapeutics (e.g., peptides, polynucleotides, TCRs, CARs, cells comprising a TCR or CAR, dendritic cells comprising a polypeptide, dendritic cells comprising a polynucleotide, antibodies, etc.), and adjuvants may be administered individually and in any suitable order.

[0172]

[0201] Lipidation can be classified into several different types, such as N-myristoylation, palmatoylation, GPI-anchor addition, prenylation, and several additional modification types. N-myristoylation is the covalent attachment of myristate, a C14 saturated acid, to a glycine residue. Palmitoylation is the thioester linkage of a long-chain fatty acid (C16) to a cysteine ​​residue. GPI-anchor addition is the attachment of glycosylphosphatidylinositol (GPI) via an amide bond. Prenylation is the thioether linkage of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to a cysteine ​​residue. Further modification types may include the attachment of S-diacylglycerol via the sulfur atom of cysteine, O-octanoyl conjugation via serine or threonine residues, S-archeol conjugation to cysteine ​​residues, and cholesterol attachment.

[0173]

[0202] Fatty acids for producing lipidated peptides can include the group of C2-C30 saturated, monounsaturated, or polyunsaturated fatty acids. Exemplary fatty acids can include palmitoyl, myristoyl, stearoyl, and decanoyl groups. In some cases, lipid moieties with adjuvant properties are attached to a polypeptide of interest to induce or enhance immunogenicity in the absence of exogenous adjuvants. Lipidated peptides, i.e., lipopeptides, can be referred to as self-adjuvanting lipopeptides. Any of the fatty acids described above or elsewhere herein can induce or enhance the immunogenicity of a polypeptide of interest. Fatty acids capable of inducing or enhancing immunogenicity can include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.

[0174]

[0203] Polypeptides, such as naked or lipidated peptides, can be incorporated into liposomes. Sometimes, lipidated peptides can be incorporated into liposomes. For example, the lipid portion of a lipidated peptide can spontaneously integrate into the lipid bilayer of a liposome. That is, the lipopeptide can be displayed on the "surface" of the liposome.

[0175]

[0204] Liposomes can also be used to deliver nucleic acids to cells. The nucleic acid of interest includes one or more sequences encoding a T cell receptor. Liposomes can be used to deliver DNA or RNA. Liposomes can be used to deliver nucleic acids incorporated into vectors. The nucleic acid can be 50 to 200,000 nucleotides in length, or 100 to 500,000 nucleotides in length, or 20 to 500,000 nucleotides in length. Exemplary liposomes suitable for incorporation into the formulation include, but are not limited to, multilamellar vesicles (MLVs), oligolamellar vesicles (OLVs), unilamellar vesicles (UVs), small unilamellar vesicles (SUVs), medium-sized unilamellar vesicles (MUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), multivesicular vesicles (MVVs), single or oligolamellar vesicles prepared by reverse phase evaporation (REVs), multilamellar vesicles prepared by reverse phase evaporation (MLV-REVs), stable multilamellar vesicles (SPLVs), freeze-thawed MLVs (FATMLVs), vesicles prepared by extrusion (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydrated-rehydrated vesicles (DRVs), and bubblesomes (BSVs).

[0176]

[0205] Depending on the method of preparation, liposomes may be unilamellar or multilamellar and may vary in size, with diameters ranging from about 0.02 μm to greater than about 10 μm. Liposomes can adsorb many types of cells and then release incorporated agents (e.g., peptides described herein). In some cases, liposomes fuse with target cells, thereby allowing the contents of the liposome to enter the target cells. Liposomes can be endocytosed by phagocytic cells. Endocytosis can be followed by intraliposomal degradation of liposomal lipids and release of the encapsulated agent.

[0177]

[0206] Liposomes provided herein can also comprise carrier lipids.In some embodiments, carrier lipids are phospholipids.Carrier lipids that can form liposomes include but are not limited to dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS). Other suitable phospholipids include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), etc., or combinations thereof. In some embodiments, the liposomes further comprise a sterol (e.g., cholesterol) that modulates liposome formation. The carrier lipid can be any known non-phosphate polar lipid.

[0178]

[0207] The pharmaceutical compositions may also be encapsulated in liposomes using well known technology.Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of the present invention.

[0179]

[0208] Pharmaceutical compositions can be administered in liposomes or microspheres (or microparticles).The method of preparing liposomes and microspheres for administration to patients is well known to those skilled in the art.Basically, material is dissolved in aqueous solution, and suitable phospholipids and lipids are added, if necessary, together with surfactants, and the material is dialyzed or sonicated as necessary.

[0180]

[0209] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be tailored for passage directly from the gastrointestinal tract into the bloodstream, or compounds can be incorporated and the microspheres or composites of microspheres can be implanted for sustained release over periods ranging from days to months.

[0181]

[0210] 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 of the well-known techniques, carriers, and excipients suitable and 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.

[0182]

[0211] 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 ex vivo or in vivo methods. Ex vivo methods may include the use of autologous DCs pulsed ex vivo with a polypeptide described herein to activate or load the DCs prior to administration to a patient. In vivo methods may include targeting specific DC receptors using an antibody linked to a polypeptide described herein. DC-based immunogenic pharmaceutical compositions may further comprise a DC activator, such as a TLR3, TLR-7-8, and CD40 agonist. DC-based immunogenic pharmaceutical compositions may further comprise an adjuvant and a pharmaceutically acceptable carrier.

[0183]

[0212] Adjuvants can be used to enhance the immune response (humoral and / or cellular) elicited in a patient receiving the immunogenic pharmaceutical composition. Sometimes, adjuvants can elicit a Th1-type response. Other times, adjuvants can elicit a Th2-type response. A Th1-type response can be characterized by the production of cytokines such as IFN-γ, whereas a Th2-type response can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0184]

[0213] In some embodiments, lipid-based adjuvants, such as MPLA and MDP, can be used with the immunogenic pharmaceutical compositions disclosed herein. For example, monophosphoryl lipid A (MPLA) is an adjuvant that enhances the presentation of liposomal antigens to specific T lymphocytes. In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in conjunction with the immunogenic pharmaceutical preparations described herein.

[0185]

[0214] Adjuvants may include stimulatory 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), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed 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, and MAbCAM-. 1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, variant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Ap o-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, 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-I, JNK, interferon response genes, 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.

[0186]

[0215] Additional adjuvants include MCP-1, MIP-la, MIP-lp, IL-8, RANTES, 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, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, and erythrocyte serine / myocyte serine / myocyte stimulating factor. Cell 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 genes, 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 functional fragments thereof.

[0187]

[0216] In some embodiments, the adjuvant can be a toll-like receptor modulator. Toll-like receptor modulators include TLR9 agonists, but are not limited to small molecule toll-like receptor modulators such as imiquimod. Sometimes, the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene block copolymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Sometimes, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion can include at least one oil and at least one surfactant, and the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion can have a diameter of less than 5 μm, or even less than 1 μm. These small dimensions can be achieved by a microfluidizer, which provides a stable emulsion. Droplets with a diameter of less than 220 nm can be subjected to filtration sterilization.

[0188]

[0217] In some cases, immunogenic pharmaceutical compositions may include carriers and excipients (including, but not limited to, buffers, carbohydrates, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils of petroleum, animal, vegetable, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like, saline, aqueous dextrose and glycerol solutions, flavorings, coloring agents, and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifiers, wetting agents, and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. In other cases, the pharmaceutical formulation is substantially free of preservatives. In other cases, the pharmaceutical formulation may contain at least one preservative. Any suitable carrier known to those skilled in the art can be employed to administer the pharmaceutical compositions described herein, although it will be recognized that the type of carrier will vary depending on the mode of administration.

[0189]

[0218] The immunogenic pharmaceutical composition may include a preservative such as thimerosal or 2-phenoxyethanol. In some cases, the immunogenic pharmaceutical composition is substantially free of mercury-based materials (e.g., less than 10 μg / mL), e.g., thimerosal-free. As an alternative to mercury compounds, α-tocopherol succinate may be used.

[0190]

[0219] Physiological salts such as sodium salts can be included in the immunogenic pharmaceutical composition to control tonicity. Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, among others.

[0191]

[0220] The immunogenic pharmaceutical composition may have an osmolality ranging from 200 mOsm / kg to 400 mOsm / kg, 240 to 360 mOsm / kg, or 290 to 310 mOsm / kg.

[0192]

[0221] The immunogenic pharmaceutical composition may include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (particularly with aluminum hydroxide adjuvant), or citrate buffer, in some cases in the range of 5 to 20 or 10 to 50 mM.

[0193]

[0222] The pH of the immunogenic pharmaceutical composition can be about 5.0 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.8.

[0223] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be non-pyrogenic, for example, containing less than 1 EU (endotoxin unit, standard measurement) per dose, and may contain less than 0.1 EU per dose. The composition may be gluten-free.

