T cell receptor constructs and uses thereof

Abstract

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

Description

[0001] Cross-references

[0002] 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 Art

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

[0004] CDR3 is the main CDR responsible for recognizing and processing antigens, although CDR1 of the α chain has also been shown to interact with the N-terminal portion of the antigen peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is believed to be used to recognize MHC. The constant region of the TCR domain consists of a short connecting sequence in which cysteine ​​residues form a disulfide bond that forms a connection between the two chains. The affinity of TCR for specific antigens makes it valuable in a variety of therapeutic methods. For example, cancer patients (such as melanoma patients) can be effectively treated by adoptive immunotherapy because TCR is extremely sensitive to its antigen and can guide immune responses on tumor cells expressing its cognate antigen. Therefore, it is necessary to develop new and effective therapeutic agents for TCRs targeting peptide-MHC complexes. Summary of the Invention

[0005] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β 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 β 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 β 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 β 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 β 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 β 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 β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR α chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 1; CDR2 has the amino acid sequence shown in SEQ ID NO: 2; and CDR3 has the amino acid sequence shown 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 α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown 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 from human RAS comprising the mutation G12V. In some embodiments, the epitope 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 from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by an HLA-A11:01 allele.

[0006] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β 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 β 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 β 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 β 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 β 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 β 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 β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR α chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 15; CDR2 has the amino acid sequence shown in SEQ ID NO: 16; and CDR3 has the amino acid sequence shown 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 α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 23. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown 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 from human RAS comprising the mutation G12V. In some embodiments, the epitope 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 from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by an HLA-C01:02 allele.

[0007] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β 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 β 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 β 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 β 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 β 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 β 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 β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCR α chain construct having CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 29; CDR2 has the amino acid sequence shown in SEQ ID NO: 30; and CDR3 has the amino acid sequence shown 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 α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 37. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown 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, (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 from human RAS comprising the mutation G12V. In some embodiments, the epitope 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 from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by an HLA-C01:02 allele.

[0008] In some embodiments, the recombinant nucleic acid is a vector. In some embodiments, the epitope is 8-25 amino acids in length. 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 a 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 TCR binds to human MHC. In some embodiments, the mutation is not present in non-cancerous cells of the subject. In some embodiments, the TCR 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 Binding to the MHC-peptide complex. In some embodiments, the recombinant nucleic acid is operably linked to a promoter.

[0009] Provided herein is a cell comprising a recombinant nucleic acid as described herein. In some embodiments, the cell is a CD4+ T cell. In some embodiments, the cell is a CD8+ T cell. In some embodiments, the cell is isolated from a subject with a RAS mutation.

[0010] Provided herein is a pharmaceutical composition 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 immunomodulator or adjuvant. In some embodiments, the adjuvant is poly I:C. In some embodiments, the pharmaceutical composition is used to treat an immune disease or cancer.

[0011] Provided herein are uses of the pharmaceutical compositions described herein for treating immune diseases or cancer.

[0012] Provided herein is a method of treating a subject having a disease or condition comprising administering to the subject a pharmaceutical composition described herein.

[0013] Provided herein is a method of treating a subject having cancer, comprising administering to the subject a pharmaceutical composition described herein.

[0014] Provided herein is a method of identifying a subject having cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as a subject expressing 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.

[0015] Provided herein is a nucleic acid encoding a TCR, wherein the TCR binds to a complex comprising (i) an epitope from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by an HLA-A11:01 allele or an HLA C01:02 allele, wherein the complementarity determining region 3 (CDR3) of the TCR does not comprise the sequence of any one of SEQ ID NOs:46-68.

[0016] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR specifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-A68:01 allele, wherein the mutant epitope from human RAS is characterized by a G12V mutation. In some embodiments, the TCR β chain construct comprises a complementary determining region 3 (CDR3), wherein CDR3 has an amino acid sequence of SEQ ID NO: 82. In some embodiments, the TCR β chain construct comprises a complementary determining region 1 (CDR1), wherein CDR1 has an amino acid sequence of SEQ ID NO: 80. In some embodiments, the TCR β chain construct comprises a complementary determining region 2 (CDR2), wherein CDR2 has an amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR further comprises a TCR α chain construct comprising a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 77; CDR2 has the amino acid sequence shown in SEQ ID NO: 78; and CDR3 has the amino acid sequence shown in SEQ ID NO: 79. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 83. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 83. In some embodiments, the epitope from human RAS comprising the mutation G12V is SEQ ID NO: 43 or 44.

[0017] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR specifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C03:03 allele, wherein the mutant epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR specifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele, wherein the mutant epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR specifically binds to a mutant epitope 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 mutant epitope from human RAS is characterized by a G12V mutation. In some embodiments, TCR(i) binds to a T cell receptor 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 binds to a mutant epitope from human RAS in complex with human MHC encoded by an HLA-C03:03 allele; and (ii) 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 DBinds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele. In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 111. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 111. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 111. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 111. In some embodiments, the TCR further comprises a TCR α chain construct having a CDR1, a CDR2, and a CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 104; CDR2 has the amino acid sequence shown in SEQ ID NO: 105; and CDR3 has the amino acid sequence shown in SEQ ID NO: 106. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 110. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 110.

[0018] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 115. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 115. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 115. In some embodiments, the TCR β 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 α chain construct comprising a CDR1, a CDR2, and a 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 α 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 α 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 α 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 shown in SEQ ID NO:114.

[0019] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 119. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 119. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 119. In some embodiments, the TCR β 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 α chain construct comprising a CDR1, a CDR2, and a 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 α 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 α 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 α 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 shown in SEQ ID NO:118.

[0020] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 125. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 125. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 125. In some embodiments, the TCR β 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 α chain construct comprising a CDR1, a CDR2, and a 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 α 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 α 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 α 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 from human RAS comprising the mutation G12V is SEQ ID NO: 133 or 134.

[0021] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β 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 β 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 β 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 β 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 β 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 β 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 β chain construct includes a variable region having the amino acid sequence shown in SEQ ID NO: 131. In some embodiments, the TCR further comprises a TCR α chain construct comprising a CDR1, a CDR2, and a CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 29; CDR2 has the amino acid sequence shown in SEQ ID NO: 30; and CDR3 has the amino acid sequence shown in SEQ ID NO: 126. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 130. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 130. In some embodiments, the TCR α chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 130. In some embodiments, the TCR α chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 130. In some embodiments, the TCR binds to an epitope from human RAS comprising mutation G12D. In some embodiments, the epitope from human RAS comprising mutation G12D is SEQ ID NO: 135. In some embodiments, the TCR binds to a complex comprising (i) an epitope from human RAS comprising mutation G12D and (ii) an MHC protein encoded by an HLA-A11:01 allele.

[0022] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is expressed with a K of at most 1000 nM. D Specifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele, wherein the mutant epitope from human RAS is characterized by a G12D mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR has a K of 1000 nM or greater. DSpecifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele, wherein the mutant epitope from human RAS is characterized by a G12D mutation. In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR further comprises a TCR α chain construct comprising a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 85; CDR2 has the amino acid sequence shown in SEQ ID NO: 86; and CDR3 has the amino acid sequence shown in SEQ ID NO: 87. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 91.

[0023] In some embodiments, the TCR β chain construct comprises a complementary determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 95. In some embodiments, the TCR β chain construct comprises a complementary determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 94. In some embodiments, the TCR β chain construct comprises a complementary determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR further comprises a TCR α chain construct comprising 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 α 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 α 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 α 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 α chain construct comprises a variable region having the amino acid sequence set forth in SEQ ID NO: 96.

[0024] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR β chain construct comprises a variable region having at least 95%, 96%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR further comprises a TCR α chain construct comprising a CDR1, a CDR2, and a CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 98; CDR2 has the amino acid sequence shown in SEQ ID NO: 99; and CDR3 has the amino acid sequence shown in SEQ ID NO: 100. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 102. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 102. In some embodiments, the TCR α chain construct comprises a variable region having at least 95%, 96%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 102. In some embodiments, the TCR α chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 102. In some embodiments, the epitope from human RAS comprising mutation G12D is SEQ ID NO: 132.

[0025] In some embodiments, the TCR is expressed with an EC of at least 1000 nM. 50 Specific binding to the mutant epitope.

[0026] Incorporation by reference

[0027] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The novel features of the present 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 referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments utilizing the principles of the present invention, wherein:

[0029] Figure 1 Depicted is the workflow of antigen-specific TCR identification and analysis. PBMC from healthy donors can be stimulated with the antigen of interest, and then antigen-specific T cells (left) can be identified with peptide-MHC polymers. Antigen-specific T cells can be separated and TCR can be sequenced and analyzed (center left). TCR can be synthesized and expressed in cell lines or PBMC, and specificity can be confirmed again by peptide-MHC polymers (center right). Then, the cell line or PBMC expressing TCR can be co-cultured with the cell line expressing the antigen, to confirm the function of TCR (right).

[0030] Figure 2 Antigen-specific CD8 + Example schematic of T cell expansion. PBMCs can be stimulated with the antigen and cytokines of interest. Following expansion, antigen-specific CD8 + T cells.

[0031] Figure 3A Schematic diagram of an example of a recombinant TCR construct and a vector design for expressing the TCR construct in cells.

[0032] Figure 3B Figure 2 is a schematic diagram of an example of a viral vector encoding a recombinant TCR for transduction or transfection into cells.

[0033] Figure 4A Delineation of RAS antigen-specific CD8 in response to RAS peptide stimulation + Example flow cytometric analysis of T cell expansion.

[0034] Figure 4B Depicted are example flow cytometric analyses of RAS antigen-specific Jurkat cells following sorting of the top 10% multimer-positive cells expressing recombinant TCRs following puromycin selection.

[0035] Figure 5 Figure 3 depicts RAS-peptide-HLA-A11:01 complex-specific CD8 in response to RAS G12V peptide stimulation.+ T cell expansion (left), RAS-peptide-HLA-A11:01 complex-specific CD8 in response to RAS G12C peptide stimulation + T cell expansion (center) and RAS-peptide-HLA-A11:01 complex-specific CD8 in response to RAS G12D peptide stimulation + Example flow cytometric analysis of T cell expansion (right).

[0036] Figure 6 Figure 3 depicts RAS-peptide-HLA-A11:01 complex-specific CD8 in response to stimulation with G12V or G12C mutant RAS peptides. + Example flow cytometric analysis of T cell expansion (left), analysis of TCR clonal abundance after sequencing TCRs from pooled hits (center), and confirmation of mutant versus wild-type specificity of the top two retrieved clones after recombinant TCR expression in Jurkat cells (right).

[0037] Figure 7 Depicted is an example flow cytometric analysis of Jurkat cells transduced to express RAS TCR-1.

[0038] Figure 8 Depicted is an example flow cytometric analysis of peripheral blood mononuclear cells transduced to express RAS TCR-1.

[0039] Figure 9 Depicted are example flow cytometric analyses of electroporated Jurkat cells expressing RAS TCR-2 or RAS TCR-3.

[0040] Figure 10 Depicts experimental results of TCR functional assays used to assess the specificity and avidity of RAS TCR-1. Graphs show CD69 activation following 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 graph) or RAS mutant 9-mer peptides (left) or increasing amounts of RAS mutant 10-mer peptides (right).

[0041] Figure 11 Depicts experimental results of TCR functional assays assessing the specificity and avidity of RAS TCR-2 and RAS TCR-3. Graphs show CD69 activation following co-culture of RAS TCR-transduced Jurkat cells with HLA-C01:02-expressing K562 cells loaded with RAS wild-type (rightmost data point in each graph) or RAS mutant peptides.

[0042] Figure 12Depicts the experimental results of a cytotoxicity assay in which PBMCs transduced with either an unrelated TCR or RAS TCR-1 were co-cultured with SW620 (RAS G12V+) target tumor cells expressing HLA-A11:01 and GFP over an increasing range of CD4+ cell to target ratios (E:T). SW620 target tumor cells co-cultured with either PBMCs transduced with an unrelated TCR were used as negative controls. The graph shows the growth of target cells over 100 hours (X-axis) as measured by GFP signal, as reflected by the total green object area of ​​the culture image (Y-axis).

[0043] Figure 13 Depicted are experimental results of a cytotoxicity assay in which CD4+ T cells (isolated from PBMCs transduced with either an irrelevant TCR or RAS TCR-1) were co-cultured with SW620 (RAS G12V+) target tumor cells expressing HLA-A11:01 and GFP at an increasing range of PBMC to target ratios. SW620 target tumor cells cultured alone or without any PBMCs were used as negative controls. The graph depicts the growth of target cells over 72 hours (X-axis) as measured by GFP signal, as reflected by the total green object area of ​​the culture image (Y-axis).

[0044] Figure 14 Depicted are exemplary data showing the percentage of lysis of the tumor cell line SNG-M over time following treatment with PBMCs transduced with an irrelevant TCR or RAS TCR-1.

[0045] Figure 15 Depicted are exemplary data showing changes in tumor volume over time in mice subcutaneously inoculated with the tumor cell line A375-HLA-A11:01-KRAS G12V following 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.

[0046] Figure 16 Depicted are exemplary data showing the functional avidity of RAS TCR-2 and RAS TCR-3.

[0047] Figure 17A Depicted are exemplary data showing the percentage of lysis of the colorectal cancer cell line SW620 engineered to express HLA C*01:02 after treatment with PBMCs transduced with RAS TCR-2 or RAS TCR-3 at the indicated effector cell to target cell ratios. The percentage of cytolysis was calculated based on tumor cells co-cultured with untransduced T cells.

[0048] Figure 17B Depicted are exemplary data showing the percentage of lysis of the pancreatic tumor cell line Panc 03.27 after treatment with PBMCs transduced with RAS TCR-2 or RAS TCR-3 at the indicated effector cell to target cell ratios. The percentage of cytolysis was calculated based on tumor cells co-cultured with untransduced T cells.

[0049] Figure 18 Depicted are exemplary data showing the percentage of CD69+ cells in engineered cells following transduction with varying concentrations of RAS TCR-5, RAS TCR-6, or RAS TCR-7 bound to G12D RAS complexed with MHC encoded by the HLA-C05:01 allele. DETAILED DESCRIPTION

[0050] The term "about" or "approximately" means that a particular value is within an acceptable error range as determined by one skilled in the art, which depends in part on how the value is measured or determined, such as limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as practiced in the art. Alternatively, "about" can mean a range of at most 20%, at most 10%, at most 5%, or at most 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold. When specific values ​​are described in the application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable error range for the specific value.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test this disclosure, only preferred methods and materials are now described. The details of one or more specific embodiments are set forth in the following description.

[0052] I. definition

[0053] Unless otherwise indicated, the term "TCR" should be understood to encompass a complete TCR and its antigen-binding portion or antigen-binding fragment (also referred to as an MHC-peptide binding fragment). In some embodiments, the TCR is a complete or full-length TCR. In some embodiments, the TCR is an antigen-binding portion that is smaller than the full-length TCR but binds to a specific antigenic peptide (e.g., an MHC-peptide complex) that is bound (e.g., in its context) to an MHC molecule. In some cases, the antigen-binding portion or fragment of a TCR may comprise only a portion of the domain of a full-length or complete TCR, but is still capable of binding to the epitope (e.g., an MHC-peptide complex) bound to the complete TCR. In some cases, the antigen-binding portion or fragment of a TCR comprises a variable domain of a TCR, such as a variable α chain and a variable β chain of a TCR, sufficient to form a binding site that binds to a specific MHC-peptide complex, such as typically each chain comprising three complementary determining regions. A polypeptide or protein having a binding domain that is an antigen-binding domain or homologous to an antigen-binding domain is included. These terms also encompass complementarity determining region (CDR) grafted TCRs and other humanized TCRs (including CDR modifications and framework region modifications). It should be noted that while only immunoglobulin chains (e.g., heavy and light chains) may be mentioned, the disclosed invention is applicable to a variety of other different types of paired sequences, such as T cell receptor chain pairs (TCRα and TCRβ chains and TCRγ and TCRδ chains), and is not limited to immunoglobulins.

[0054] The terms "complementarity determining region" and "CDR" are known in the art and are synonymous with "hypervariable region" or "HVR" and refer to non-contiguous amino acid sequences within the TCR variable region that confer specificity and / or binding affinity to the MHC-peptide complex. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each α chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each β chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the α and β chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, FR-H4) in each full-length α chain variable region and four FRs (FR-L1, FR-L2, FR-L3, FR-L4) in each full-length β chain variable region.

[0055] "Variable region" or "variable domain" refers to the domain of the TCR α, β, γ, or δ chain that is involved in binding the TCR to the antigen-MHC complex. The variable domains of the α and β chains (Vα and Vβ, respectively), as well as the variable domains of the γ and δ chains (Vγ and Vδ, respectively) of native TCRs generally have similar structures, with each domain comprising 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.

[0056] TCR fragments are also provided herein, including antigen binding fragments. In some embodiments, TCR is its antigen binding portion, such as a variant of a full-length TCR without its transmembrane region and / or cytoplasmic region, which can be referred to as a fully soluble TCR. In some embodiments, TCR is a dimeric TCR (dTCR). In some embodiments, TCR is a single-chain TCR (scTCR), such as an scTCR with a structure described in PCT Patent Publication No. WO2003 / 020763, WO2004 / 033685 or WO2011 / 044186. In certain embodiments, TCR is a single-chain TCR fragment comprising an α chain variable region connected to a β chain variable region, such as scTv. In some embodiments, scTv is also referred to as scFv. In some aspects, single-chain Tv or scTv refers to a TCR fragment comprising a variable α chain or γ chain (Vα or Vγ) and a variable β chain or δ chain (Vβ or Vδ) domain of TCR, wherein these domains are present on a single polypeptide chain. Typically, the Tv polypeptide further comprises a polypeptide linker between the Vα and Vβ domains or the Vγ and Vδ domains, which enables scTv to form the desired antigen binding structure. In some aspects, diabodies refer to TCR fragments with two antigen binding sites, which fragments comprise Vα (Vα-Vβ) connected to Vβ in the same polypeptide chain or Vγ (Vγ-Vδ) connected to Vδ in the same polypeptide chain. By using a linker that is too short so that the two domains on the same chain cannot pair, these domains are forced to pair with the complementary domains of the other chain and form two antigen binding sites. Exemplary diabodies are more fully described in, for example, EP404097 and WO93111161. In some aspects, Fv refers to a TCR fragment comprising complete peptide-MHC complex recognition and peptide-MHC complex binding sites. This region is composed of a dimer of a TCRα chain and a TCRβ chain or a TCRγ chain and a TCRδ chain in a 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 dimer or Vγ-Vδ dimer. Collectively, the combination of one or more CDRs from each Vα-Vβ chain or Vγ-Vδ chain confers TCR peptide-MHC complex binding specificity. For example, it is understood that, for example, when transferred to the Vα and Vβ chains or Vγ-Vδ chains of a receptor-selective TCR or antigen-binding fragment thereof, CDRα3 and CDRβ3 or CDRγ3 and CDRδ3 may be sufficient to confer TCR antigen binding specificity, and such CDR combinations can be tested for binding, affinity, etc.In addition, although the two domains of the Tv fragment (Vα and Vβ or Vγ and Vδ) are encoded by separate genes, they can be joined by synthetic linkers using recombinant methods to make them into a single protein chain, in which the Vα and Vβ or Vγ and Vδ chain regions are paired to form a monovalent molecule (called single-chain Tv (scTv)). Such scTv is also intended to be included in the peptide-MHC complex binding portion of the TCR.

[0057] In some aspects, "bispecific TCR" refers to a TCR that exhibits specificity for two different peptide-MHC complexes or two different types of peptide-MHC complexes. As used herein, the term particularly includes, but is not limited to, a TCR that exhibits binding specificity for a target peptide-MHC complex and another peptide-MHC complex that facilitates delivery to a specific tissue. Similarly, a multispecific TCR has two or more binding specificities. In some aspects, a linear TCR refers 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.

[0058] In some aspects, an "antigen binding domain" refers 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 included in such terms include, but are not limited to: (i) Tab fragments, monovalent fragments consisting of Vβ, Vα, Cβ, and Cα domains; (ii) T(ab')2 fragments, bivalent fragments comprising two Tab fragments connected by a disulfide bond in the hinge region; (iii) Td fragments consisting of Vα and Cα1 domains; (iv) Tv fragments comprising the Vβ and Vα domains of a single arm of a TCR, including scTv; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which comprise a Vα domain; and (vi) isolated CDRs. This definition includes TCRs with a single α chain or a single β chain.

[0059] The TCR provided includes humanized TCR and human TCR. "Humanized" TCR refers to that all or substantially all CDR amino acid residues are derived from non-human CDR, and all or substantially all FR amino acid residues are derived from the TCR of human FR. Humanized TCR may optionally include at least a portion of the TCR constant region derived from human TCR. "Humanized form" of non-human TCR refers to a variant of humanized non-human TCR, which is generally used to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human TCR. In some embodiments, some FR residues in the humanized TCR are replaced by corresponding residues from non-human TCR (e.g., TCR derived from CDR residues), for example, to restore or improve TCR specificity or affinity. "Human TCR" is a TCR corresponding to the amino acid sequence of a TCR produced by humans or human cells or utilizing a non-human source of human TCR libraries or other human TCR encoding sequences (including human TCR libraries). The term does not include humanized forms of non-human TCRs (such as all or substantially all CDRs are non-human) comprising non-human peptide-MHC complex binding regions. Human TCR can be prepared by administering immunogens to transgenic animals, which have been modified to produce complete human TCRs or complete TCRs with human variable regions in response to antigenic attack. Such animals typically include all or part of human TCR loci, which replace endogenous TCR loci, or are present in extrachromosomal or randomly integrated into the chromosome of an animal. In such transgenic animals, endogenous TCR loci are typically inactivated. Human TCR can also be derived from human TCR libraries, including phage display and cell-free libraries, which include TCR encoding sequences derived from human libraries.

[0060] The term "cancer neoantigen" or "neoantigen" or "neoepitope" may refer to an antigen that is not encoded in a normal, non-mutated host genome. A neoantigen may relate to an antigen that comprises one or more amino acid modifications compared to the parent antigen. For example, a neoantigen may be a tumor-associated neoantigen, wherein the term "tumor-associated neoantigen" may include peptides or proteins that comprise amino acid modifications due to tumor-specific mutations. In some cases, a neoantigen represents an oncogenic viral protein or an abnormal protein produced by somatic mutation. For example, a neoantigen may be produced by the activity of a viral protein that disrupts the cellular machinery. Another example is exposure to carcinogenic compounds, which in some cases may lead to somatic mutations. Such somatic mutations may ultimately lead to the formation of tumors / cancers. A neoantigen may be a class of tumor antigens caused by tumor-specific changes in proteins. Neoantigens include, but are not limited to, tumor antigens that arise from, for example, protein sequence substitutions, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and expression of endogenous retroviral polypeptides. A neoepitope may be an epitope that is not present in a reference cell (such as a non-diseased cell, e.g., a non-cancerous cell or germ cell) but is found in a diseased cell (e.g., a cancer cell). This includes situations where the corresponding epitope is found in normal, non-diseased cells or germ cells, but due to one or more mutations in a diseased cell (e.g., a cancer cell), the sequence of the epitope has changed, resulting in a neo-epitope.

[0061] In some aspects, an "epitope" refers to a portion of an antigen or other macromolecule that is capable of forming a binding interaction with the variable region binding pocket of a TCR. In some aspects, an epitope refers to a portion of a peptide-MHC complex that is capable of forming a binding interaction with the variable region binding pocket of a TCR. Such binding interactions can be manifested as intermolecular contacts with one or more amino acid residues of one or more CDRs. Peptide-MHC complex binding can involve, for example, CDR3, a CDR3 pair, or in some cases, the interaction of up to all six CDRs of a Vα and Vβ chain or a Vγ or Vδ chain. An epitope can be a linear peptide sequence (e.g., "continuous"), or can be composed of a non-continuous amino acid sequence (e.g., "conformational" or "discontinuous"). A TCR can recognize one or more amino acid sequences. Therefore, an epitope can define multiple different amino acid sequences. In some aspects, a TCR can recognize one or more amino acid sequences or epitopes in the context of an MHC. The epitope recognized by a TCR can be determined by peptide mapping and sequence analysis techniques well known to those skilled in the art. The binding interaction is manifested 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 the molecule that is recognized by the immune system, e.g., by a T cell, particularly when presented in the context of an MHC molecule. An epitope of a protein such as a tumor antigen can comprise a continuous or discontinuous portion of the protein and can be 5 to 100, 5 to 50, 8 to 30, or 10 to 25 amino acids in length, e.g., an epitope can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0062] The term "binding" refers to the direct association between two molecules under physiological conditions due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, and includes interactions such as salt bridges and water bridges, as well as any other conventional binding means.

[0063] In some embodiments, reference to a TCR having "specific binding" refers to situations where the TCR does not exhibit any significant binding to molecules other than peptide-MHC complexes containing the epitope recognized by the TCR. The term also applies, for example, to situations where an antigen binding domain is specific for a particular epitope carried by multiple peptide-MHC complexes, in which case a selected TCR or peptide-MHC complex binding fragment thereof carrying that peptide-MHC complex binding domain will be able to bind to various peptide-MHC complexes carrying that epitope. The term "preferentially binds" or "specifically binds" means that the TCR or fragment thereof binds to the epitope with a higher affinity than it binds to unrelated amino acid sequences and, if cross-reactive with other polypeptides containing the epitope, is non-toxic at levels formulated for administration in humans. In one aspect, such affinity is at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, or at least 1000 fold greater than the affinity of the TCR or fragment thereof for an unrelated amino acid sequence.

[0064] 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 class I or class II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). Affinity can be expressed as the equilibrium constant for reversible binding of two agents and can be expressed as K D , K A , K off or K on .K D Refers to the dissociation constant between two members of a binding pair and has units of molar concentration. K A K is the affinity constant between two members of a binding pair and is the reciprocal of the dissociation constant. Affinity can be determined experimentally, for example by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. off K refers to the dissociation rate constant of two members of a binding pair (e.g., the dissociation rate constant of an HLA-binding peptide from class I or class II HLA or a peptide-HLA complex from a TCR). onRefers to the association rate constant of the two members of a binding pair (e.g., the association rate constant of an HLA binding peptide with a class I or class II HLA or a peptide-HLA complex with a TCR). The affinity of a binding protein for a ligand, such as a TCR for an epitope, can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). The term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution.