[0194]

[0224] The immunogenic pharmaceutical composition may contain a surfactant, such as polyoxyethylene sorbitan ester surfactants (known as Tweens) or octoxynol (e.g., octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol). The surfactant may be present in trace amounts. The immunogenic pharmaceutical composition may contain less than 1 mg / mL of octoxynol-10 and polysorbate 80, each. Other trace residual components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0195]

[0225] The immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension in a suitable vehicle known in the art. The pharmaceutical composition can be sterilized by conventional, well-known sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged for immediate use or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.

[0196]

[0226] Pharmaceutical compositions comprising an active agent, such as immune cells described herein, combined with one or more adjuvants can be formulated to include a molar ratio. For example, a molar ratio of about 99:1 to about 1:99 can be used for an active agent, such as immune cells described herein, combined with one or more adjuvants. In some cases, the molar ratio range for an active agent, such as immune cells described herein, combined with one or more adjuvants can be selected from the following: about 80:20 to about 20:80; about 75:25 to about 25:75; about 70:30 to about 30:70; about 66:33 to about 33:66; about 60:40 to about 40:60; about 50:50; or about 90:10 to about 10:90. The molar ratio of an active agent, such as immune cells described herein, combined with one or more adjuvants can be about 1:9, and in some cases, about 1:1. Active agents, such as immune cells described herein, combined with one or more adjuvants can be formulated together in the same dosage unit, for example, in one vial, suppository, tablet, capsule, aerosol spray, or each agent, form, and / or compound can be formulated in separate units, for example, two vials, a suppository, a tablet, two capsules, a tablet and a vial, an aerosol spray, etc.

[0197]

[0227] In some cases, the immunogenic pharmaceutical composition can be administered with an additional agent. The choice of additional agent can depend, at least in part, on the condition being treated. The additional agent can include any agent that has a therapeutic effect against pathogen infection (e.g., a viral infection), including, for example, a checkpoint inhibitor such as an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agent (e.g., an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibody), or a drug used to treat an inflammatory condition, such as an NSAID, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, the checkpoint inhibitor can be a PD-1 / PD-L1 antagonist selected from the group consisting of nivolumab (ONO-4538 / BMS-936558, MDX1 106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulation may further include one or more supplements, such as vitamins C, E, or other antioxidants.

[0198]

[0228] Pharmaceutical compositions containing active agents, such as the immune cells described herein, combined with one or more adjuvants can be formulated in a conventional manner using one or more physiologically acceptable carriers, including, for example, excipients, diluents, and / or auxiliary agents that facilitate processing of the active agent into an administrable formulation. The appropriate formulation may depend, at least in part, on the selected route of administration. The agents described herein can be delivered to a patient using several routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular application, as well as by inhalation.

[0199]

[0229] Active agent can be formulated for parenteral administration (for example, by injection, for example, bolus injection or continuous infusion), and can be presented in the unit dosage form of ampule, pre-filled syringe, small volume injection, or multi-dose container with added preservative.Composition can take the form of suspension, solution, or emulsion in oily or aqueous vehicle, for example, solution in aqueous polyethylene glycol, etc.

[0200]

[0230] For injectable formulations, the vehicle can be selected from vehicles known in the art to be suitable, including aqueous solutions or oil suspensions, or emulsions containing sesame, corn, cottonseed, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles. The formulation may also contain biocompatible, biodegradable polymer compositions, such as poly(lactic-co-glycolic acid). These materials can be fabricated into microspheres or nanospheres, loaded with drugs, and further coated or derivatized to provide superior sustained-release performance. Suitable vehicles for periocular or intraocular injection include, for example, injection-grade water, liposomes, and suspensions of therapeutic agents in a vehicle suitable for lipophilic substances. Other vehicles for periocular or intraocular injections are known in the art.

[0201]

[0231] In some cases, pharmaceutical compositions are formulated according to routine procedures as pharmaceutical compositions adapted for intravenous administration to humans.Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.If necessary, compositions may contain solubilizing agents and local anesthetics such as lidocaine to alleviate pain at the injection site.Generally, the ingredients are supplied individually or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active ingredient.When the composition is administered by injection, the composition can be dispensed into an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be prepared, allowing the ingredients to be mixed before administration.

[0202]

[0232] When administration is by injection, the active agent can be formulated in aqueous solutions, particularly in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. In another embodiment, the pharmaceutical composition does not contain an adjuvant or any other substance added to enhance the immune response.

[0203]

[0233] In addition to the formulations described above, active agents can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation or transdermal delivery (for example, subcutaneous or intramuscular), intramuscular injection, or by using a transdermal patch.That is, for example, the agent can be formulated with a suitable polymeric or hydrophobic material (for example, as an emulsion in an acceptable oil), or with an ion exchange resin, or as a poorly soluble derivative, for example, a poorly soluble salt.

[0204]

[0234] In some cases, pharmaceutical compositions containing one or more agents exert a localized and regional effect when applied topically or injected at or near a specific site of infection. Direct topical application of, for example, viscous liquids, solutions, suspensions, dimethyl sulfoxide (DMSO)-based solutions, liposomal formulations, gels, jellies, creams, lotions, ointments, suppositories, foams, or aerosol sprays can be used for local administration to produce a localized and / or regional effect. Pharmaceutically suitable vehicles for such formulations include, for example, lower aliphatic alcohols, polyglycols (e.g., glycerol or polyethylene glycol), esters of fatty acids, oils, fats, silicones, and the like. Such formulations may also contain preservatives (e.g., p-hydroxybenzoic acid esters) and / or antioxidants (e.g., ascorbic acid and tocopherol). See also Dermatological Formulations: Percutaneous absorption, Barry (ed.), Marcel Dekker Inc., 1983. In another embodiment, topical / external formulations containing delivery agents, carriers, or ion channel inhibitors are used to treat viral infections of the epidermis or mucosa.

[0205]

[0235] The pharmaceutical composition may contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, light-blocking agents, odor absorbers, and dyes. The amounts of these various adjuvants are those conventionally used in the field under consideration, for example, from about 0.01% to about 20% of the total weight of the composition. Depending on the nature of the adjuvants, these adjuvants can be introduced into the fatty phase, the aqueous phase, and / or the lipid vesicles.

[0206] IX. Treatment Methods

[0236] Provided herein are methods of using any of the above-disclosed nucleic acids, a vector comprising any of the above-disclosed nucleic acid sequences, a protein encoded by any of the above-disclosed nucleic acids, or a host cell disclosed above for the manufacture of a medicament for treating an immune disease or cancer.

[0207]

[0237] Also provided herein are methods of treating a subject having a disease, disorder, or condition. The method of treatment may include administering a pharmaceutical composition disclosed herein to a subject having the disease, disorder, or condition. The present disclosure provides methods of treatment including immunogenic therapy. Methods of treating a disease (e.g., cancer or viral infection) are provided. The method may include administering to the subject an effective amount of a pharmaceutical composition comprising T cells specific for an immunogenic antigen. In some embodiments, the antigen comprises a tumor antigen.

[0208]

[0238] In some embodiments, a method for treating a subject having a disease or condition comprises administering a pharmaceutical composition disclosed herein to the subject. In some embodiments, the method is a method for preventing resistance to cancer therapy, comprising administering a pharmaceutical composition disclosed herein to a subject in need of such prevention. In some embodiments, the method is a method for eliciting an immune response, comprising administering a pharmaceutical composition disclosed herein to a subject in need of such prevention. In some embodiments, the immune response is a humoral response. In some embodiments, the immune response is a cytotoxic T cell response.

[0209]

[0239] In some embodiments, the subject has cancer, and the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL).

[0210]

[0240] In some embodiments, the subject has breast cancer that is resistant to anti-estrogen therapy. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has CLL that is resistant to ibrutinib therapy. In some embodiments, the CLL expresses a Bruton's tyrosine kinase with a mutation, for example, a C481S mutation. In some embodiments, the subject has lung cancer that is resistant to tyrosine kinase inhibitors. In some embodiments, the lung cancer expresses an epidermal growth factor receptor (EGFR) with a mutation, such as a T790M, L792F, or C797S mutation.

[0211]

[0241] In some embodiments, the method further comprises administering at least one additional therapeutic agent or treatment. In some embodiments, the at least one additional therapeutic agent or treatment is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, 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 administration of the pharmaceutical composition disclosed herein.

[0212]

[0242] In some other aspects, provided herein is the use of a pharmaceutical composition for the manufacture of a medicament for use in therapy. In some embodiments, the method of treatment comprises administering to a subject an effective amount of T cells that specifically recognize the immunogenic neo-antigenic peptide. In some embodiments, the method of treatment comprises administering to a subject an effective amount of TCRs, e.g., TCRs expressed in T cells, that specifically recognize the immunogenic neo-antigenic peptide.

[0213]

[0243] In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial or uterine carcinoma, salivary gland cancer, kidney or renal cancer, and the like. 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's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel proliferation with phakomatosis, edema, Meigs' syndrome, and combinations thereof.