[0065] Throughout this disclosure, “combined data” results may be referred to as “IC 50 Affinity can also be expressed as inhibitory concentration 50 (IC 50 ), or the concentration at which 50% of the first member of the binding pair (e.g., peptide) is displaced. Similarly, ln(IC 50 ) refers to IC 50 For example, IC 50 It can be the concentration of the test peptide at which 50% inhibition of the binding of the labeled reference peptide is observed in the binding assay. Taking into account the conditions under which the assay is run (e.g., limiting HLA protein concentration and / or labeled reference peptide concentration), these values ​​can be close to K DValues. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney et al., J. Immunol. 154:247 (1995); and Sette et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding of a reference standard peptide. Binding can also be determined using other assay systems, including those using living cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del. Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-throughput soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and 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)).

[0066] The term "major histocompatibility complex" and the abbreviation "MHC" can include MHC class I and MHC class II molecules and refers to a complex of genes present in all vertebrates. MHC proteins or molecules can be important for signaling between lymphocytes and antigen-presenting cells or diseased cells in immune responses, where MHC proteins or molecules bind peptides and present them to T cell receptors for recognition. Proteins encoded by MHC can be expressed on the cell surface and display self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. The MHC region can be divided into three subgroups: class I, class II, and class III. MHC class I proteins can include α chains and β2 microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T cells. MHC class II proteins can include α chains and β chains, and they can present antigen fragments to T helper cells. The MHC class III region can encode other immune components, such as complement components and cytokines. The MHC can be polygenic (several MHC class I and MHC class II genes exist) and polymorphic (multiple alleles exist for each gene).

[0067] The term "haplotype" can refer to the human leukocyte antigen (HLA) alleles found on a chromosome and the proteins encoded by them. A haplotype can also refer to the alleles present at any locus within the MHC. Each class of MHC is represented by several loci: for example, class I has 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, and class II has HLA-DRA, HLA-DRB1-9, HLA-DQA1, HLA-DQB1, HLA-DPAl, HLA-DPB1, HLA-DPB2, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB. The terms "HLA allele" and "MHC allele" are used interchangeably herein.

[0068] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. They may refer to a polymeric form of nucleotides of any length, which may be deoxyribonucleotides, ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, 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. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If modifications are present, modifications to the nucleotide structure may be performed before or after polymer assembly. A polynucleotide may contain non-standard nucleotides, such as nucleotide analogs or modified nucleotides. In some embodiments, non-standard nucleotides may stabilize hybrid formation. In some embodiments, non-standard nucleotides can destroy the stability of hybrid formation. In some embodiments, non-standard nucleotides can enhance hybridization specificity. In some embodiments, non-standard nucleotides can reduce hybridization specificity. Examples of non-standard nucleotide modifications include 2'O-methyl, 2'O-allyl, 2'O-propargyl, 2'O-alkyl, 2'fluoro, 2'arabinose, 2'xylose, 2'fluoroarabinose, phosphorothioate, phosphorodithioate, phosphoramidate, 2'amino, 5-alkyl substituted pyrimidine, 3'deoxyguanosine, 5-halopyrimidine, alkyl substituted purine, halopurine, bicyclic nucleotide, 2'MOE, PNA molecules, LNA molecules, LNA-like molecules, diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chloro Uracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6-Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queuosine, 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 its derivatives. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

[0069] "Complementarity" can refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick or other non-traditional types. The complementarity percentage can indicate the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 residues out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Complete complementarity" can mean that all consecutive residues in a nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. "Substantially complementary" refers to the degree of complementarity of 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 (such as to assess complementarity percentage) can be measured by any suitable alignment algorithm, including but not limited to Needleman-Wunsch algorithm (e.g., EMBOSS Needle alignment tool, available from www.ebi.ac.uk / Tools / psa / emboss_needle / nucleo.html, optionally using default settings), BLAST algorithm (see, e.g., BLAST alignment tool, available from blast.ncbi.nlm.nih.gov / Blast.cgi, optionally using default settings), or Smith-Waterman algorithm. Optimal alignment can be assessed using any suitable parameters (including default parameters) of the selected algorithm.

[0070] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. For example, a polypeptide can comprise 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, polypeptide sugars, 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 binding peptides, can comprise 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, and the like. In some aspects, the polypeptide may comprise modifications relative to the native or natural sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or due to errors caused by PCR amplification.

[0071] As used herein, the twenty conventional amino acids known to those skilled in the art and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), non-natural amino acids (e.g., α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid), and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-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 symbols used herein, according to standard usage and convention, the left side is the amino terminal direction and the right side is the carboxyl terminal direction. The percentage of sequence identity relative to a reference polypeptide sequence (or nucleic acid sequence) is the percentage that the amino acid residue in the candidate sequence (or in the case of a nucleic acid sequence, nucleotide) is identical with the amino acid residue (or nucleotide) in the reference polypeptide sequence (or nucleic acid sequence) after comparing the sequences and introducing room as needed to reach maximum sequence identity (and not considering any conservative replacement as a part for sequence identity). In order to determine the amino acid sequence identity percentage, alignment can be achieved in a variety of ways known to those skilled in 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 the appropriate parameters for aligning sequences, including any algorithm required for the maximum alignment of the full length of the compared sequences. However, for purposes herein, amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code has been submitted to the U.S. Copyright Office (US Copyright Office, Washington DC, 20559) along with user documentation. The program is registered with the U.S. Copyright Office under registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.), or can be compiled from the source code. The ALIGN-2 program should be compiled for UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.In the case of amino acid sequence comparisons using ALIGN-2, the percent amino acid sequence identity of a given amino acid sequence A with, for, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain percent amino acid sequence identity with, for, or against a given amino acid sequence B) is calculated as follows: 100 multiplied by 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 will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A. Unless expressly stated otherwise, all percent amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0072] "Germline sequences" refer to the gene sequences from haploid gametes and the diploid cells they form. Germline DNA contains multiple gene segments that encode a single TCRα or TCRβ chain, or a single TCRγ or TCRδ chain. These gene segments are present 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 generated to produce more than 10 8 Random reshuffling of a specific dynamic genetic system.

[0073] "Inhibit," "treat," and "treat" are used interchangeably and refer to, for example, stagnation of symptoms, prolongation of survival, partial or complete improvement of symptoms, and partial or complete elimination of conditions, diseases, or disorders associated with excess levels of a protein or associated with protein activity. For example, treatment of cancer includes, but is not limited to, stagnation, partial or complete elimination of cancerous growth or tumors. Treatment or partial elimination includes, for example, a multiple reduction in growth or tumor size and / or volume, such as about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or any multiple reduction therebetween. Similarly, treatment or partial elimination can include a percentage reduction in growth or tumor size and / or volume of about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or any percentage reduction therebetween. Prevention refers to preventing, preventing the occurrence of symptoms, preventing the progression of a disease or disorder associated with excess levels of a protein or associated with protein activity.

[0074] "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., rhesus monkeys), humans, etc.; non-mammals, including, for example, 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 some aspects, a subject refers to a single organism (e.g., a human). In some aspects, a group of individuals is provided that constitute a small group with a common immune factor and / or disease for study, and / or a group of individuals that do not have the disease (e.g., a negative / normal control). The subject from whom the sample is obtained may have a disease and / or condition (e.g., one or more allergies, infections, cancers, or autoimmune conditions, etc.) and may be compared to a negative control subject that is not affected by the disease.

[0075] "Kit" refers to a delivery system for delivering materials or reagents to implement the methods disclosed herein. In some embodiments, the kit includes a system that allows reaction reagents (e.g., probes, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for measuring, etc.) to be stored, transported, or delivered from one location to another. For example, the kit includes one or more containers (e.g., boxes) containing relevant reaction reagents and / or support materials. Such contents can be delivered to the intended recipient together or individually. For example, a first container can include an enzyme for measuring, while a second container includes multiple primers. Packaging material refers to the physical structure that holds the kit components. Packaging material can keep the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). Labels or package inserts can include appropriate written instructions. Therefore, the kit can additionally include labels or instructions for using the kit components in any method of the present invention. The kit can include a compound in a package or dispenser, and instructions for using the compound in the methods described herein.

[0076] The term "resistance mutation" refers to a gene mutation that renders the gene or a host cell containing the gene resistant to drug treatment. For example, the BTK C481S mutation is a resistance mutation that confers resistance to ibrutinib.

[0077] II. Overview

[0078] The present disclosure provides T cell receptors (TCRs) against neoantigens, isolated nucleic acid molecules encoding TCRs against neoantigens, T cells expressing the TCRs, and pharmaceutical compositions for treating diseases involving malignant cells expressing the neoantigens.

[0079] Provided herein is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 of SEQ ID NO: 31, and / or wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 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.

[0080] Provided herein is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specific binding to an epitope from RAS in complex with human MHC, wherein the CDR3 of the TCR does not comprise any one of SEQ ID NOs: 46-68. Provided herein is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specific binding to an epitope from RAS in complex with human MHC, wherein the epitope 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 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 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 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 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 from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 45.

[0081] Provided herein is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid 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 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid 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 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid encoding at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 wherein 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 is a nucleic acid 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 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 NO: 79, 87, 93, 100, 106, 112, 116, 120 or 126.Provided herein is a nucleic acid 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 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 NO: 82, 90, 95, 101, 109, 113, 117, 123 or 129.

[0082] Provided herein is a nucleic acid 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 from RAS in complex with a human MHC, wherein the epitope 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 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 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 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 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 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.

[0083] Provided herein is a separated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope from RAS that is complexed with a human MHC encoded by an HLA-A11:01 allele or HLA-C01:02. In some embodiments, the TCR specifically binds to an epitope from RAS that is complexed with a human MHC encoded by an HLA-A11:01 allele. In some embodiments, the TCR specifically binds to an epitope from RAS that is complexed with a human MHC encoded by an HLA-C01:02.

[0084] Provided herein is a separated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR), the TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct, wherein the TCR specifically binds to an epitope from RAS that is compounded with a human MHC encoded by an HLA-A68:01 allele, HLA-C05:01, HLA-C03:04 or HLA-C03:03. In some embodiments, the TCR specifically binds to an epitope from RAS that is compounded with a human MHC encoded by an HLA-A68:01 allele. In some embodiments, the TCR specifically binds to an epitope from RAS that is compounded with a human MHC encoded by an HLA-C05:01. In some embodiments, the TCR specifically binds to an epitope from RAS that is compounded with a human MHC encoded by an HLA-C03:04. In some embodiments, the TCR specifically binds to an epitope from RAS complexed with human MHC encoded by HLA-C03:03.

[0085] In some embodiments, the TCR α 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 β 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 α chain construct and / or the TCR β chain construct comprises a complementary 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 β chain construct comprises a complementary 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 α chain construct comprises a complementary 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 β chain construct comprises a complementary 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.

[0086] In some embodiments, the TCR α 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 β 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 α chain construct and / or the TCR β chain construct comprises a complementary 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 β chain construct comprises a complementary 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 α chain construct comprises a complementary 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 β chain construct comprises a complementary 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.

[0087] In some embodiments, the TCR α 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 β 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 α chain construct and / or the TCR β chain construct comprises a complementary 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 β chain construct comprises a complementary 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 α chain construct comprises a complementary 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 β chain construct comprises a complementary 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.

[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: 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 β 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 α 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 β 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.

[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: 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 β 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 α 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 β 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.

[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: 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 β 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 α 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 β 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.

[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: 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 β 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 α 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 β 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 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.

[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: 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 β 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 α 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 β 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.

[0093] 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 β 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 α 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 β 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.

[0094] 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 β 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 α 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 β 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.

[0095] 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 β 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 α 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 β 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.

[0096] 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 β 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 α 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 β 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.

[0097] In yet another aspect, the present disclosure provides a host cell comprising a nucleic acid encoding a TCR for a neoantigen provided herein, a vector containing 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 cell is a CD8 + T cells. The host cell can be a natural killer (NK) cell or a B cell. The host cell can be an immortalized cell line.

[0098] In yet another aspect, the present disclosure provides pharmaceutical compositions comprising nucleic acids encoding TCRs for provided neoantigens, host cells comprising nucleic acids encoding TCRs for provided neoantigens, vectors containing the nucleic acid sequences, or proteins encoded by nucleic acids provided herein. In some embodiments, methods of using the pharmaceutical compositions disclosed herein are also provided herein.

[0099] Also provided herein 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.

[0100] III. T cell receptor (TCR)

[0101] The ability of T cells to recognize antigens associated with various cancers or infectious organisms is conferred by their TCR, which is composed of either an alpha (α) chain and a beta (β) chain or a gamma (γ) chain and a delta (δ) chain. The proteins comprising these chains are encoded by DNA, which employs unique mechanisms to generate the vast diversity of TCRs. This multi-subunit immune recognition receptor associates with the CD3 complex and binds to 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 the APC is the central event in T cell activation, which occurs at the immunological synapse, the point of contact between the T cell and APC.

[0102] Each TCR comprises a variable complementary determining region (CDR) and a framework region (FR) 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 mice. 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 humans. 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 sequence of the third complementary determining region (CDR3) loop of the α and β chain variable regions largely determines the sequence diversity of αβ T cells, which is derived from recombination between the 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 within the TCR α and β chain loci allows for the encoding of a vast array of different CDR3 sequences. During TCR gene rearrangement, independent additions and deletions of nucleotides at the Vβ-Dβ, Dβ-Jβ, and Vα-Jα junctions further increase CDR3 sequence diversity. In this regard, immune competence is reflected in the diversity of TCRs. γδTCRs differ from αβTCRs in that they encode receptors that interact closely with the innate immune system. TCRγδ is expressed early in development, has a specialized anatomical distribution, possesses unique pathogen and small molecule specificities, and engages in a broad repertoire of innate and adaptive cellular interactions. Early in ontogeny, as a limited subset of TCRγδ cells colonizes various tissues before birth, a biased pattern of TCRγV and J segment expression is established.