[0214]

[0244] The methods of the present disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers to be treated by the methods of the present disclosure can include melanoma (e.g., metastatic malignant melanoma), renal cancer (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, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

[0215]

[0245] Furthermore, the diseases or conditions provided herein include refractory or recurrent malignant diseases whose growth can be inhibited using the treatment methods of the present disclosure. In some embodiments, the cancer to be treated by the treatment methods of the present disclosure is selected from the group consisting of carcinoma, squamous cell carcinoma, adenoma, sarcoma, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital 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, blood cancer, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, cancers to be treated by the methods of the present disclosure include, for example, carcinoma, squamous cell carcinoma (e.g., cervix, eyelid, conjunctiva, vagina, lung, oral cavity, skin, bladder, tongue, larynx, and esophagus), and adenocarcinoma (e.g., prostate, small intestine, endometrium, cervix, large intestine, lung, pancreas, esophagus, rectum, uterus, stomach, breast, and ovary). In some embodiments, cancers to be treated by the methods of the present disclosure further include sarcoma (e.g., myogenic sarcoma), leukemia, neuroma, melanoma, and lymphoma. In some embodiments, cancers to be treated by the methods of the present disclosure are breast cancer. In some embodiments, cancers to be treated by the treatment methods of the present disclosure are triple-negative breast cancer (TNBC). In some embodiments, cancers to be treated by the treatment methods of the present disclosure are ovarian cancer. In some embodiments, cancers to be treated by the treatment methods of the present disclosure are colorectal cancer.

[0216]

[0246] In some embodiments, the patient or patient population to be treated with the pharmaceutical composition of the present 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 population to be treated with the pharmaceutical composition of the present disclosure has a hematological cancer. In some embodiments, the patient has a hematological cancer such as diffuse large B-cell lymphoma ("DLBCL"), Hodgkin's lymphoma ("HL"), non-Hodgkin's lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML"), or multiple myeloma ("MM"). In some embodiments, the patient or patient population to be treated has a cancer selected from the group consisting of ovarian cancer, lung cancer, and melanoma.

[0217]

[0247] Specific examples of cancers that can be prevented and / or treated in accordance with the present disclosure include, but are not limited to, the following: renal cancer, kidney cancer, glioblastoma multiforme, metastatic breast cancer, breast cancer, mammary sarcoma, neurofibroma, neurofibromatosis, childhood tumors, neuroblastoma, malignant melanoma, epithelial carcinoma, leukemias, including but not limited to acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, including myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia and myclodysplastic syndromes, chronic leukemias, including but not limited to chronic Myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, polycythemia vera, lymphomas, including but not limited to Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, including but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma, Waldenstrom's macroglobulinemia, monoclonal gammopathy of undetermined significance, benign monoclonal gammopathy, heavy chain disease, bone cancer and bone marrow disease Mixed tissue sarcomas, including but not limited to osteosarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors, including but not limited to glioblastoma, astrocytoma, brain stem glioma, ependyma breast cancer, including but not limited to, cell tumors, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; adenoma, lobular (small cell) carcinoma, intraductal 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 gland cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, including but not limited to papillary or follicular thyroid cancer;medullary and anaplastic thyroid cancer, pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors, pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetic urinary retention, eye cancer, including but not limited to ocular melanoma, e.g., iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma, vaginal cancer, e.g., squamous cell carcinoma, adenocarcinoma, and melanoma, vulvar cancer, e.g., Cancers of the esophagus, 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; cancers of the stomach, including but not limited to adenocarcinoma, fungus ( polypoid), ulcerative, superficial spreading, diffuse spreading, 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, including adenocarcinoma, cholangiocarcinoma, 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 (squamous cell carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer, lung cancer, testicular cancer, including but not limited to germinal tumor, seminoma, undifferentiated cancer, including but not limited to, androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile 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 warts; skin cancer, including but not limited to, basal cell carcinoma;Included are squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, keratinizing patchy melanoma, renal cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal gland tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus), renal carcinoma, Wilms' tumor, bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovial tumor, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma.

[0218]

[0248] Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha, gamma, and mu chain diseases, benign monoclonal gammopathy and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendicitis cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancers of the blood tissue, and others. Other non-limiting examples of cancer types to which the methods encompassed by the present disclosure can be applied include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, 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 adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, Cancers include testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias such as acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyeloblastic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the methods of the present disclosure includes an epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer, hi other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers are characterized in various ways, including but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or anaplastic. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including but not limited to mantle cell lymphoma. Lymphoproliferative disorders are also considered proliferative diseases.

[0219]

[0249] In some embodiments, the subject has breast cancer that is 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, or any combination thereof.

[0220]

[0250] In some embodiments, the breast cancer expresses a mutated estrogen receptor.

[0251] In some embodiments, the cancer is recurrent or metastatic breast cancer. In some embodiments, the cancer to be treated by the treatment methods of the present disclosure is triple-negative breast cancer (TNBC).

[0221]

[0252] The pharmaceutical compositions provided herein may be used alone or in combination with conventional treatment regimens, such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0222]

[0253] In some embodiments, at least one or more chemotherapeutic agents may be administered in addition to the pharmaceutical composition comprising the immunogen therapy. In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.

[0223]

[0254] In carrying out the methods of treatment or use provided herein, a therapeutically effective amount of the pharmaceutical composition can be administered to a subject with a disease or condition. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors.

[0224]

[0255] The subject may be, for example, a mammal, a human, a pregnant woman, an elderly person, an adult, a juvenile, a pre-adolescent, a child, a toddler, an infant, a newborn or neonate. The subject may be a patient. In some cases, the subject may be a human. In some cases, the subject may be a child (e.g., a pre-adolescent). In some cases, the subject may be an infant. In some cases, the subject may be a formula-fed infant. In some cases, the subject may be an individual enrolled in a clinical trial. In some cases, the subject may be a laboratory animal, such as a mammal or a rodent. In some cases, the subject may be a mouse. In some cases, the subject may be an obese or overweight subject.

[0225]

[0256] In some embodiments, the subject has been previously treated with one or more different cancer treatment methods.In some embodiments, the subject has been previously treated with one or more radiation therapy, chemotherapy or immunotherapy.In some embodiments, the subject has been previously treated with one, two, three, four or five previous therapies.In some embodiments, the previous therapy is cytotoxic therapy.

[0226]

[0257] In some embodiments, the disease or condition that can be treated by the methods disclosed herein is abnormal cell proliferation. In some embodiments, the disease or condition that can be treated by the methods disclosed herein is cancer. In some embodiments, the cancer is a malignant cancer. In some embodiments, the cancer is a benign cancer. In some embodiments, the cancer is an invasive cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid cancer.

[0227]

[0258] The methods of the present disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers to be treated by the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (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, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other de novo malignancies.

[0228]

[0259] Furthermore, the diseases or conditions provided herein include refractory or recurrent malignant diseases whose growth can be inhibited using the methods of treatment of the present disclosure. In some embodiments, the cancer to be treated by the methods of treatment of the present disclosure is selected from the group consisting of 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 anogenital 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, blood cancer, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, cancers to be treated by the methods of the present disclosure include, for example, carcinoma, squamous cell carcinoma (e.g., cervix, eyelid, conjunctiva, vagina, lung, oral cavity, skin, bladder, tongue, larynx, and esophagus), and adenocarcinoma (e.g., prostate, small intestine, endometrium, cervix, large intestine, lung, pancreas, esophagus, rectum, uterus, stomach, breast, and ovary). In some embodiments, cancers to be treated by the methods of the present disclosure further include sarcoma (e.g., myogenic sarcoma), leukemia, neuroma, melanoma, and lymphoma. In some embodiments, cancers to be treated by the methods of the present disclosure are breast cancer. In some embodiments, cancers to be treated by the treatment methods of the present disclosure are triple-negative breast cancer (TNBC). In some embodiments, cancers to be treated by the treatment methods of the present disclosure are ovarian cancer. In some embodiments, cancers to be treated by the treatment methods of the present disclosure are colorectal cancer.

[0229]

[0260] In some embodiments, the patient or patient population to be treated with the pharmaceutical composition of the present 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 population to be treated with the pharmaceutical composition of the present disclosure has a hematological cancer. In some embodiments, the patient has a hematological cancer such as diffuse large B-cell lymphoma ("DLBCL"), Hodgkin's lymphoma ("HL"), non-Hodgkin's lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML"), or multiple myeloma ("MM"). In some embodiments, the patient or patient population to be treated has a cancer selected from the group consisting of ovarian cancer, lung cancer, and melanoma.