[0103] TCR targets provided herein can be engineered TCRs, such as chimeric antigen receptors (CARs). CARs can be composed of three regions: an extracellular domain, a transmembrane domain, and an intracellular domain.

[0104] The extracellular domain can be the region where the receptor is exposed to the extracellular fluid and can be composed of an antigen recognition region. In some embodiments, the extracellular domain further comprises a spacer. In some embodiments, the extracellular domain further comprises a signal peptide. The signal peptide can guide the nascent protein into the endoplasmic reticulum. The signal protein in CAR can be a single-chain variable region fragment (scFv). Fusion proteins can be proteins formed by fusing two or more genes that originally encode different proteins, but when they are translated in a cell, translation produces one or more polypeptides with functional properties derived from each original gene. scFv is a chimeric protein that is formed by connecting a light chain domain and a heavy chain variable domain via a short linker peptide. The linker can include a hydrophilic residue with a glycine and / or serine residue segment. The linker can include a glutamic acid and lysine residue segment, which can improve solubility.

[0105] The transmembrane domain can be a hydrophobic domain that spans the membrane. In some embodiments, the transmembrane domain comprises an alpha helical domain. The transmembrane domain can play a functional role in the stability of the entire receptor. In some embodiments, the transmembrane domain comprises the transmembrane domain of the component closest to the membrane from the endodomain. In some embodiments, the transmembrane domain comprises the CD3-ζ transmembrane domain. In some embodiments, the transmembrane domain allows artificial TCRs to be incorporated into natural TCR complexes. In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain.

[0106] Intracellular domain can be the functional intracellular part of receptor (such as TCR or CAR).After antigen recognition, receptor aggregation and can transmit signal to cell.In some embodiments, intracellular domain comprises CD3-ζ intracellular domain.In some embodiments, intracellular domain comprises at least one ITAM.In some embodiments, intracellular domain comprises at least 3 or at least 3 ITAM.In some embodiments, intracellular domain comprises CD28 intracellular domain.In some embodiments, intracellular domain comprises OX40 intracellular domain.In some embodiments, intracellular domain comprises chimeric intracellular domain.For example, intracellular domain can comprise CD28 intracellular domain, OX40 intracellular domain and CD3-ζ intracellular domain.

[0107] IV. T cells

[0108] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. T cells include CD4 + T cells (helper T cells) and CD8 + T cells (cytotoxic T cells). CD4 +T cells can assist other white blood cells in the immune process, including the maturation of B cells and the activation of cytotoxic T cells and macrophages. + T cells, CD4 + T cells are activated. Once activated, T cells divide rapidly and secrete cytokines that regulate active immune responses. + T cells can destroy virus-infected cells and tumor cells and may be involved in transplant rejection. CD8 + T cells can recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of nearly all cells 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 either an α and β chain or a γ and δ chain. The proteins comprising these chains are encoded by DNA, which employs a unique mechanism to generate the diversity of TCRs. This multi-subunit immune recognition receptor can associate with the CD3 complex and bind to peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The first signal for T cell activation can be provided by 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 the peptide are activated. The companion cell is typically an antigen-presenting cell, such as a professional antigen-presenting cell, or, in the case of naive responses, a dendritic cell, although B cells and macrophages can also be important APCs. Binding of TCR to antigenic peptides on APCs may be a core event in T cell activation, which occurs at the immunological synapse, the point of contact between T cells and APCs.

[0109] T cells can be prepared according to methods known in the art. The T cells can be an enriched T cell preparation, an APC-depleted cell preparation, or a substantially purified T cell preparation. The T cells can be a mixed T cell population or a purified T cell subset. The T cells can be an enriched T cell preparation that contains an increased number or percentage of T cells relative to an isolated T cell population.

[0110] T cell or T cell subsets can be obtained from various lymphoid tissues. T cell can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen tissue, lymphoid tissue and tumor. As used herein, the term "peripheral blood lymphocyte" (PBL) and its grammatical equivalents can refer to lymphocytes circulating in blood (e.g., peripheral blood). Peripheral blood lymphocytes can refer to lymphocytes that are not located in organs. Peripheral blood lymphocytes can include T cells, NK cells, B cells or any combination thereof.

[0111] The method may include isolating T cells from a subject. The method may include obtaining T cells from a subject. T cells may be obtained from a T cell line. T cells may be obtained from an autologous source. T cells may be obtained from an allogeneic source. T cells may also be obtained from a xenogeneic source, such as from a mouse, rat, non-human primate, or pig.

[0112] T cells can be cell products depleted of APCs. T cells can be substantially free of APCs. For example, T cells can comprise T cells separated from more than 75% of APCs. In an exemplary embodiment, peripheral blood mononuclear cells (PBMCs) can be obtained from blood (e.g., in heparinized vials). PBMCs can be separated from red blood cells by centrifugation and recovered from the interface. The recovered PBMCs can be optionally washed (e.g., with PBS).

[0113] T cell purification can be achieved, for example, by positive or negative selection, including but not limited to the use of antibodies against CD2, CD3, CD4, CD5, CD8, CD14, CD19 and / or MHC class II molecules. Specific T cell subsets, such as CD28 + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO + T cells can be isolated by positive or negative selection techniques. For example, CD3 / CD28 conjugated magnetic beads can be used to positively select CD3 + 、CD28 + T cells. In one aspect 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.

[0114] For example, a T cell sample can comprise cells from the subject's circulating blood and can be obtained by apheresis or leukapheresis. The T cell sample can comprise lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets. Unwanted components in the T cell sample can be removed, and the remaining T cells can be suspended in culture medium. For example, the cells can be washed to remove the plasma portion. For example, the T cell sample can be prepared by lysing red blood cells and passing them through a PERCOLL TM T cells were isolated from peripheral blood lymphocytes by gradient centrifugation.

[0115] In some embodiments, the T cell comprises at least one T cell receptor (TCR), the TCR comprising a TCR α chain construct and / or a TCR β chain construct capable of specifically binding to an epitope from RAS in complex with human MHC. In some embodiments, the T cell comprises at least one T cell receptor (TCR), the TCR comprising a TCR α chain construct and / or a TCR β chain construct capable of specifically binding to an epitope from TMPRSS2:ERG in complex with human MHC. In some embodiments, the T cell comprises at least one T cell receptor (TCR), the TCR comprising a TCR α chain construct and / or a TCR β chain construct capable of specifically binding to an epitope from GATA3 in complex with human MHC. In some embodiments, the host cell comprises at least one TCR disclosed herein, wherein the host cell is a CD4+ T cell. In some embodiments, the host cell is a CD8+ T cell. In some embodiments, the host cell is an autologous cell. In some embodiments, the host cell is an allogeneic cell. In some embodiments, the host cell is a human cell. In some other cases, the host cell can be a natural killer (NK) cell, a B cell, or an immortalized cell line.

[0116] In some embodiments, T cells can be obtained by positive selection and / or negative selection. In positive selection, affinity agents (such as antibodies, antibody fragments and aptamers) can be used to bind cell surface markers expressed on cell colonies, for example, CD3 of T cells. Affinity agents can be used to mark T cells. The T cells of various methods well known in the art can then be enriched and labeled. The non-limiting examples of these methods include fluorescence activated cell sorting (FACS) and cell separation based on (para) magnetic particles (for example, MACS cell separation kit from Miltenyi Biotec).

[0117] In negative selection, affinity reagents can be used to bind to cell surface markers expressed by blood cells other than the desired population. For example, when attempting to isolate T cells, a cocktail of affinity reagents can be used to label B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and red blood cells. The labeled cells are then consumed to enrich the T cells. Exemplary methods for consuming labeled cells include FACS and cell separation based on (para)magnetic particles.

[0118] In addition to marker-based isolation, specialized growth conditions can be used to promote the growth of a specific cell population. For example, specific cytokines or growth factors can be used to achieve specific growth conditions. For example, T cells can be preferentially proliferated in culture medium containing phytohemagglutinin (PHA), IL-2, and / or IL-15.

[0119] In some cases, non-functional markers can be used to isolate T cells because binding of functional markers (such as binding of anti-CD3 antibodies (alone or conjugated to magnetic particles) to CD3) may trigger unwanted signaling events on T cells. Therefore, a cocktail of antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (glycophorin A) can be used to isolate T cells.

[0120] Detailed protocols can be found in published literature (e.g., see Lefort et al., J Vis Exp. 2010;(40):2017), which is incorporated herein by reference. T cell isolation kits are available from, for example, STEMCELL Technologies, Thermofisher, and Miltenyi Biotec.

[0121] The T cells described herein may be allogeneic T cells.

[0122] In some embodiments, the T cell can be a genetically modified cell comprising a modified human T cell receptor (TCR) α chain gene and / or a modified human TCR β chain gene in its genome, wherein the cell has reduced cell surface expression of an endogenous TCR.

[0123] Gene-editing nucleases can be used to disrupt components of the TCR. The TCRα chain (TCRα) is encoded by a single TRAC gene and pairs with the TCRβ chain (TCRβ) encoded by two TCRB genes. Because the TCRα / β dimer can generate a fully functional TCR complex, disrupting TCRα and / or TCRβ function can reduce (or even eliminate) endogenous TCR expression.

[0124] A variety of methods can be used to disrupt endogenous TCRα or TCRβ genes. For example, there are four types of gene editing proteins that have a common mode of action, namely binding to a user-defined DNA sequence and mediating double-stranded DNA breaks (DSBs). Zinc finger nucleases (ZFNs) are heterodimeric arrays that co-localize at the target DNA site. ZFNs contain separate finger subunits that bind to DNA and are tethered to the FokI nuclease domain that cuts DNA. Transcription activator-like effector nucleases (TALENs) contain repeating units that bind to DNA through a highly variable diamino acid sequence (repeated variable diresidues; RVD) that controls DNA base recognition. Similar to ZFNs, TALENs also function as dimeric proteins that are fused to the FokI nuclease domain to produce DSBs. Meganucleases (MNs) are monomeric proteins with intrinsic nuclease activity that are derived from bacterial homing nucleases and engineered to target unique target sites. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the related Cas9 nuclease platform involves a small guide RNA (gRNA) transcript that contacts a target DNA sequence via Watson-Crick base pairing, and the Cas9 nuclease that cleaves the DNA.

[0125] In some embodiments, introducing the genome-editing nuclease into the T cell comprises introducing a polynucleotide encoding the genome-editing nuclease into the T cell.

[0126] In some embodiments, introducing the 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.

[0127] 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.

[0128] In some embodiments, the TALEN nuclease or megaTAL nuclease is encoded by an RNA with an exogenous polyadenylation signal.

[0129] 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.

[0130] In some embodiments, disruption of TCRα and / or TCRβ expression further disrupts the assembly of TCRα and TCRβ. In some embodiments, disruption of TCRα expression further disrupts the formation of a complex between the TCR and CD3. In some embodiments, disruption of TCRα expression involves further disrupting the assembly of TCRα and TCRβ.

[0131] In some embodiments, the genetically modified T cells comprise a disrupted TCR alpha chain and / or beta chain and an inactivated gene encoding an immune checkpoint protein such as PD1 and CTLA-4. This may be achieved by combining gene inactivation of specific TALE nucleases for TCR alpha or TCR beta with gene inactivation of the gene encoding an immune checkpoint protein such as PD1 and CTLA-4.

[0132] 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, TCRα, and TCRβ. In some embodiments, the genetic modification depends on the inactivation of two genes selected from the group consisting of PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG 3 and TCRα, LAG 3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ. In some embodiments, the genetic modification depends on the inactivation of more than two genes. The genetic modification can be performed ex vivo.

[0133] V. TCR specific for RAS peptide-MHC complex

[0134] Provided herein is a nucleic acid 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 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 wherein 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 is a nucleic acid encoding at least one TCR, comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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 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: 20. Provided herein is a nucleic acid encoding at least one TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct capable of specifically binding to an epitope 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: 31 and / or 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: 34.

[0135] Provided herein is a nucleic acid 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 from RAS in complex with a human MHC, wherein the epitope 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 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 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 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 from RAS comprises a region having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 45.

[0136] Provided herein is an isolated nucleic acid or a 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 from RAS in complex with a human MHC encoded by an 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 from RAS in complex with a human MHC encoded by an 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 from RAS in complex with a human MHC encoded by an HLA-A11:01 allele. Provided herein is an isolated nucleic acid or a 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 from RAS in complex with a human MHC encoded by an 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, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an 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, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-C01:02 allele. Provided herein is an isolated nucleic acid or a 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 from RAS in complex with a human MHC encoded by an 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, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an 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, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-C01:02 allele.

[0137] In some embodiments, the TCR α chain construct as described above comprises a complementary 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 β chain construct as described above comprises a complementary 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 α chain construct comprises a complementary 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 β chain construct comprises a complementary 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 α chain construct comprises a complementary 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 β chain construct comprises a complementary 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 α chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 9; and the TCR β chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the TCR α 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 β 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 α chain construct as described above 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 β chain construct as described above 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 α 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 β 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 α 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 β 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 α chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 23; and the TCR β chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, the TCR α 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 β 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 TCR α chain construct as described above comprises a CDR1 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 29. In some embodiments, the TCR β chain construct as described above comprises a CDR1 having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 32. In some embodiments, the TCR α 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 β 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 α 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 β 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 α chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 37; and the TCR β chain construct comprises a variable region having at least 80% sequence identity to SEQ ID NO: 40. In some embodiments, the TCR α 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 β 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.

[0138] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is capable of specifically binding to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-A68:01 allele. The mutant epitope from human RAS may be characterized by a G12V mutation. In some embodiments, the TCR β chain construct comprises a complementary determining region 3 (CDR3), wherein CDR3 has the amino acid sequence of SEQ ID NO: 82. In some embodiments, the TCR β chain construct comprises a complementary determining region 1 (CDR1), wherein CDR1 has the amino acid sequence of SEQ ID NO: 80. In some embodiments, the TCR β chain construct comprises a complementary determining region 2 (CDR2), wherein CDR2 has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR β 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 shown in SEQ ID NO: 84. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the TCR further comprises a TCR α chain construct comprising a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 77; CDR2 has the amino acid sequence shown in SEQ ID NO: 78; and CDR3 has the amino acid sequence shown in SEQ ID NO: 79. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 83. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 83.In some embodiments, the epitope from human RAS comprising mutation G12V has 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity to SEQ ID NO:43 or 44.

[0139] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is capable of specifically binding to a mutant epitope from human RAS that is compounded with a human MHC encoded by an HLA-C03:03 allele. The mutant epitope from human RAS may be characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is capable of specifically binding to a mutant epitope from human RAS that is compounded with a human MHC encoded by an HLA-C03:04 allele. The mutant epitope from human RAS may be characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is capable of specifically binding to a mutant epitope 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. The mutant epitope from human RAS may be characterized by a G12V mutation. In some embodiments, TCR (i) is expressed 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 binds to a mutant epitope from human RAS in complex with human MHC encoded by an HLA-C03:03 allele; and (ii) 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 DBinds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele. In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 111. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 111. In some embodiments, the TCR β 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 β 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 α chain construct having a 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 α 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 shown 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 shown in SEQ ID NO: 110. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 110.

[0140] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 115. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 115. In some embodiments, the TCR β 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 β 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 α chain construct comprising a 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 α 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 shown 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 shown in SEQ ID NO: 114. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 114.

[0141] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 119. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 119. In some embodiments, the TCR β 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 β 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 α chain construct comprising a 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 α 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 shown 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 shown in SEQ ID NO: 118. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 118.

[0142] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 125. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 125. In some embodiments, the TCR β 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 β 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 α chain construct comprising a 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 α 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 shown 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 shown in SEQ ID NO: 124. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 124. In some embodiments, the epitope 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.

[0143] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β 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 β 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 β 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 β 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 β 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 β 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 shown in SEQ ID NO: 131. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 131. In some embodiments, the TCR further comprises a TCR α chain construct having a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 29; CDR2 has the amino acid sequence shown in SEQ ID NO: 30; and CDR3 has the amino acid sequence shown in SEQ ID NO: 126. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 130. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 130. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 130. In some embodiments, the TCR binds to an epitope from human RAS comprising mutation G12D.In some embodiments, the epitope from human RAS comprising 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 from human RAS comprising mutation G12D and (ii) an MHC protein encoded by an HLA-A11:01 allele.

[0144] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is capable of expressing a T cell receptor with 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. D Specifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele. The mutant epitope from human RAS may be characterized by a G12D mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR), the TCR comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR is capable of binding 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. DSpecifically binds to a mutant epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele. The mutant epitope from human RAS may be characterized by a G12D mutation. In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR β 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 shown in SEQ ID NO: 92. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the TCR further comprises a TCR α chain construct having a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 85; CDR2 has the amino acid sequence shown in SEQ ID NO: 86; and CDR3 has the amino acid sequence shown in SEQ ID NO: 87. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 91. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 91.

[0145] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR β 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 shown in SEQ ID NO: 97. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, the TCR further comprises a TCR α chain construct having a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 15; CDR2 has the amino acid sequence shown in SEQ ID NO: 16; and CDR3 has the amino acid sequence shown in SEQ ID NO: 93. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 96. In some embodiments, the TCR α chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown 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 shown in SEQ ID NO: 96. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 96.

[0146] In some embodiments, the TCR β 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 β 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 β 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 β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR β 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 shown in SEQ ID NO: 103. In some embodiments, the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the TCR further comprises a TCR α chain construct having a CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 98; CDR2 has the amino acid sequence shown in SEQ ID NO: 99; and CDR3 has the amino acid sequence shown in SEQ ID NO: 100. In some embodiments, the TCR α chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 102. In some embodiments, the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO: 102. In some embodiments, the epitope from human RAS comprising 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.

[0147] In some embodiments, the TCR is expressed with an EC of at least 1000 nM. 50Specific binding to the mutant epitope.

[0148] In various embodiments, the nucleic acid sequence encoding the TCR is codon optimized.

[0149] Mutations in any of the three ras genes, H-ras, K-ras, and N-ras, are among the most common events in human tumorigenesis. Approximately 30% of all human tumors are found to carry at least one mutation in any of the canonical ras genes. Ras mutations are evident in, for example, biliary tract adenocarcinoma, bladder transitional cell carcinoma, breast cancer, cervical adenocarcinoma, colon adenocarcinoma, colon adenoma, neuroblastoma (autonomic ganglia), acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, plasma cell myeloma, hepatocellular carcinoma, large cell carcinoma, non-small cell carcinoma, ductal carcinoma, endocrine tumors, prostate adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, angiosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, myxoma, malignant fibrous histiocytoma, pleomorphic sarcoma, germ cell tumor, seminoma, anaplastic carcinoma, follicular carcinoma, papillary carcinoma, and Hurthle cell carcinoma. Ras mutations are found in cancers affecting many tissues and organs of the body, such as the lung, liver, breast, bladder, colon, cervix, pancreas, prostate, stomach, thyroid, testicles, soft tissue, skin, and blood.

[0150] In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the RAS peptide comprises a G12V mutation. In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the RAS peptide comprises a G12C mutation. In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the RAS peptide comprises a G12D mutation. In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the RAS peptide comprises a mutation at Q61. In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the RAS peptide comprises a sequence of 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, wherein the RAS peptide comprises a sequence of VVGAVGVGK (SEQ ID NO: 43) or VVVGAVGVGK (SEQ ID NO: 44). In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the RAS peptide comprises the sequence of AVGVGKSAL (SEQ ID NO: 45).

[0151] In various embodiments, the TCR binds to an MHC:RAS peptide complex wherein the human MHC is encoded by the HLA-A02:01 allele. In various embodiments, the TCR binds to an MHC:RAS peptide complex wherein the human MHC is encoded by the HLA-A03:01 allele. In various embodiments, the TCR binds to an MHC:RAS peptide complex wherein the human MHC is encoded by the HLA-A11:01 allele. In various embodiments, the TCR binds to an MHC:RAS peptide complex wherein the human MHC is encoded by the HLA-C01:02 allele.

[0152] In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the human MHC is encoded by the HLA-A11:01 allele, and wherein the RAS peptide comprises the sequence of VVGAVGVGK (SEQ ID NO:43) or VVVGAVGVGK (SEQ ID NO:44). In various embodiments, the TCR binds to an MHC:RAS peptide complex, wherein the human MHC is encoded by the HLA-C01:02 allele, and wherein the RAS peptide comprises the sequence of AVGVGKSAL (SEQ ID NO:45).

[0153] 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 complexed with MHC encoded by a specific allele; and additionally, the TCR may exhibit different binding affinity for another epitope peptide containing a different point mutation of the same oncoprotein when the epitope peptide is complexed with MHC encoded by a specific allele, but not for a WT peptide that does not contain any mutation.

[0154] VI. Delivery of nucleic acids or vectors

[0155] Nucleic acids encoding the TCR, or vectors containing such nucleic acids, can be delivered to host cells for expression and processing.

[0156] Terms such as "transfer," "introduction," or "transfection" are used interchangeably herein and relate to the introduction of nucleic acid, particularly exogenous or heterologous nucleic acid, into a cell.

[0157] Can use any carrier transfection cell that can associate with nucleic acid, for example, by forming complex with nucleic acid or forming vesicle (wherein surround or encapsulate nucleic acid), thereby improve the stability of nucleic acid compared with naked nucleic acid.According to the carrier available of the present disclosure, comprise for example lipid-containing carrier, such as cationic lipid, liposome (particularly cationic liposome) and micelle and nanoparticle.Cationic lipid can form complex with negatively charged nucleic acid.Can use any cationic lipid according to the present disclosure.

[0158] In various embodiments, the nucleic acid encoding the TCR disclosed herein is operably linked to a promoter. In addition, the present disclosure provides a vector, such as a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector or a particle and / or a vector for gene therapy, comprising one or more nucleic acids as described above. A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid. A vector comprises a nucleic acid insert encoding a polypeptide or protein desired to be expressed in a cell (such as a host cell). For the purposes of this disclosure, an insert may be a nucleic acid encoding a TCR; an α chain, a β chain, or both of the TCR. The term "incorporating" a nucleic acid sequence into a vector may mean preparing a suitable expression vector, wherein the insert comprises the nucleic acid sequence. An "expression vector" is a vector that, when present in an appropriate environment, can direct the expression of a protein encoded by one or more genes carried by the vector. A "retrovirus" refers to a virus with an RNA genome. A "γ-retrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include, but are not limited to, mouse 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. Several examples of lentiviruses include HIV (human immunodeficiency virus: including 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 the core virus are also referred to as "viral vectors." There are a large number of available viral vectors suitable for use in the present invention, including vectors that have been identified for human gene therapy applications, such as the vectors described by Pfeifer and Verma (Pfeifer, A. and IM Verma. 2001. Ann. Rev. Genomics Hum. Genet. 2: 177-211). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors (e.g., Moloney murine leukemia virus (MLV)-derived vectors), and include 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 virus.Methods for using retroviral and lentiviral vectors and packaging cells for transducing viral particles containing TCR transgenes into mammalian target cells 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 6:327930; Zhao et al., J. Immunol., 2005, 174:4415-4423; Engels et al., 2003, Hum. Gene Ther. 14:1155-68; Frecha et al., 2010, Mol. Ther. 18:1748-57; Verhoeyen et al., 2009, Methods Mol. Biol. 506:97-114. Retroviral and lentiviral vector constructs and expression systems are also commercially available. In some embodiments, a viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a TCR alpha chain specific for a peptide antigen into hematopoietic progenitor cells. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a transduction marker. The transduction marker of the viral vector is known in the art and includes a selective marker that can confer drug resistance, or a detectable marker that can be detected by methods such as flow cytometry, such as a fluorescent marker or a cell surface protein. When the viral vector genome includes more than one nucleic acid sequence to be expressed as a separate transcript in a host cell, the viral vector can also include additional sequences between two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, and viral 2A peptides. Other vectors can also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., 1998, Gene Ther. 5: 1517-30). Other vectors include those derived from baculovirus and alphavirus (Jolly DJ. 1999. Emerging viral vectors. pp. 209-40, in Friedmann T. ed. 1999. The development of human gene therapy. New York: Cold Spring Harbor Lab).

[0159] The vector can comprise a nucleotide sequence, such as an origin of replication, that allows the nucleic acid to replicate in the host cell. The vector can also comprise one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. The vector is preferably an expression vector, which comprises a nucleic acid according to the present invention, and which is operably linked to a sequence that allows expression of the nucleic acid.

[0160] In some embodiments, a vector comprising a nucleic acid encoding a TCR disclosed herein is provided herein. In some embodiments, the vector is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus or 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 herpes virus, 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 microvesicle, a cell penetrating peptide or a lipid sphere.

[0161] Constructs are presented herein, for example, nucleic acid constructs encoding α and β chains of TCRs for expression in cells. In some embodiments, the construct comprises one or more polynucleotides encoding TCR α chains and TCR β chains. In some embodiments, the polynucleotides are incorporated into a suitable vector. In some embodiments, the polynucleotides encoding the α and β chains are incorporated into the same vector. In some embodiments, the polynucleotides encoding the α and β chains are incorporated into different vectors, and both vectors are delivered for expression in a single cell.

[0162] In some embodiments, cells can be transduced or transfected with nucleic acids encoding TCR, wherein the cells can express TCR, and the cells are used as therapeutic agents. In some embodiments, the cells are derived from subjects or hosts, wherein the subjects or hosts are humans. In some embodiments, the subjects or hosts comprise cells with mutations in epitopes, and the TCR expressed in the cells can specifically bind to epitopes with mutations. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T lymphocytes. In some embodiments, the cells are lymphocyte precursor cells. In some embodiments, the cells are T lymphocyte precursor cells. In some embodiments, the cells are T lymphocyte progenitor cells. In some embodiments, the cells are thymocytes.

[0163] In some embodiments, the T cells are immature T cells. In some embodiments, the T cells are antigen naive T cells. The host cells can be cultured ex vivo for 1, 2, 3, 4, 5 or more days for monitoring and recovery after transfection or transduction with a polynucleotide encoding a TCR.

[0164] VII. Neoantigens

[0165] The TCR disclosed herein is specific for immunogenic neoantigens. In some embodiments, the neoantigenic peptide is from RAS. In some embodiments, the neoantigenic peptide is from GATA3. In some embodiments, the neoantigenic peptide is from BTK. In some embodiments, the neoantigenic peptide is from TMPRSS2:ERG. In some embodiments, one or more neoantigenic peptides are loaded onto APCs, wherein the peptide-loaded APCs are subsequently used to stimulate T cells to produce antigen-specific T cells. In some embodiments, the APCs used for peptide loading are dendritic cells. Immunogenic neoantigen sequences can be identified by any suitable method known in the art.