[0230]

[0261] Specific examples of cancers that can be prevented and / or treated in accordance with the present disclosure include, but are not limited to, the following: renal cancer, kidney cancer, glioblastoma multiforme, metastatic breast cancer, breast cancer, breast sarcoma, neurofibroma, neurofibromatosis, childhood tumors, neuroblastoma, malignant melanoma, epithelial carcinoma, leukemias, including but not limited to acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, including myeloblastic, promyelocytic, myelomonocytic, mononuclear, erythroleukemia and myelodysplastic syndromes, chronic leukemias, including but not limited to chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, and myelodysplastic syndromes. leukemia, hairy cell leukemia, polycythemia vera, lymphomas, including but not limited to Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, including but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma, Waldenstrom's macroglobulinemia, monoclonal gammopathy of undetermined significance, benign monoclonal gammopathy, heavy chain disease, bone cancer and connective tissue sarcoma, including but not limited to bone Sarcomas, myeloma bone disease, multiple myeloma, cholesteatoma-induced osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma, brain tumors, including but not limited to glioblastoma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma breast cancer, including but not limited to, thyroid cancer, including but not limited to, thyroid carcinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; adenoma, lobular (small cell) carcinoma, intraductal 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 gland cancer, including but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, including but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer;cancers of the pituitary gland, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors; cancers of the pituitary gland, including but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetic urinary retention; cancers of the eye, including but not limited to, ocular melanoma, e.g., iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; cancers of the vagina, e.g., squamous cell carcinoma, adenocarcinoma, and melanoma; cancers of the vulva, e.g., squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma and Paget's disease, cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma, uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma, ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors, cervical cancer, 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, fungiform (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma, colon cancer, colorectal cancer, RAS-mutated colorectal cancer, colon cancer, rectal cancer, liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, including adenocarcinoma, cholangiocarcinoma, including but not limited to papillary, nodular, and diffuse, lung cancer, including RAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer, lung cancer, testicular cancer, including but not limited to embryonal tumors, seminoma, undifferentiated, classic (typical), spermatocytic, non-seminocytic, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, including but not limited to androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma, penile 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 warts, skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma,Keratinizing patchy melanoma, renal cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal nephroma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus), kidney carcinoma, Wilms' tumor, bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Further cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovial tumor, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma.

[0231]

[0262] In some embodiments, a subject with a mutation in a RAS gene is treated for cancer by administering a pharmaceutical composition comprising a TCR that recognizes a RAS mutant epitope in complex with an MHC protein encoded by the subject's HLA allele, wherein the TCR has a TCR alpha chain variable region and a TCR beta chain variable region with the amino acid sequence disclosed herein. In some embodiments, the pharmaceutical composition comprises a nucleic acid sequence encoding the TCR. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is messenger RNA encoding a TCR having a TCR alpha chain variable region and a TCR beta chain variable region with the amino acid sequence disclosed herein. In some embodiments, the pharmaceutical composition comprises a vector capable of driving expression of the TCR, comprising a nucleic acid sequence encoding the TCR, wherein the TCR recognizes a RAS mutant epitope in complex with an MHC protein encoded by the subject's HLA allele, and wherein the TCR has a TCR alpha chain variable region and a TCR beta chain variable region with the amino acid sequence disclosed herein. In some embodiments, the pharmaceutical composition comprises cells comprising a nucleic acid sequence encoding a TCR that recognizes a RAS mutant epitope in complex with an MHC protein encoded by a subject's HLA allele, wherein the TCR has a TCR alpha chain variable region and a TCR beta chain variable region having the amino acid sequences disclosed herein.In some embodiments, a subject with cancer is administered a pharmaceutical composition comprising a TCR that recognizes a RAS mutant epitope in complex with an MHC protein encoded by the subject's HLA allele, wherein the TCR has a TCR alpha chain variable region and a TCR beta chain variable region having an amino acid sequence disclosed herein, and the cancer is selected from the group consisting of adenocarcinoma of the bile duct, transitional cell carcinoma of the bladder, breast cancer, cervical adenocarcinoma, colon adenocarcinoma, colon adenoma, neuroblastoma (autonomic ganglion), acute myeloid leukemia, chronic myeloid leukemia, and the like. leukemia, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's 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, germinoma, seminoma, anaplastic carcinoma, follicular adenocarcinoma, papillary carcinoma, and Hürthle cell carcinoma.

[0232]

[0263] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0233] Example 1: Workflow for identification and validation of antigen-specific T cells

[0264] This example describes an exemplary workflow for generating and using antigen peptide-specific TCRs. Figure 1 shows a schematic representation of the workflow. Figure 2 details an exemplary timeline of the workflow process. The process begins with obtaining and culturing PBMCs from a subject, followed by stabilization in overnight culture and incubation in the presence of peptide and cytokines. Antigen-presenting cells (APCs) derived from monocytes and DCs help elicit T cells in response to the peptide antigen presented by the APCs. For 10 days to 2 weeks, the antigen-specific T cells are expanded and selected for antigen specificity and activation (e.g., expression of CD8+ markers indicates the generation of cytotoxic T lymphocytes). Activated antigen-responsive T cells contain antigen-specific TCRs.

[0234]

[0265] Briefly, a mutated peptide containing a mutant target epitope (e.g., a mutant RAS peptide) was synthesized using a peptide synthesizer. The synthesized peptide was used to load APCs and stimulate T cells derived from a sample of peripheral blood mononuclear cells (PBMCs) from a healthy donor. Neoantigen-specific T cells can also be obtained using PBMCs from patients with the neoantigen of interest. After incubation with peptide-loaded APCs, the T cell population was analyzed by flow cytometry. Antigen-specific T cells were isolated using flow cytometry (Figures 1, 2, 4A, 5, and 6). Single-cell TCR sequencing was performed on the isolated antigen-specific T cells using the 10x Genomics Single Cell V(D)J system to profile the sequences of the isolated antigen-specific TCRs. Sequencing reads were analyzed using the Cell Ranger™ analysis pipeline, and candidate TCR sequences were selected for further analysis and functional assays (Figures 1 and 6). Candidate TCR sequences can also be removed if the beta chain CDR3 sequence comprises any one of SEQ ID NOs: 46 to 68 before the candidate is subjected to any further analysis and functional assays described herein. For example, if a candidate TCR comprises a beta chain CDR3 sequence of any one of SEQ ID NOs: 46 to 68, the candidate TCR can be removed without being subjected to further analysis or functional assays so that the ultimately identified TCR does not comprise a beta chain CDR3 sequence of any one of SEQ ID NOs: 46 to 68.

[0235]

[0266] To analyze the functionality of TCRs, HEK293T or A375 cells were transduced with vectors or mRNA encoding RAS peptides containing the target mutant epitopes to generate antigen-producing cell lines. Jurkat cells, TCRβ-deficient Jurkat cells, or PBMCs from healthy donors were transduced with nucleic acids encoding candidate TCRs (Figure 3A) in lentiviral vectors. Exemplary vectors are shown in Figure 3B. Figures 4B and 6 show functional assays of cells transduced with vectors expressing TCRs. For antigen recognition assays to analyze the functionality of candidate TCRs, cell lines expressing RAS antigens were cocultured with TCR-transduced Jurkat cells, TCRβ-deficient Jurkat cells, or PBMCs from healthy donors (Figures 10 or 11).

[0236] Example 2: Obtaining antigen-specific T cells

[0267] Antigen-specific T cells were expanded using in vitro T cell induction. Healthy human donor PBMCs were seeded into each well of a 24-well plate in AIM V medium (Invitrogen). After 24 hours of incubation, mutant RAS peptide, TNF-α, IL-1β, PGE1, and IL-7 were added to the wells. The culture medium was replaced with fresh medium every two days. Antigen-specific T cells were evaluated and isolated on days 10–20 (Figure 2).

[0237] Example 3: Staining and sorting of HLA multimers

[0268] Neoantigen-specific T cells were detected by combining HLA multimer staining with two different fluorochrome-conjugated recombinant HLA (e.g., HLA-A11.01 or HLA-C01.02) multimers and neoantigen peptides. For cell surface staining, anti-CD8, anti-CD4, anti-CD19, anti-CD16, anti-CD14, anti-CD56 antibodies, and Live / Dead IR dye (Invitrogen) were used. + T cells are CD8 + CD4 - CD19 - CD16 - CD14 - CD56 -IR - (Figures 1, 2, 4A, 5, and 6). For selection, 5 × 10 6 Cells were incubated with 1-20 μg of multimer in 100 μL of PBS + 0.5% human serum. Cells were stained with antibodies and Live / Dead IR dye for an additional 30 minutes. After staining, cells were washed twice and diluted with PBS + 0.5% human serum. Live / Dead IR dye-negative cells were gated and analyzed by FACSAria cell sorter (BD Biosciences). + / multimer + T cells were sorted.

[0238] Example 4: Deep sequencing of antigen-specific T cells

[0269] Antigen-specific T cells were sorted into a mixture of PBS and 2% FBS (Hyclone Defined), and RNA from single T cells was genetically barcoded using the 10x Genomics V(D)J kit. RNA libraries were prepared according to the manufacturer's protocol. The resulting libraries were sequenced using the IllumiNova MiSeq platform. The sequencing data were then analyzed using 10x Genomics analysis software to obtain paired TCR alpha and beta sequences. Dominant clones were identified based on the frequency of cells sharing a common TCR alpha and / or TCR beta (Figures 1 and 6).