[0166] In various embodiments, the neoantigen comprises an epitope. In animals and humans, the mutated epitope may be effective in inducing an immune response or activating T cells. In some embodiments, the epitope comprises a mutation. In some embodiments, the mutation is selected from a point mutation, a splice site mutation, a frameshift mutation, a readthrough mutation, a gene fusion mutation, and any combination thereof.

[0167] 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.

[0168] In certain embodiments, a neoantigen or epitope thereof can include, 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 specific embodiments, the neoantigen or epitope thereof is equal to or less than 100 amino acids.

[0169] In some embodiments, the length of the neoantigen or epitope thereof of MHC class I is 13 residues or less, and generally consists of about 8 to about 11 residues, particularly 9 or 10 residues. In some embodiments, the length of the neoantigen or epitope thereof of MHC class II is 9-24 residues.

[0170] In some embodiments, the neoantigen binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the neoantigen binds to the HLA protein with greater affinity than the corresponding wild-type peptide. In specific embodiments, the IC of the neoantigenic peptide or polypeptide is 50 At least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM or less.

[0171] In some embodiments, the epitope binds to human MHC with a greater affinity than the corresponding wild-type peptide. 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, 10 nM, or 5 nM. D or IC 50 Binding to human MHC. In some embodiments, the epitope is expressed 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 Binding to human MHC. In some embodiments, the epitope is expressed at an EC of less than 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 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. 50 In some embodiments, the epitope comprises a mutation, wherein the mutation is not present in the non-cancerous cell of the subject. In some embodiments, the epitope is encoded by a gene or expressed gene of a cancer cell of the subject. In some embodiments, the TCR binds to the 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 TCR binds to the 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 IC50 In some embodiments, the TCR binds to the HLA-peptide complex with an EC of less than 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 In some embodiments, the TCR binds to the HLA-peptide complex 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. 50 or affinity binding to the HLA-peptide complex.

[0172] In some embodiments, the length of the neoantigenic peptide can be about 8 to about 50 amino acid residues, or about 8 to about 30 amino acid residues, about 8 to about 20 amino acid residues, about 8 to about 18 amino acid residues, about 8 to about 15 amino acid residues, or about 8 to about 12 amino acid residues. In some embodiments, the length of the neoantigenic peptide can be about 8 to about 500 amino acid residues, or about 8 to about 450 amino acid residues, about 8 to about 400 amino acid residues, about 8 to about 350 amino acid residues, about 8 to about 300 amino acid residues, about 8 to about 250 amino acid residues, about 8 to about 200 amino acid residues, about 8 to about 150 amino acid residues, about 8 to about 100 amino acid residues, about 8 to about 50 amino acid residues, or about 8 to about 30 amino acid residues.

[0173] In some embodiments, the neoantigenic 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 neoantigenic 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 neoantigenic peptides may 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 neoantigenic peptides may 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.

[0174] In some embodiments, the neoantigenic peptide has a total 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.

[0175] In some embodiments, the neoantigenic peptide has a total 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.

[0176] In some embodiments, the neoantigenic 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 neoantigenic 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 neoantigenic peptides may have a pi value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0177] In some embodiments, the neoantigenic peptides may have an HLA binding affinity of between 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 neoantigenic 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 neoantigenic peptides may have an HLA binding affinity of at most 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 μΜ.

[0178] In some embodiments, the neoantigenic peptides described herein may comprise carriers, such as those well known in the art, for example, 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, etc.

[0179] In some embodiments, the neoantigenic peptides described herein can be modified by terminal -NH2 acylation (e.g., by alkanoyl (C1-C20) or thioglycolic acid acetylation), terminal carboxyl amidation (e.g., ammonia, methylamine, etc.). In some embodiments, these modifications can provide sites for attachment to supports or other molecules.

[0180] In some embodiments, the neoantigenic peptides described herein may include modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surfactants (e.g., lipids), or chemical modifications (e.g., acetylation, etc.). In addition, the bonds in the peptides may be other bonds besides peptide bonds, such as covalent bonds, ester bonds or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0181] In some embodiments, the neoantigenic peptides described herein may comprise substitutions to change the physical properties (e.g., stability or solubility) of the resulting peptide. For example, the neoantigenic peptides can be modified by replacing cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical properties, cysteine ​​has a tendency to form disulfide bonds and structurally alters the peptide sufficiently to reduce binding ability. Replacing C with α-aminobutyric acid not only alleviates this problem, but in some cases actually improves binding and cross-binding ability. The replacement of cysteine ​​with α-aminobutyric acid can occur at any residue of the neoantigenic peptide, for example, at an anchored or non-anchored position of an epitope or analog within the peptide, or at other positions of the peptide.

[0182] In some embodiments, the neoantigenic peptides described herein may include amino acid mimetics or non-natural amino acid residues, such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, 2, 3, or 4-pyrenylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine. The present invention also provides the present invention relates to a kind of amino acid, D- (trifluoromethyl) -phenylglycine; D- (trifluoromethyl) -phenylalanine; D- p -fluorophenylalanine; D- or L- p -biphenyl -phenylalanine; D- or L- p -methoxybiphenylalanine; D- or L-2-indole (allyl) alanine; and D- or L-alkyl alanine, wherein the alkyl group can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl (sec-isotyl), isopentyl or non-acidic amino acid residue. The aromatic ring of the non-natural amino acid includes, for example, thiazolyl, thienyl, pyrazolyl, benzimidazolyl, naphthyl, furyl, pyrrolyl and pyridyl aromatic rings. Modified peptides with various amino acid mimetics or non-natural amino acid residues are particularly useful because they tend to exhibit increased in vivo stability. Such peptides can also have improved shelf life or preparation properties.

[0183] The stability of the peptide can be measured in a variety of ways. For example, peptidases and various biological culture media (such as human plasma and serum) have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab. Pharmacokinetics 11: 291 (1986). The half-life of the peptides described herein can be conveniently determined using 25% human serum (v / v) assay. The protocol is as follows: before use, the combined human serum (AB type, non-heat inactivated) is broken by centrifugation. The serum is then diluted to 25% with RPMI-1640 or other suitable tissue culture media. At predetermined time intervals, a small amount of reaction solution is taken out and added to a 6% trichloroacetic acid (TCA) aqueous solution or ethanol. The turbid reaction sample is cooled (4 ° C) for 15 minutes and then rotated to precipitate the precipitated serum protein. The presence of the peptide is then determined by reverse phase HPLC using stability-specific chromatographic conditions.

[0184] In some embodiments, the neoantigenic peptides described herein can be prepared synthetically by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources (such as natural tumors or pathogenic organisms). The epitope can be synthesized separately or directly or indirectly conjugated to the peptide. Although the neoantigenic peptides described herein are substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments, the peptide can be conjugated to natural fragments or particles by synthesis.

[0185] In some embodiments, the neoantigenic peptides described herein can be prepared in a variety of ways. In some embodiments, the peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. (For example, see Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2nd Edition, Pierce Chemical Co., 1984). In addition, chemical ligation can be used to join individual peptides to produce larger peptides that are still within the scope of the present invention.

[0186] Alternatively, recombinant DNA technology can be used, wherein the nucleotide sequence encoding the peptide is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultured under conditions suitable for expression. These procedures are generally known in the art, as generally described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Therefore, recombinant peptides comprising one or more epitopes as described herein or consisting thereof can be used to present appropriate T cell epitopes.

[0187] VIII. Pharmaceutical Compositions

[0188] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, which include excipients and adjuvants that assist in processing the active agent into a pharmaceutically acceptable product. Suitable formulations may depend on the selected route of administration. Any known techniques, carriers, and excipients may be used as suitable techniques, carriers, and excipients as understood in the art.

[0189] In some cases, the pharmaceutical composition is formulated as a cell-based therapeutic agent, such as a T cell therapeutic agent. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic agent or a nucleic acid-based therapeutic agent, wherein the nucleic acid encodes a polypeptide. The composition may comprise T cells specific for two or more immunogenic antigens or neoantigenic peptides. In some embodiments, T cell-specific therapeutics may be supplemented by one or more additional therapies.

[0190] In some embodiments, the pharmaceutical composition comprises: a nucleic acid encoding a TCR targeting a neoantigen disclosed herein, a vector containing the nucleic acid, a protein encoded by the nucleic acid, or a host cell comprising the nucleic acid, the protein, or the vector; and a pharmaceutically acceptable excipient or diluent. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the immunomodulator is a cytokine. In some embodiments, the adjuvant is poly I:C.

[0191] Also provided herein is the use of the pharmaceutical composition in treating immune diseases or cancer.

[0192] In addition to the active ingredient, the pharmaceutical composition may also include a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other substances well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration. Acceptable carriers, excipients or stabilizers are those that are non-toxic to the recipient at the dosage and concentration used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol ); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as or polyethylene glycol (PEG).

[0193] An acceptable carrier is physiologically acceptable to the patient being administered and maintains the therapeutic properties of the compound being administered therewith / therein. Acceptable carriers and their formulations are generally described in, for example, Remington's Pharmaceutical Sciences (18th edition A. Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is normal saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition or vehicle that participates in carrying or transporting the subject compound from the site of administration of an organ or body part to another organ or body part, or in an in vitro assay system, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. An acceptable carrier is compatible with the other ingredients of the preparation and is harmless to the subject being administered. An acceptable carrier should not change the specific activity of the other ingredients either.

[0194] In one aspect, provided herein are pharmaceutically acceptable or physiologically acceptable compositions comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating materials, isotonic agents, and absorption enhancers or retarder compatible with drug administration. Therefore, pharmaceutical compositions or pharmaceutical preparations refer to compositions suitable for use as medicines in subjects. Compositions can be formulated to be compatible with specific routes of administration (i.e., systemic or local). Therefore, compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0195] In some embodiments, the pharmaceutical composition may further include an acceptable additive to improve the stability of the composition. Acceptable additives may not change the active agent, i.e., the specific activity of 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 may 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, used to increase the stability of the peptide and reduce the gelation of the solution. The surfactant may be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0196] In some embodiments, the pharmaceutical composition comprises a therapeutic agent that is a T cell expressing one or more polynucleotides encoding a T cell receptor. In some embodiments, the pharmaceutical composition comprises a physiologically acceptable carrier suitable for cell suspension.

[0197] The pharmaceutical composition can be administered, for example, by injection. Pharmaceutical compositions for injection include aqueous solutions (which are water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using coatings such as lecithin, in the case of dispersions, by maintaining the required particle size, and by using a surfactant to maintain fluidity. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, such as sugars, polyols such as mannitol, sorbitol and sodium chloride, can be included in the composition. The resulting solution can be packaged for use as is, or lyophilized; the lyophilized product can then be merged with a sterile solution before administration. For intravenous injection, or injection at the affected part, the active ingredient will be in the form of an acceptable aqueous solution for parenteral administration, which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can use, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection to prepare suitable solutions. As needed, preservatives, stabilizers, buffers, antioxidants and / or other additives can be included. Sterile injectable solutions can be prepared by incorporating the required amount of the active ingredient into a suitable solvent together with a combination of the above-listed ingredients or ingredients (if necessary), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing an alkaline dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods can be vacuum drying and freeze drying, which obtain the active ingredient plus any additional required ingredient powder from its previously filtration-sterilized solution.

[0198] The pharmaceutical composition can be routinely administered intravenously, such as by injection of a unit dose. For injection, the active ingredient can be in the form of a parenteral acceptable aqueous solution that is substantially pyrogen-free and has a suitable pH, isotonicity, and stability. Suitable solutions can be prepared using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Additionally, the composition can be administered via atomization.

[0199] When a pharmaceutical composition is contemplated for use in medicine or any of the methods provided herein, it is contemplated that the composition may be substantially free of pyrogens such that the composition does not induce an inflammatory response or an unsafe allergic reaction when administered to a human patient. Testing a composition for pyrogens and preparing a composition that is substantially free of pyrogens is well known to those of ordinary skill in the art and can be accomplished using commercially available kits.

[0200] Acceptable carriers may contain compounds that stabilize, increase or delay absorption, or increase or delay clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize or increase or decrease absorption of pharmaceutical compositions (including liposome carriers). To prevent digestion, the compound may be complexed with a composition to render it resistant to acid and enzymatic hydrolysis, or the compound may be complexed in 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).

[0201] The pharmaceutical composition can be used in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be used depends on the ability of the subject to be treated, the subject's immune system to utilize the active ingredient and the degree of desired binding capacity. The precise amount of the active ingredient that needs to be used depends on the practitioner's judgment and is specific for each individual. The appropriate regimen for initial administration and booster injection is also different, but typically, initial administration is first followed by repeated administration at intervals of one or more hours by subsequent injection or other administration. Alternatively, continuous intravenous infusion that is enough to maintain blood concentration is considered.

[0202] In some embodiments, the present disclosure relates to an immunogenic composition, such as a pharmaceutical composition capable of eliciting a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent described herein (e.g., a peptide, a polynucleotide, a TCR, a CAR, a cell containing a TCR or a CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.) corresponding to a tumor-specific antigen or a neoantigen.

[0203] 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.

[0204] In some embodiments, the antigen polypeptide or polynucleotide can be provided as an antigen presenting cell (e.g., dendritic cell) containing such polypeptide or polynucleotide. In other embodiments, such antigen presenting cells are used to stimulate T cells for patients. In some embodiments, the antigen presenting cell is a dendritic cell. In related embodiments, the dendritic cell is an autologous dendritic cell pulsed with a neoantigen peptide or nucleic acid. The neoantigen peptide can be any suitable peptide that produces an appropriate T cell response. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is a HTL. Therefore, one embodiment of the present disclosure is an immunogenic composition containing at least one antigen presenting cell (e.g., dendritic cell) pulsed or loaded with one or more neoantigen 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 neoantigen peptides or polynucleotides in vitro. In related embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide can be any suitable polynucleotide that can transduce dendritic cells, thereby resulting in the presentation of the neoantigen peptide and the induction of immunity. In some embodiments, such antigen presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells or allogeneic T cells). In 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, 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 any therapeutic agent can be performed simultaneously or sequentially and in any order.

[0205] In some embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) for therapeutic treatment described herein may be formulated for parenteral, topical, nasal, oral, or local administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the composition can be administered intratumorally. The composition can be administered at the site of surgical resection to induce a local immune response to the tumor. In some embodiments, a composition for parenteral administration is described herein, which comprises a solution of a neoantigenic peptide, and the immunogenic composition is dissolved or suspended in an acceptable carrier such as an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterilized by filtration. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized product is combined with a sterile solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0206] The ability of an adjuvant to increase the immune response to an antigen is generally manifested as a significant increase in immune-mediated reactions or a decrease in disease symptoms. For example, an increase in humoral immunity can be manifested as a significant increase in antibody titers against the antigen, and an increase in T cell activity can be manifested as increased cell proliferation or cytotoxicity or cytokine secretion. Adjuvants can also alter the immune response, for example, by changing a response that is primarily humoral or type 2 T helper cells to one that is primarily cellular or type 1 T helper cells.

[0207] Suitable adjuvants are known in the art (see WO2015 / 095811) and include, but are not limited to, poly(I:C), poly-ICLC, STING agonists, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, Delivery systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulator derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several immunoadjuvants specific for dendritic cells have been described (e.g., MF59) (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 the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent 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, which is incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich D I et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0208] It has also been reported that CpG immunostimulatory oligonucleotides enhance the effect of adjuvants in therapeutic settings. Without being bound by theory, CpG oligonucleotides work by activating innate (non-adaptive) immune systems via Toll-like receptors (TLRs) (mainly TLR9). The TLR9 activation of CpG triggers enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live viruses or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cell immunogenic pharmaceutical compositions, and polysaccharide conjugates in preventive and therapeutic immunogenic pharmaceutical compositions. Importantly, it enhances the maturation and differentiation of dendritic cells, thereby leading to the activation of enhanced TH1 cells and the generation of strong cytotoxic T lymphocytes (CTL), even in the absence of CD4+T cell help. Even in the presence of adjuvants that usually promote TH2 bias, such as alum or incomplete Freund's adjuvant (IFA), the TH1 bias induced by TLR9 stimulation is also maintained. When formulated with other adjuvants or co-administered or in preparations such as microparticles, nanoparticles, lipid emulsions or similar formulations, CpG oligonucleotides demonstrate even greater adjuvant activity, which is particularly useful for inducing strong responses when antigens are relatively weak. They can also accelerate immune responses and enable antigen dosages to be reduced, and in some experiments, have suitable antibody responses (Arthur M.Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484) with full dose immunogenic pharmaceutical compositions that do not contain CpG. U.S. Patent number 6,406,705 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, DE), which is a component of pharmaceutical compositions as described herein. Other TLR binding molecules can also be used, such as RNA in conjunction with TLR7, TLR8 and or TLR9.

[0209] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., poly I:CI2U), poly IC:LC, non-CpG bacterial DNA or RNA, ssRNA against TLR840, and immunoactive 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 adjuvants. The amounts and concentrations of adjuvants and additives that can be used in the context of the present invention can be readily determined by the skilled person without undue experimentation. Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0210] In some embodiments, the immunogenic composition according to the present disclosure may include more than one different adjuvant. In addition, the present invention includes a pharmaceutical composition comprising any adjuvant substance, including any of the above-mentioned adjuvants and combinations thereof. In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent (e.g., a peptide, a polynucleotide, a TCR, a CAR, a cell containing a TCR or a CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.), and the adjuvant can be administered separately in any suitable order.

[0211] Lipidation can be divided into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation and several other types of modification. N-myristoylation is the covalent attachment of myristate (C14 saturated acid) to glycine residues. Palmitoylation is the thioester connection of long-chain fatty acids (C16) to cysteine ​​residues. GPI-anchor addition is a glycosyl-phosphatidylinositol (GPI) connection via an amide bond. Prenylation is the thioether connection of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to a cysteine ​​residue. Other types of modification may include attachment of S-diacylglycerols via the sulfur atom of cysteine, conjugation via the O-octanoyl group of a serine or threonine residue, conjugation to an S-archaeol of a cysteine ​​residue, and cholesterol attachment.

[0212] The fatty acid for generating the lipidated peptide can comprise C2 to C30 saturated, monounsaturated or polyunsaturated fatty acyl groups. Exemplary fatty acids can comprise palmitoyl, myristoyl, stearoyl and decanoyl. In some cases, the lipid moiety with adjuvant properties is attached to the polypeptide of interest to trigger or enhance immunogenicity in the absence of an exogenous adjuvant. Lipidated peptides or lipopeptides can be referred to as self-adjuvant lipopeptides. Any fatty acid described above and elsewhere herein can trigger or enhance the immunogenicity of the polypeptide of interest. The fatty acid that can trigger or enhance immunogenicity can comprise palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl and decanoyl.

[0213] Polypeptides such as naked peptides or lipidated peptides can be incorporated into liposomes. Sometimes, lipidated peptides can be incorporated into liposomes. For example, the lipid portion of the lipidated peptide can spontaneously integrate into the lipid bilayer of the liposome. Thus, the lipopeptide can be presented on the "surface" of the liposome.

[0214] Liposomes can also be used to deliver nucleic acids into cells. The nucleic acid of interest comprises one or more sequences encoding T cell receptors. Liposomes can be used to deliver DNA or RNA. Liposomes can be used to deliver nucleic acids in vectors. The nucleic acid length can be 50-200,000 nucleotides, or 100-500,000 nucleotides, or 20-500,000 nucleotides. 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), unilamellar or oligolamellar vesicles prepared by reverse phase evaporation (REVs), multilamellar vesicles prepared by reverse phase evaporation (MLV-REVs), stable multilamellar vesicles (SPLVs), frozen and thawed MLVs (FATMLVs), vesicles prepared by extrusion (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydration-rehydration vesicles (DRVs), and foam bodies (BSVs).

[0215] Depending on the method of preparation, liposomes can be unilamellar or multilamellar and can vary in size, ranging from about 0.02 μm to greater than about 10 μm in diameter. Liposomes can adsorb many types of cells and then release the incorporated agent (e.g., a peptide as described herein). In some cases, the liposomes fuse with the target cells, whereupon the contents of the liposomes are subsequently emptied into the target cells. The liposomes can be endocytosed by phagocytes. Endocytosis can be followed by intralysosomal degradation of the liposome lipids and release of the encapsulating agent.

[0216] Liposomes provided herein may also include a carrier lipid. In some embodiments, the carrier lipid is a phospholipid. The carrier lipid capable of forming liposomes includes but is not limited to dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS). Other suitable phospholipids also 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 its combination. In some embodiments, liposomes also include a sterol (such as cholesterol) that regulates liposome formation. Carrier lipids can be any known non-phosphate polar lipids.

[0217] The pharmaceutical composition can be encapsulated in liposomes using known techniques. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present invention.

[0218] The pharmaceutical composition can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are well known to those skilled in the art. Basically, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids and surfactants (if necessary) are added, and the material is dialyzed or sonicated as needed.

[0219] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be adapted for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, a compound can be incorporated and implanted into the microspheres or microsphere complexes for slow release over a period of days to months.

[0220] Cell-based immunogenic pharmaceutical compositions can also be administered to a subject. For example, an immunogenic pharmaceutical composition based on antigen presenting cells (APCs) can be formulated using any known techniques, carriers, and excipients suitable and as understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, an APC-based immunogenic pharmaceutical composition can be an immunogenic pharmaceutical composition based on dendritic cells.

[0221] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared by in vitro or in vivo methods. In vitro methods can include the use of autologous DC pulsed with the polypeptides described herein to activate or load the DCs prior to administration to the patient. In vivo methods can include the use of antibodies coupled to the polypeptides described herein to target specific DC receptors. DC-based immunogenic pharmaceutical compositions can further include DC activators, such as TLR3, TLR-7-8, and CD40 agonists. DC-based immunogenic pharmaceutical compositions can further include adjuvants and pharmaceutically acceptable carriers.

[0222] Adjuvants can be used to enhance the immune response (humoral and / or cellular response) elicited in a patient receiving an immunogenic pharmaceutical composition. Sometimes, an adjuvant can elicit a Th1 type response. Other times, an adjuvant can elicit a Th2 type response. A Th1 type response can be characterized by the production of cytokines such as IFN-γ, while a Th2 type response can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0223] In some aspects, 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 increases the presentation of liposomal antigens to specific T lymphocytes. In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant with the immunogenic pharmaceutical formulations described herein.

[0224] Adjuvants may also include stimulatory molecules such as cytokines. Non-limiting examples of cytokines include: CCL20, alpha-interferon (IFNα), beta-interferon (IFNβ), gamma-interferon (IFNγ), platelet-derived growth factor (PDGF), TNFα, GM-CSF, epidermal growth factor (EGF), skin T cell-attracting chemokine (CTACK), epidermal 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, MadCAM-1, LFA-1, VLA -1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, 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.

[0225] Other adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-1, JNK, interferon response 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 their functional fragments.

[0226] In some aspects, the adjuvant can be a modulator of toll-like receptors. Examples of modulators of toll-like receptors include TLR9 agonists and are not limited to small molecule modulators of toll-like receptors, such as imiquimod. Sometimes, the adjuvant is selected from bacterial toxins, polyoxypropylene-polyethylene glycol block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or a combination thereof. Sometimes, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion may comprise at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplet diameter in the emulsion may be less than 5 μm, or even have a submicron diameter, and these small sizes are achieved by a microfluidizer to provide a stable emulsion. Droplets less than 220 nm in size can be sterilized by filtration.

[0227] In some cases, the immunogenic pharmaceutical composition may include carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and / or preservatives), water, oils (including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), salt solutions, dextrose and glycerol aqueous solutions, flavorings, coloring agents and other acceptable additives, adjuvants or binders, other pharmaceutically acceptable auxiliary substances required to achieve approximate physiological conditions, such as pH buffers, tonicity regulators, emulsifiers, wetting agents, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. In another case, the pharmaceutical product is substantially free of preservatives. In other cases, the pharmaceutical product may contain at least one preservative. It will be appreciated that while any suitable carrier known to those of ordinary skill in the art may be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the mode of administration.

[0228] The immunogenic pharmaceutical composition may include a preservative, such as thimerosal or 2-phenoxyethanol. In some cases, the immunogenic pharmaceutical composition is substantially free (e.g., <10 μg / mL) of mercury species, e.g., free of thimerosal. Alpha-tocopheryl succinate may be used as a substitute for mercury compounds.

[0229] To control tonicity, the immunogenic pharmaceutical composition may contain physiological salts, such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride.

[0230] The immunogenic pharmaceutical composition may have an osmolality within the range of 200 mOsm / kg to 400 mOsm / kg, 240-360 mOsm / kg, or 290-310 mOsm / kg.

[0231] The immunogenic pharmaceutical composition may comprise one or more buffers, such as Tris buffer; borate buffer; succinate buffer; histidine buffer (particularly containing aluminum hydroxide adjuvant); or citrate buffer. In some cases, the buffer is included in the range of 5-20 or 10-50 mM.

[0232] 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.

[0233] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be pyrogen-free, for example, containing <1 EU (endotoxin unit, a standard measure) per dose, and may be <0.1 EU per dose. The composition may be gluten-free.

[0234] The immunogenic pharmaceutical composition may include a detergent, for example, a polyoxyethylene sorbitan ester surfactant (known as a "Tween") or an octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol). The detergent may be present only in trace amounts. The immunogenic pharmaceutical composition may include less than 1 mg / mL of each of octoxynol-10 and polysorbate 80. Other residual components in trace amounts may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0235] 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, known sterilization techniques, or can be sterilized by filtration. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized product combined with the sterile solution before administration.

[0236] For example, a pharmaceutical composition comprising an active agent (such as an immune cell disclosed herein) can be formulated with one or more adjuvant combinations to have a certain mol ratio. For example, an active agent such as an immune cell as described herein and one or more adjuvant combinations can be used. The mol ratio of about 99:1 to about 1:99 can be used. In some cases, the mol ratio of an active agent such as an immune cell as described herein and one or more adjuvant combinations can be selected from 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; and about 90:10 to about 10:90. The mol ratio of an active agent such as an immune cell as described herein and one or more adjuvant combinations can be about 1:9, and in some cases can be about 1:1. Active agents, such as the immune cells described herein, in combination with one or more adjuvants, can be formulated together in the same dosage unit, e.g., one vial, suppository, tablet, capsule, aerosol spray; or each agent, form and / or compound can be formulated in a separate unit, e.g., two vials, two suppositories, two tablets, two capsules, one tablet and one vial, aerosol spray, etc.

[0237] In some cases, the immunogenic pharmaceutical composition may be administered with an additional agent. The selection of additional agents may depend at least in part on the condition being treated. Additional agents may include, for example, checkpoint inhibitors such as anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agents (e.g., anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibodies); or any agent having a therapeutic effect on pathogen infection (e.g., viral infection), including, for example, drugs used to treat inflammatory conditions, such as NSAIDs, for example, ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, the checkpoint inhibitor can be a PD-1 / PD-L1 agonist 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 can additionally contain one or more supplements, such as vitamin C, E, or other antioxidants.