[0239] Example 5: Synthesis and cloning of antigen-specific TCR genes

[0270] Figure 3A shows a schematic diagram of an exemplary vector design incorporating TCR alpha and beta chain constructs, which contain nucleic acid sequences encoding the variable (V), diversity (D, in the beta chain construct only), joining (J), and constant (C) regions for the TCR alpha (α) and beta (β) chains, respectively, as shown in the diagram. In the vector, upstream regulatory elements contain elements required for expression of the incorporated nucleic acid sequences, including, for example, a promoter (e.g., the EF1a region shown in the diagram), and further incorporate sequences encoding F2A and P2A proteolytic cleavage sites and puromycin resistance (Puro) for puromycin-mediated selection. Lentiviral vectors were constructed from pCDH-EF1-T2A-Puro (SBI System Bioscience). Antigen-specific TCR lentiviral vectors were generated by inserting the TCR beta variable region, followed by the TCR beta murine constant region, a furin cleavage site, an SGSG linker, an F2A site, a TCR alpha variable region, a TCR alpha constant region, a T2A site, and a puromycin resistance gene (Figure 3A and Figure 3B).

[0240] Example 6: Transfection and transduction

[0271] Lentiviruses encoding antigen-specific TCRs were prepared by transient transfection of 293T cells. 7 × 10 cells were transfected into 10 cm plates. 6 Cells were seeded with 1000 kJ / ml and transfected 16 hours later with 7 μg of lentiviral vector, 7 μg of packaging plasmid mixture (pPAX and pMD2.G), 28 μL of Fugene (Promega), and 1 mL of Opti-MEM (Gibco). One day after transfection, the culture medium was replaced. 72 hours after transfection, the supernatant was harvested and concentrated 10-fold.

[0241]

[0272] Jurkat cells or PBMCs were transduced with concentrated lentivirus encoding TCR sequences. Jurkat cells were washed and resuspended in RPMI-1640 containing polybrene and 10% FBS. 5 x 10 cells were transduced per well in 100 μL of medium. 5 CD8-Jurkat cells were seeded into a 96-well plate and 25 μL of concentrated lentivirus was added. The cells were centrifuged at 2400 revolutions per minute (rpm) for 1 hour and incubated in a CO2 incubator. The cells were transduced again with fresh medium containing polybrene and FBS and 25 μL of concentrated lentivirus. 24 hours after the second transduction, the medium was replaced with RPMI 1640 containing 10% FBS and Pen / Strep. Puromycin treatment (1 μg / ml) was initiated 4 days after transduction.

[0242] Example 7: Binding of TCR-transduced Jurkats to HLA peptides

[0273] To assess TCR transduction, transduced Jurkat cells or PBMCs were stained with fluorochrome-conjugated multimers (HLA-neoantigens), anti-CD8 antibodies, anti-mTCR constant region antibodies, and live / dead IR dye. Cells positive for mTCR and multimers were measured by flow cytometry (Figure 4B and Figures 6-8). Figure 7 shows that Jurkat cells transduced with a TCR specific for the RAS G12V peptide differentially expressed a TCR (i.e., RAS-TCR-1) compared with untransduced Jurkat cells. Figure 8 shows that four different PBMC lines, each transduced with a TCR specific for the RAS G12V peptide, differentially expressed a TCR compared with untransduced PBMC lines. Figure 9 shows that Jurkat cells electroporated with TCRs specific for two other RAS G12V peptides (i.e., RAS-TCR-2 and RAS-TCR-3) differentially expressed TCRs compared to non-electroporated Jurkat cells.

[0243] Example 8: Peptide loading of target cells and functional affinity of TCRs specific for RAS mutant peptides

[0274] For pulse stimulation with exogenous peptides, 1 × 10 6 T2, 293T, or A375 cells were cultured in 1×10 PBS containing 10 pg / mL human β2-microglobulin (Calbiochem) and 1×10 -5 M~1×10 -12 The cells were incubated with varying amounts of RAS peptide ranging from 10 μM for 2 hours at 37°C in 5% CO2. -5 M influenza peptide GIL (influenza matrix protein 58-66 T2 cells pulsed with GILGFVTL (Metabion) served as a negative control. After washing, peptide-loaded T2 cells were used in TCR activation assays.

[0244]

[0275] As shown in Figure 10, to examine the affinity and specificity of the TCR specific for the RAS mutant peptide, Jurkat cells transduced with and expressing a TCR specific for the mutant RAS peptide were cocultured with HLA-A11:01+ target cells pulsed with the RAS G12V 9mer (VVGAVGVGK (SEQ ID NO: 43); left panel), the RAS G12V 10mer (VVVGAVGVGK (SEQ ID NO: 44); right panel), or the RAS WT peptide (data points on the far right of both panels). CD69 was measured as a marker of activation (shown on the Y axis in Figure 10 as the percentage of CD69+ cells relative to surviving TCR+CD3+ cells). Only the mutant G12V peptide, but not the WT RAS peptide, was able to induce activation of the transduced Jurkat cells, demonstrating the specificity of the TCR for the mutant RAS peptide. Furthermore, the affinities of the 9mer and 10mer were found to be 3.8 nM and 9.6 nM, respectively.

[0245]

[0276] Similarly, as shown in Figure 18, Jurkat cells transduced with and expressing TCRs (TCR-5, TCR-6, TCR-7) specific for mutant RAS peptides were cocultured with HLA-C05:01+ target cells pulsed with Ras G12D 10-mer (GADGVGKSAL (SEQ ID NO: 132)) or WT peptide. CD69 was measured as a marker of activation (shown on the Y-axis in Figure 18 as the percentage of CD69+ cells relative to total surviving cells). The results indicated that the TCRs were reactive.

[0246]

[0277] Furthermore, to examine the affinity and specificity of TCRs specific for other RAS mutant peptides, Jurkat cells electroporated with and expressing TCRs specific for each mutant RAS peptide (RAS-TCR-2 and RAS-TCR-3, left and right panels, respectively) were cocultured with HLA-C01:02+ target cells pulsed with the RAS G12V mutant peptide (AVGVGKSAL (SEQ ID NO: 45)) or the RAS WT peptide (data points on the far right of both panels). CD69 and PD1 were measured as markers of activation (shown on the Y axis in Figure 11 as the percentage of CD69+PD1+ cells relative to surviving TCR+CD3+ cells). Only the mutant G12V peptide, but not the WT RAS peptide, was able to induce activation of transduced Jurkat cells, demonstrating the specificity of the TCR for the mutant RAS peptide.

[0247]

[0278] Figure 16 illustrates exemplary data demonstrating the functional affinity of RAS TCR-2 and RAS TCR-3. Transduced Jurkat NFAT luciferase reporter cells expressing RAS TCR-2 or RAS TCR-3 were pulsed with KRAS G12V 9mer or KRAS WT peptide and then stimulated with HLA-C * These T cells were co-cultured with 01:02+ target cells. Relative luciferase units were measured as a marker of T cell activation. Both RAS TCR-2 and RAS TCR-3 were specific and showed dose-dependent activation.

[0248] Example 9: Target cells expressing neoantigens

[0279] 1 x 10 for target cells expressing endogenous antigens 6 HEK293 or A375 cells were transduced with lentiviral vectors encoding the RAS mutant peptides, and the transduced target cells were selected with puromycin (1 μg / ml) in culture medium (DMEM containing 10% FBS).

[0249] Example 10: IL-2 release assay

[0280] To examine specificity, T cell clones (2.5 × 10 in 50 μL) were used. 5 cells) were transfected with HEK293T, HLA-A03.01-transduced A375, or HLA-A11.01-transduced A375 cell lines (5 × 10 in 50 μL). 4 Cells) and RAS mutant peptide neoantigens. After 24 hours of co-culture, culture supernatants are harvested and IL-2 concentrations are assessed by standard MSD using the V-PLEX Human IL-2 assay (Meso Scale Discovery).

[0250] Example 11: IFN-γ release assay

[0281] To examine specificity, T cell clones (2 × 10 in 100 μL) were used. 3 Cells) are incubated with a cell line expressing a RAS mutant peptide neoantigen. After 24 hours of co-culture, culture supernatants are harvested and IFN-γ secretion is assessed by standard ELISA using the OptEIA™ Human IFN-γ Set (BD Biosciences Pharmingen).

[0251] Example 12: Cytotoxicity assay

[0282] The cytotoxic activity of T cell clones can be analyzed by a standard 4-hour chromium 51 release assay. Briefly, 1 x 10 6 target cells with 100 μCi of Na 51Labeling can be performed with CrO4 (ICN Biochemicals) for 1 to 1.5 hours. 51 Cr-labeled target cells can be cultured with T cells in RPMI 1640 containing 12% FCS. To determine functional avidity, 1 × 10 4 1 × 10 T cells were loaded with varying amounts of RAS mutant peptide neoantigens and pulsed with peptide. 3 In addition to the T2 cells, a constant E:T ratio of 10:1 can be achieved.

[0252]

[0283] After 4 hours of co-incubation at 37°C, 50 μL of supernatant can be collected and radioactivity can be measured in a gamma counter. The percentage of specific cell lysis can be calculated as 100 × (experimental release - spontaneous release) / (maximum release - spontaneous release). Spontaneous release can be assessed by incubating target cells in the absence of effector cells. To calculate the percentage of relative cell lysis, the maximum percentage of specific cell lysis is set as a reference value of 100%. Corresponding values ​​can be calculated relative to this reference value. To determine half-maximal cell lysis, the percentage of relative cell lysis can be plotted against peptide concentration. The peptide concentration at which the curve intersects with 50% relative cell lysis can be considered as the value of half-maximal cell lysis.

[0253] Example 13: Cell killing assay

[0284] PBMCs were transduced with RAS neoantigen-specific recombinant TCRs and their ability to kill cancer cell lines was analyzed (Figure 12). Cells expressing the recombinant TCR (rTCR) showed significantly higher killing of cancer cell lines compared to controls. The SW620 cell line, which naturally expresses the KRAS G12V mutation, was used as the target cell. HLA-A11:01 was introduced into the SW620 cell line by lentiviral transduction. rTCR-transduced PBMCs were cocultured with two different SW620 cell lines, with or without HLA-A11:01 expression, for 100 hours. Supernatants and cells were then harvested at various time points, and cytokine secretion (data not shown) and SW620 cell proliferation were analyzed.

[0254]

[0285] Cytotoxic activity was assessed by co-culturing cells expressing a TCR specific for a mutant RAS peptide on a specific HLA with target cancer cells transduced with the mutant RAS peptide and expressing the corresponding HLA. The target cancer cells were specifically measured for Annexin V, an apoptosis marker, and the relative proliferation of the target cells was determined (Figure 12). Target cancer SW620 cells were engineered to express the mutant peptide along with the appropriate MHC-I allele. SW620 target tumor cells cultured alone or co-cultured with PBMCs transduced with an irrelevant TCR served as negative controls. Mock-transduced target cells (e.g., not expressing the mutant peptide) also served as negative controls. Cells were also transduced to stably express GFP, allowing target cell proliferation to be tracked. PBMCs from healthy donors used as effector cells were transduced to express a TCR specific for the mutant RAS peptide. Target and effector cells were co-cultured at a ratio of 10:1 for 100 hours in medium containing Annexin V detection reagent. GFP and Annexin V signals were measured over time using an IncuCyte S3 instrument. Annexin V signals originating from effector cells were excluded by size exclusion. Target cell proliferation and death were measured as the area (mm ) of GFP and Annexin V over time, respectively. 2 ) (Y-axis in Figure 12).

[0255]

[0286] Specific killing of target cells was also observed when T cells were used in killing assays. Cytotoxic activity was assessed by co-culturing CD4+ T cells isolated from PBMCs expressing TCRs specific for mutant RAS peptides on specific HLA-associated antigens with target cancer cells transduced with the mutant RAS peptides and expressing the corresponding HLA-associated antigens, specifically measuring Annexin V, an apoptosis marker, in the target cancer cells and determining the relative proliferation of the target cells (Figure 13). Target cancer SW620 cells were engineered to express the mutant peptides along with the appropriate MHC-I alleles. SW620 target tumor cells cultured alone or co-cultured with CD4+ T cells isolated from PBMCs not transduced with TCRs specific for the RAS mutant peptides served as negative controls. Mock-transduced target cells (e.g., not expressing the mutant peptides) could also be used as negative controls. Target and effector cells were co-cultured at ratios of 10:1, 5:1, 3:1, and 1:1 for 100 hours in medium containing Annexin V detection reagent. As shown in Figure 13, increasing the amount of CD4+ T cells relative to target cells induced increased cell killing in target cells expressing mutant RAS peptides. Because target cell killing was induced by isolated CD4+ T cells co-cultured with target cancer cells, this data also suggests that killing is not due to CD8 protein.

[0256]

[0287] In some cases, after co-culture, HLA-A11:01 or HLA-C01:02 transduced SW620 cell lines (Ras mut For the cell line + HLA-A11:01 or HLA-C01:02 group, untransduced SW620 (Ras mutSignificantly higher levels of IFNγ, IL-2, and TNFα can be detected in the rTCR-transduced SW620 cell lines compared to the control. Furthermore, a significantly higher percentage of caspase-3 (a marker of apoptosis) positive cells can be observed for the HLA-A11:01- or HLA-C01:02-transduced SW620 cell lines compared to the control. Thus, rTCR-transduced PBMCs are not only capable of secreting cytokines (IFNγ, IL-2, and TNFα), but are also functionally capable of specifically killing target cells.

[0257]

[0288] In some cases, cytotoxic activity is assessed prior to TCR cloning. T cells elicited against mutant RAS peptides on a specific HLA are co-cultured with mutant target cancer cells expressing the corresponding HLA loaded with a range of RAS mutant peptide concentrations. Both the relative proliferation of the target cancer cells and Annexin V, an apoptosis mar...

Claims

1. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:

6.

2. 2. The recombinant nucleic acid of claim 1, wherein the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:

4.

3. 3. The recombinant nucleic acid of claim 1 or 2, wherein the TCR beta chain construct further comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence set forth in SEQ ID NO:

5.

4. 4. A recombinant nucleic acid according to any one of claims 1 to 3, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

12.

5. 5. A recombinant nucleic acid according to any one of claims 1 to 4, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:

12.

6. 6. A recombinant nucleic acid according to any one of claims 1 to 5, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

12.

7. 7. A recombinant nucleic acid according to any one of claims 1 to 6, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

12.

8. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 1; CDR2 has the amino acid sequence set forth in SEQ ID NO:2, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 3; A recombinant nucleic acid according to any one of claims 1 to 7.

9. (a) a sequence having at least 80% sequence identity to SEQ ID NO: 10 or 11; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 7 or 8 9. The recombinant nucleic acid of claim 1 , comprising:

10. 10. A recombinant nucleic acid according to any one of claims 1 to 9, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

9.

11. 11. A recombinant nucleic acid according to any one of claims 1 to 10, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:

9.

12. 12. A recombinant nucleic acid according to any one of claims 1 to 11, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

9.

13. 13. A recombinant nucleic acid according to any one of claims 1 to 12, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

9.

14. TCR is, (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 14 or 70, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or 70; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13 or 69, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13 or 69.

14. The recombinant nucleic acid of any one of claims 1 to 13, comprising:

15. 15. The recombinant nucleic acid of claim 1, wherein the TCR binds to an epitope derived from human RAS containing the mutation G12V.

16. 16. The recombinant nucleic acid of claim 15, wherein the epitope derived from human RAS containing the mutation G12V is SEQ ID NO: 43 or 44.

17. 17. The recombinant nucleic acid of claim 15 or 16, wherein the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12V and (ii) an MHC protein encoded by the HLA-A11:01 allele.

18. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:

20.

19. 19. The recombinant nucleic acid of claim 18, wherein the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:

18.

20. 20. The recombinant nucleic acid of claim 18 or 19, wherein the TCR beta chain construct further comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence set forth in SEQ ID NO:

19.

21. 21. A recombinant nucleic acid according to any one of claims 18 to 20, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

26.

22. 22. A recombinant nucleic acid according to any one of claims 18 to 21, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:

26.

23. 23. A recombinant nucleic acid according to any one of claims 18 to 22, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

26.

24. 24. A recombinant nucleic acid according to any one of claims 18 to 23, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

26.

25. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 15; CDR2 has the amino acid sequence set forth in SEQ ID NO: 16; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 17; 25. A recombinant nucleic acid according to any one of claims 18 to 24.

26. (a) a sequence having at least 80% sequence identity to SEQ ID NO: 24 or 25; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 21 or 22 26. The recombinant nucleic acid of any one of claims 18 to 25, comprising:

27. 27. A recombinant nucleic acid according to any one of claims 18 to 26, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

23.

28. 28. A recombinant nucleic acid according to any one of claims 18 to 27, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

23.

29. 29. A recombinant nucleic acid according to any one of claims 18 to 28, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

23.

30. 30. The recombinant nucleic acid of any one of claims 18 to 29, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

23.

31. TCR is, (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 28 or 72, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 28 or 72; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 27 or 71, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 27 or 71.

31. The recombinant nucleic acid of any one of claims 18 to 30, comprising:

32. 32. The recombinant nucleic acid of any one of claims 18 to 31, wherein the TCR binds to an epitope derived from human RAS containing the mutation G12V.

33. 33. The recombinant nucleic acid of any one of claims 18 to 32, wherein the epitope derived from human RAS containing the mutation G12V is SEQ ID NO:

45.

34. 34. The recombinant nucleic acid of claim 32 or 33, wherein the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12V and (ii) an MHC protein encoded by the HLA-C01:02 allele.

35. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:

34.

36. 36. The recombinant nucleic acid of claim 35, wherein the TCR beta chain construct further comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence set forth in SEQ ID NO:

32.

37. 37. The recombinant nucleic acid of claim 35 or 36, wherein the TCR beta chain construct further comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence set forth in SEQ ID NO:

33.

38. 38. A recombinant nucleic acid according to any one of claims 35 to 37, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

40.

39. 39. A recombinant nucleic acid according to any one of claims 35 to 38, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

40.

40. 40. A recombinant nucleic acid according to any one of claims 35 to 39, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

40.

41. 41. A recombinant nucleic acid according to any one of claims 35 to 40, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

40.

42. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO:29; CDR2 has the amino acid sequence set forth in SEQ ID NO: 30; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 31; 42. A recombinant nucleic acid according to any one of claims 35 to 41.

43. (a) a sequence having at least 80% sequence identity to SEQ ID NO: 38 or 39; and (b) a sequence having at least 80% sequence identity with SEQ ID NO: 35 or 36 43. The recombinant nucleic acid of any one of claims 35 to 42, comprising:

44. 44. A recombinant nucleic acid according to any one of claims 35 to 43, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

37.

45. 45. A recombinant nucleic acid according to any one of claims 35 to 44, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

37.

46. 46. ​​A recombinant nucleic acid according to any one of claims 35 to 45, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

37.

47. 47. A recombinant nucleic acid according to any one of claims 35 to 46, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

37.

48. TCR is, (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 41 or 74, or an amino acid sequence which is at least 80% identical to SEQ ID NO: 41 or 74; (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 42 or 73, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 42 or 73.

48. The recombinant nucleic acid of any one of claims 35 to 47, comprising:

49. 49. The recombinant nucleic acid of any one of claims 35 to 48, wherein the TCR binds to an epitope derived from human RAS containing the mutation G12V.

50. 50. The recombinant nucleic acid of any one of claims 35 to 49, wherein the epitope derived from human RAS containing the mutation G12V is SEQ ID NO:

45.

51. 51. The recombinant nucleic acid of claim 49 or 50, wherein the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12V and (ii) an MHC protein encoded by the HLA-C01:02 allele.

52. 52. The recombinant nucleic acid of any one of claims 1 to 51, which is a vector.

53. 53. The recombinant nucleic acid of any one of claims 1 to 52, wherein the epitope has a length of 8 to 25 amino acids.

54. 54. The recombinant nucleic acid of any one of claims 1 to 53, wherein the epitope comprises a mutation that differs from the corresponding wild-type epitope by at least one amino acid.

55. 55. The recombinant nucleic acid of any one of claims 1 to 54, wherein the epitope binds to human MHC with greater affinity than the corresponding wild-type epitope.

56. The epitope has a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM for human MHC. Ｄ or IC ５０ 56. The recombinant nucleic acid of any one of claims 1 to 55, wherein the nucleic acid is linked by

57. 57. The recombinant nucleic acid of any one of claims 1 to 56, wherein the mutation is not present in non-cancer cells of the subject.

58. The TCR binds to the MHC-peptide complex with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. Ｄ or IC ５０ 58. The recombinant nucleic acid of any one of claims 1 to 57, wherein the nucleic acid is linked by

59. 59. The recombinant nucleic acid of any one of claims 1 to 58, operably linked to a promoter.

60. 60. A cell comprising a recombinant nucleic acid of any one of claims 1 to 59.

61. A cell comprising a TCR encoded by a recombinant nucleic acid of any one of claims 1 to 59.

62. 62. The cell of claim 60 or 61, which is a CD4+ T cell.

63. 62. The cell of claim 60 or 61, which is a CD8+ T cell.

64. 62. The cell of claim 60 or 61, isolated from a subject with a RAS mutation.

65. 60. A pharmaceutical composition comprising: (a) a recombinant nucleic acid according to any one of claims 1 to 59; and (b) a pharmaceutically acceptable excipient or diluent.

66. 66. The pharmaceutical composition of claim 65, further comprising an immunomodulatory agent or adjuvant.

67. 67. The pharmaceutical composition of claim 66, wherein the adjuvant is poly I:C.

68. 65. A pharmaceutical composition comprising: (a) a cell according to any one of claims 60 to 64; and (b) a pharmaceutically acceptable excipient or diluent.

69. 68. A pharmaceutical composition according to any one of claims 65 to 67 for use in the treatment of an immune disease or cancer.

70. 69. The pharmaceutical composition of claim 68 for use in the treatment of an immune disease or cancer.

71. 68. Use of the pharmaceutical composition of any one of claims 65 to 67 for treating an immune disease or cancer.

72. 69. Use of the pharmaceutical composition of claim 68 for treating an immune disease or cancer.

73. 68. A method of treating a subject having a disease or condition, comprising administering to the subject the pharmaceutical composition of any one of claims 65 to 67.

74. 69. A method of treating a subject having a disease or condition, comprising administering to the subject the pharmaceutical composition of claim 68.

75. 68. A method of treating a subject having cancer, comprising administering to the subject the pharmaceutical composition of any one of claims 65 to 67.

76. 69. A method of treating a subject having cancer, comprising administering to the subject the pharmaceutical composition of claim 68.

77. 68. A method of identifying a subject having cancer as a candidate for a therapeutic agent, comprising determining the subject as a subject that expresses a protein encoded by an HLA-A11:01 allele or an HLA CO1:02 allele, wherein the therapeutic agent is a pharmaceutical composition selected from any one of claims 65-67.

78. 69. A method of identifying a subject having cancer as a candidate for a therapeutic agent, comprising determining the subject as a subject that expresses a protein encoded by an HLA-A11:01 allele or an HLA CO1:02 allele, wherein the therapeutic agent is a pharmaceutical composition of any one of claims 64 to 68.

79. A nucleic acid encoding a TCR, wherein the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12V and (ii) an MHC protein encoded by an HLA-A11:01 allele or an HLA-C01:02 allele, and wherein complementarity determining region 3 (CDR3) of the TCR does not comprise any of the sequences of SEQ ID NOs: 46-68.

80. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-A68:01 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation.

81. 81. The recombinant nucleic acid of claim 80, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

82.

82. 82. The recombinant nucleic acid of claim 80 or 81, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

80.

83. 83. A recombinant nucleic acid according to any one of claims 80 to 82, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

81.

84. 84. A recombinant nucleic acid according to any one of claims 80 to 83, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

84.

85. 85. A recombinant nucleic acid according to any one of claims 80 to 84, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

84.

86. 86. A recombinant nucleic acid according to any one of claims 80 to 85, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

84.

87. 87. A recombinant nucleic acid according to any one of claims 80 to 86, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

84.

88. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 77; CDR2 has the amino acid sequence set forth in SEQ ID NO: 78; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 79; 88. A recombinant nucleic acid according to any one of claims 80 to 87.

89. 89. A recombinant nucleic acid according to any one of claims 80 to 88, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

83.

90. 90. A recombinant nucleic acid according to any one of claims 80 to 89, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

83.

91. 91. A recombinant nucleic acid according to any one of claims 80 to 90, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

83.

92. 92. A recombinant nucleic acid according to any one of claims 80 to 91, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

83.

93. 93. A recombinant nucleic acid according to any one of claims 80 to 92, wherein the epitope derived from human RAS containing the mutation G12V is SEQ ID NO: 43 or 44.

94. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope derived from human RAS in complex with human MHC encoded by an HLA-C03:03 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation.

95. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope derived from human RAS in complex with human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation.

96. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope derived from human RAS in complex with a human MHC encoded by an HLA-C03:03 allele and a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope derived from human RAS is characterized by a G12V mutation.

97. (i) a K of at most 500 nM, at most 250 nM, at most 50 nM, at most 25 nM, at most 10 nM, or at most 5 nM to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:03 allele; Ｄ and (ii) a K of at most 500 nM, at most 250 nM, at most 50 nM, at most 25 nM, at most 10 nM, or at most 5 nM to a mutated epitope derived from human RAS in complex with human MHC encoded by the HLA-C03:04 allele. Ｄ 97. The recombinant nucleic acid of claim 96, wherein the nucleic acid is linked to

98. 98. A recombinant nucleic acid according to any one of claims 95 to 97, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

109.

99. 99. A recombinant nucleic acid according to any one of claims 95 to 98, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

107.

100. 100. A recombinant nucleic acid according to any one of claims 95 to 99, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

108.

101. 101. A recombinant nucleic acid according to any one of claims 95 to 100, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

111.

102. 102. A recombinant nucleic acid according to any one of claims 95 to 101, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

111.

103. 103. A recombinant nucleic acid according to any one of claims 95 to 102, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

111.

104. 104. A recombinant nucleic acid according to any one of claims 95 to 103, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

111.

105. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 104; CDR2 has the amino acid sequence set forth in SEQ ID NO: 105; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 106; 105. A recombinant nucleic acid according to any one of claims 95 to 104.

106. 106. A recombinant nucleic acid according to any one of claims 95 to 105, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

110.

107. 107. A recombinant nucleic acid according to any one of claims 95 to 106, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

110.

108. 108. A recombinant nucleic acid according to any one of claims 95 to 107, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

110.

109. 109. A recombinant nucleic acid according to any one of claims 95 to 108, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

110.

110. 98. A recombinant nucleic acid according to any one of claims 95 to 97, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

113.

111. 111. A recombinant nucleic acid according to any one of claims 95 to 97 and 110, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

88.

112. 112. A recombinant nucleic acid according to any one of claims 95 to 97 and 110 to 111, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

89.

113. A recombinant nucleic acid described in any one of claims 95 to 97 and 110 to 111, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

115.

114. A recombinant nucleic acid described in any one of claims 95 to 97 and 110 to 113, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:

115.

115. A recombinant nucleic acid described in any one of claims 95 to 97 and 110 to 114, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

115.

116. 116. A recombinant nucleic acid according to any one of claims 95 to 97 and 110 to 115, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

115.

117. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 104; CDR2 has the amino acid sequence set forth in SEQ ID NO: 105; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 112; 117. A recombinant nucleic acid according to any one of claims 95 to 97 and 110 to 116.

118. A recombinant nucleic acid described in any one of claims 95 to 97 and 110 to 117, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

114.

119. A recombinant nucleic acid described in any one of claims 95 to 97 and 110 to 118, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

114.

120. A recombinant nucleic acid described in any one of claims 95 to 97 and 110 to 119, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

114.

121. 121. A recombinant nucleic acid according to any one of claims 95 to 97 and 110 to 120, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

114.

122. 98. A recombinant nucleic acid according to any one of claims 95 to 97, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

117.

123. 123. The recombinant nucleic acid of any one of claims 95 to 97 and 122, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

107.

124. 124. A recombinant nucleic acid according to any one of claims 95 to 97 and 122 to 123, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

108.

125. 125. A recombinant nucleic acid according to any one of claims 95 to 97 and 122 to 124, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

119.

126. A recombinant nucleic acid described in any one of claims 95 to 97 and 122 to 125, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

119.

127. A recombinant nucleic acid described in any one of claims 95 to 97 and 122 to 126, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

119.

128. 128. A recombinant nucleic acid according to any one of claims 95 to 97 and 122 to 127, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

119.

129. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 104; CDR2 has the amino acid sequence set forth in SEQ ID NO: 105; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 116; 129. A recombinant nucleic acid according to any one of claims 95 to 97 and 122 to 128.

130. A recombinant nucleic acid described in any one of claims 95 to 97 and 122 to 129, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

118.

131. A recombinant nucleic acid described in any one of claims 95 to 97 and 122 to 130, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

118.

132. A recombinant nucleic acid described in any one of claims 95 to 97 and 122 to 131, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

118.

133. 133. A recombinant nucleic acid according to any one of claims 95-97 and 122-132, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

118.

134. 98. A recombinant nucleic acid according to any one of claims 95 to 97, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

123.

135. 135. A recombinant nucleic acid according to any one of claims 95 to 97 and 134, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

121.

136. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 135, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

122.

137. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 136, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

125.

138. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 137, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

125.

139. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 138, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

125.

140. 140. A recombinant nucleic acid according to any one of claims 95 to 97 and 134 to 139, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

125.

141. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 104; CDR2 has the amino acid sequence set forth in SEQ ID NO: 105; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 120; 141. A recombinant nucleic acid according to any one of claims 95 to 97 and 134 to 140.

142. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 141, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

124.

143. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 142, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

124.

144. A recombinant nucleic acid described in any one of claims 95 to 97 and 134 to 143, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

124.

145. 145. A recombinant nucleic acid according to any one of claims 95 to 97 and 134 to 144, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

124.

146. 146. A recombinant nucleic acid according to any one of claims 95 to 145, wherein the epitope derived from human RAS containing the mutation G12V is SEQ ID NO: 133 or 134.

147. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO:

129.

148. 148. The recombinant nucleic acid of claim 147, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

127.

149. 149. The recombinant nucleic acid of claim 147 or 148, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

128.

150. 150. A recombinant nucleic acid according to any one of claims 147 to 149, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

131.

151. 151. A recombinant nucleic acid according to any one of claims 147 to 150, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

131.

152. 152. A recombinant nucleic acid according to any one of claims 147 to 151, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

131.

153. 153. A recombinant nucleic acid according to any one of claims 147 to 152, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

131.

154. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO:29; CDR2 has the amino acid sequence set forth in SEQ ID NO: 30; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 126; 154. A recombinant nucleic acid according to any one of claims 147 to 153.

155. 155. A recombinant nucleic acid according to any one of claims 147 to 154, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

130.

156. 156. A recombinant nucleic acid according to any one of claims 147 to 155, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

130.

157. A recombinant nucleic acid described in any one of claims 147 to 156, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

130.

158. 158. A recombinant nucleic acid according to any one of claims 147 to 157, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

130.

159. 159. A recombinant nucleic acid according to any one of claims 147 to 158, wherein the TCR binds to an epitope derived from human RAS containing the mutation G12D.

160. 160. The recombinant nucleic acid of claim 159, wherein the epitope derived from human RAS containing the mutation G12D is SEQ ID NO:

135.

161. 161. The recombinant nucleic acid of claim 159 or 160, wherein the TCR binds to a complex comprising (i) an epitope derived from human RAS containing the mutation G12D and (ii) an MHC protein encoded by the HLA-A11:01 allele.

162. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR binds to a mutated epitope derived from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele with a K of up to 1000 nM. Ｄ A recombinant nucleic acid that specifically binds to a mutated epitope derived from human RAS characterized by a G12D mutation.

163. A recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR binds to a mutated epitope derived from human RAS in complex with human MHC encoded by an HLA-C05:01 allele with a K of 1000 nM or greater. Ｄ A recombinant nucleic acid that specifically binds to a mutated epitope derived from human RAS characterized by a G12D mutation.

164. 164. The recombinant nucleic acid of claim 162 or 163, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

90.

165. 165. A recombinant nucleic acid according to any one of claims 162 to 164, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

88.

166. 166. A recombinant nucleic acid according to any one of claims 162 to 165, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

89.

167. 167. A recombinant nucleic acid according to any one of claims 162 to 166, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

92.

168. 168. A recombinant nucleic acid according to any one of claims 162 to 167, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

92.

169. 169. A recombinant nucleic acid according to any one of claims 162 to 168, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

92.

170. 170. A recombinant nucleic acid according to any one of claims 162 to 169, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

92.

171. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 85; CDR2 has the amino acid sequence set forth in SEQ ID NO: 86; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 87; 171. A recombinant nucleic acid according to any one of claims 162 to 170.

172. 172. A recombinant nucleic acid according to any one of claims 162 to 171, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

91.

173. 173. A recombinant nucleic acid according to any one of claims 162 to 172, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

91.

174. 174. A recombinant nucleic acid according to any one of claims 162 to 173, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

91.

175. 175. A recombinant nucleic acid according to any one of claims 162 to 174, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

91.

176. 164. The recombinant nucleic acid of claim 162 or 163, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

95.

177. 177. A recombinant nucleic acid according to any one of claims 162, 163 and 176, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

94.

178. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 177, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO:

81.

179. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 178, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

97.

180. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 179, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

97.

181. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 180, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

97.

182. 182. A recombinant nucleic acid according to any one of claims 162 to 163 and 176 to 181, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

97.

183. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 15; CDR2 has the amino acid sequence set forth in SEQ ID NO: 16; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 93; 183. A recombinant nucleic acid according to any one of claims 162 to 163 and 176 to 182.

184. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 183, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

96.

185. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 184, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

96.

186. A recombinant nucleic acid described in any one of claims 162 to 163 and 176 to 185, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

96.

187. 187. A recombinant nucleic acid according to any one of claims 162 to 163 and 176 to 186, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

96.

188. 164. The recombinant nucleic acid of claim 162 or 163, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

101.

189. 189. A recombinant nucleic acid according to any one of claims 162 to 163 and 188, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO:

94.

190. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 189, wherein the TCR beta chain construct comprises a complementarity determining region 2 (CDR2), and CDR2 has the amino acid sequence of SEQ ID NO:

81.

191. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 190, wherein the TCR beta chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

103.

192. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 191, wherein the TCR beta chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

103.

193. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 192, wherein the TCR beta chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

103.

194. 194. A recombinant nucleic acid according to any one of claims 162 to 163 and 188 to 193, wherein the TCR beta chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

103.

195. further comprising a TCR alpha chain construct having CDR1, CDR2, and CDR3; CDR1 has the amino acid sequence set forth in SEQ ID NO: 98; CDR2 has the amino acid sequence set forth in SEQ ID NO:99; and CDR3 has the amino acid sequence set forth in SEQ ID NO: 100; 195. A recombinant nucleic acid according to any one of claims 162 to 163 and 188 to 194.

196. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 195, wherein the TCR alpha chain construct comprises a variable region having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:

102.

197. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 196, wherein the TCR alpha chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

102.

198. A recombinant nucleic acid described in any one of claims 162 to 163 and 188 to 197, wherein the TCR alpha chain construct comprises a variable region having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

102.

199. 199. A recombinant nucleic acid according to any one of claims 162-163 and 188-198, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO:

102.

200. 200. A recombinant nucleic acid according to any one of claims 162 to 199, wherein the epitope derived from human RAS containing the mutation G12D is SEQ ID NO:

132.

201. TCR binds the mutated epitope with an EC ５０ 201. The recombinant nucleic acid of any one of claims 162 to 200, which specifically binds to

Images