[0238] The pharmaceutical composition comprising the combination of an active agent such as an immune cell as described herein and one or more adjuvants can be prepared in a conventional manner using one or more physiologically acceptable carriers, which include excipients, diluents and / or adjuvants, such as carriers that promote the processing of active agents into products that can be used. Suitable formulations can rely at least in part on the selected route of administration. Medicaments as described herein can be delivered to patients using a variety of routes of administration or modes (including oral, buccal, surface, rectal, transdermal, mucosal, subcutaneous, intravenous and intramuscular administration, and by inhalation).

[0239] The active agent can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and can be presented in unit dosage form in ampoules, prefilled syringes, small volume infusions, or in multi-dose containers with added preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, for example, a solution in aqueous polyethylene glycol.

[0240] For injectable preparations, vehicle can be selected from those suitable known in the art, including aqueous solution or oily suspension or emulsion (with sesame oil, corn oil, cottonseed oil or peanut oil), and elixir, mannitol, dextrose or sterile aqueous solution, and similar pharmaceutical vehicles. Preparation can also include biocompatible, biodegradable polymer compositions, such as poly (lactic acid-co-glycolic acid). These materials can be made into microspheres or nanospheres, loaded drugs, and further coated or derivatized to provide excellent sustained release performance. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agents in injection-grade water, liposomes and vehicles suitable for lipophilic substances. Other vehicles used for periocular or intraocular injection are well known in the art.

[0241] In some cases, pharmaceutical composition is formulated into a pharmaceutical composition suitable for intravenous administration to human body according to conventional procedures. Generally speaking, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. When needed, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to alleviate the pain at the injection site. Usually, the composition is supplied separately or mixed together with a unit dosage form supply, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a sachet (sachette) indicating the amount of the active agent. When the composition is administered by infusion, it can be distributed with 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 provided so that the composition can be mixed before administration.

[0242] When administered by injection, the active agent can be formulated in an aqueous solution, particularly in a physiologically compatible buffer such as Hanks solution, Ringer's solution, or physiological saline buffer. The solution may contain formulation agents such as suspending agents, stabilizers, 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.

[0243] In addition to the preparations described above, active agents can also be formulated into depot products. Such long-acting preparations can be administered by implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or using a transdermal patch. Thus, for example, the medicament can be formulated with a suitable polymeric material or hydrophobic material (e.g., an emulsion in an acceptable oil) or an ion exchange resin, or formulated as a slightly soluble derivative, e.g., as a slightly soluble salt.

[0244] In some cases, the pharmaceutical composition comprising one or more medicaments exerts local and regional effects when applied topically or injected into a specific infection site or its vicinity. For example, viscous liquids, solutions, suspensions, solutions based on dimethyl sulfoxide (DMSO), liposomal preparations, gels, jellies, creams, lotions, ointments, suppositories, foams or aerosol sprays, etc., can be used for topical application directly through surface application to produce, for example, local and / or regional effects. Pharmaceutically suitable vehicles for such preparations include, for example, lower fatty alcohols, polyethylene glycols (such as glycerol or polyethylene glycol), fatty acid esters, oils, fats, polysiloxanes, etc. Such preparations can also include preservatives (such as parabens) and / or antioxidants (such as ascorbic acid and tocopherol). See also Dermatological Formulations:Percutaneous absorption, Barry (ed.), Marcel Dekker Incl, 1983. In another embodiment, a topical / surface preparation comprising a transporter, a carrier or an ion channel inhibitor is used to treat epidermal or mucosal viral infections.

[0245] The pharmaceutical composition may contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, sunscreens, odor absorbers, and dyes. The amount of these various adjuvants is commonly used in the relevant field, for example, from about 0.01% to about 20% of the total weight of the composition. Depending on their properties, these adjuvants can be introduced into the fat phase, the aqueous phase, and / or the lipid vesicles.

[0246] IX. Treatment

[0247] Provided herein are methods of using any of the nucleic acids disclosed above, vectors containing any of the nucleic acid sequences disclosed above, proteins encoded by any of the nucleic acids disclosed above, or host cells disclosed above to prepare a medicament for treating an immune disease or cancer.

[0248] Also provided herein are methods for treating subjects suffering from a disease, disorder, or condition. The therapeutic method may include administering a composition or pharmaceutical composition disclosed herein to a subject suffering from a disease, disorder, or condition. The present disclosure provides methods for treating a subject including immunogenic therapy. Methods for treating a disease (such as cancer or viral infection) are provided. The method may include administering to a subject an effective amount of a pharmaceutical composition comprising immunogenic antigen-specific T cells. In some embodiments, the antigen comprises a tumor antigen.

[0249] In some embodiments, a method of 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 of preventing resistance to cancer therapy, wherein the method comprises administering a pharmaceutical composition disclosed herein to a subject in need thereof. In some embodiments, the method is a method of inducing an immune response, wherein the method comprises administering a pharmaceutical composition disclosed herein to a subject in need thereof. In some embodiments, the immune response is a humoral response. In some embodiments, the immune response is a cytotoxic T cell response.

[0250] In some embodiments, the subject has cancer, wherein the cancer is selected from melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL).

[0251] In some embodiments, the subject suffers from breast cancer that is resistant to estrogen therapy. In some embodiments, breast cancer expresses an estrogen receptor with a mutation. In some embodiments, the subject suffers from CLL that is resistant to ibrutinib therapy. In some embodiments, CLL expresses a Bruton tyrosine kinase with a mutation (such as a C481S mutation). In some embodiments, the subject suffers from lung cancer that is resistant to tyrosine kinase inhibitors. In some embodiments, lung cancer expresses an epidermal growth factor receptor (EGFR) with a mutation (such as a T790M, L792F, or C797S mutation).

[0252] In some embodiments, the method further comprises administering at least one additional therapeutic agent or treatment modality. In some embodiments, at least one additional therapeutic agent or treatment modality 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 APC, a polynucleotide, an oncolytic virus, or any combination thereof. In some embodiments, 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 administering the pharmaceutical composition disclosed herein.

[0253] In some other aspects, provided herein is a use of a pharmaceutical composition in the preparation of a medicament for therapy. In some embodiments, the treatment method comprises administering to a subject an effective amount of T cells that specifically recognize an immunogenic neoantigenic peptide. In some embodiments, the treatment method comprises administering to a subject an effective amount of TCRs that specifically recognize an immunogenic neoantigenic peptide, such as TCRs expressed in T cells.

[0254] In some embodiments, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, colorectal cancer, rectal cancer, soft tissue sarcoma, Kaposi's sarcoma (Kaposi's sarcoma), B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunogenic NHL, high-grade lymphocytic NHL, high-grade small non-lytic cell NHL, 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 vascular proliferation associated with nevus hamartomatosis, edema, Meigs syndrome, and combinations thereof.

[0255] The methods of the present disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers that will 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, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

[0256] In addition, the diseases or conditions provided herein include refractory or recurrent malignancies whose growth can be inhibited using the methods of treatment disclosed herein. In some embodiments, the cancer to be treated by the methods of treatment disclosed herein is selected from carcinoma, squamous carcinoma, adenocarcinoma, sarcoma, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, anorectal squamous cell carcinoma, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, head and neck squamous cell carcinoma, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, the cancers to be treated by the methods of the present disclosure include, for example, carcinomas, squamous carcinomas (e.g., cancers of the cervix, eyelids, conjunctiva, vagina, lung, oral cavity, skin, bladder, tongue, larynx, and esophagus) and adenocarcinomas (e.g., cancers of the prostate, small intestine, endometrium, cervix, large intestine, lung, pancreas, esophagus, rectum, uterus, stomach, breast, and ovary). In some embodiments, the cancers to be treated by the methods of the present disclosure also include sarcomas (e.g., myogenic sarcomas), leukemias, neuromas, melanomas, and lymphomas. In some embodiments, the cancer to be treated by the methods of the present disclosure is breast cancer. In some embodiments, the cancer to be treated by the methods of the present disclosure is triple-negative breast cancer (TNBC). In some embodiments, the cancer to be treated by the methods of the present disclosure is ovarian cancer. In some embodiments, the cancer to be treated by the methods of the present disclosure is colorectal cancer.

[0257] In some embodiments, the patient or patient colony treated with a pharmaceutical composition of the present invention suffers from solid tumors. In some embodiments, solid tumors are 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 colony treated with a pharmaceutical composition of the present invention suffers from hematologic cancer. In some embodiments, the patient suffers from hematologic cancer, such as diffuse large B-cell lymphoma ("DLBCL"), Hodgkin lymphoma ("HL"), non-Hodgkin lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML") or multiple myeloma ("MM"). In some embodiments, the cancer of the patient to be treated or patient colony is selected from ovarian cancer, lung cancer and melanoma.

[0258] Specific examples of cancers that may be prevented and / or treated according to 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; pediatric tumors; neuroblastoma; malignant melanoma; epidermal cancer; leukemias, such as, but not limited to, acute leukemias, acute lymphoblastic leukemias, acute myeloid leukemias, such as myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemias, and myelodysplastic syndromes; chronic leukemias, such as, but not limited to, chronic myelogenous (granulocytic) leukemia, Chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as but not limited to Hodgkin's disease and non-Hodgkin's disease; multiple myeloma, such as 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 sarcomas, such as but not limited to skeletal sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced osteosarcoma, Paget's disease of bone, bone), osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as, but not limited to, gliomas, astrocytomas, brain stem gliomas, ependymomas, oligodendrogliomas, non-gliomas, acoustic neurothema, craniopharyngiomas, medulloblastomas, meningiomas, pineal cell tumors, pineoblastomas, and primary brain lymphomas; breast adenocarcinoma, including but not limited to adenocarcinoma, 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 cancer, such as but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, such as but not limited to insulinoma, gastrinoma, glucagonoma, VIPoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancer, such as but not limited to Cushing's disease disease), prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers, such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma;Uterine cancer, such as but not limited to endometrial cancer and uterine sarcoma; ovarian cancer, such as but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor and stromal tumor; esophageal cancer, such as but not limited to squamous carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma and oat cell (small cell) carcinoma; gastric cancer, such as but not limited to adenocarcinoma, fungoid (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma tumors and carcinosarcoma; colon cancer; colorectal cancer; KRAS mutant colorectal cancer; colon cancer; rectal cancer; liver cancer, such as, but not limited to, hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, such as adenocarcinoma; bile duct cancer, such as, but not limited to, papillary, nodular, and diffuse; lung cancer, such as KRAS mutant non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; lung cancer; testicular cancer, such as, but not limited to, germ cell tumors, seminoma, anaplastic ovarian, ovarian, androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; pineal cancer; oral cancer, such as, but not limited to, squamous cell carcinoma; basal cell carcinoma; salivary gland cancer, such as, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as, but not limited to, squamous cell carcinoma and verrucous; dermatophyte cancer, such as, but not limited to, ovarian, androgen-independent prostate cancer, adenocarcinoma, leiomyoma, and rhabdomyosarcoma; pelvic, ovarian, and urethral cancers. Skin cancer, such as but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, nevus-like malignant melanoma, acral pigmented melanoma; kidney cancer, such as but not limited to renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); renal cancer; Wilms' tumor; bladder cancer, such as but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancer includes myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinoma.

[0259] Cancers include, but are not limited to, B cell cancers, such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases, such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, etc. Other non-limiting examples of cancer types suitable for the methods encompassed by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, hepatocellular carcinoma, bile duct cancer, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as 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 disclosed method is an epithelial cancer, such as but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer or colon cancer. In 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. The characteristics of epithelial cancer can be in multiple other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner (brenner) or undifferentiated. In some embodiments, the disclosure is used to treat, diagnose and / or prognose lymphoma or its subtype, including but not limited to mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0260] In some embodiments, the subject has breast cancer that is resistant to antiestrogen therapy, which is 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.

[0261] In some embodiments, the breast cancer expresses an estrogen receptor with a mutation.

[0262] In some embodiments, the cancer is recurrent or metastatic breast cancer.In some embodiments, the cancer to be treated by the methods of treatment of the present disclosure is triple-negative breast cancer (TNBC).

[0263] The pharmaceutical compositions provided herein can 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).

[0264] In some embodiments, in addition to the pharmaceutical composition comprising the immunogenic therapy, at least one or more chemotherapeutic agents may be administered.In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.

[0265] In practicing the treatment or use methods provided herein, a therapeutically effective amount of the pharmaceutical composition can be administered to a subject suffering from 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.

[0266] The subject can be, for example, a mammal, a human, a pregnant woman, an elderly person, an adult, a teenager, a prepubescent child, a child, a toddler, an infant, a neonate or a newborn baby. The subject can be a patient. In some cases, the subject can be a human. In some cases, the subject can be a child (e.g., a young person below the age of puberty). In some cases, the subject can be an infant. In some cases, the subject can be a formula-fed infant. In some cases, the subject can be an individual participating in a clinical study. In some cases, the subject can be an experimental animal, e.g., a mammal or a rodent. In some cases, the subject can be a mouse. In some cases, the subject can be an obese or overweight subject.

[0267] In some embodiments, the subject has been previously treated with one or more different cancer treatment modalities. In some embodiments, the subject has been previously treated with one or more of radiation therapy, chemotherapy, or immunotherapy. In some embodiments, the subject has been treated with one, two, three, four, or five lines of prior therapy. In some embodiments, the prior therapy was cytotoxic therapy.

[0268] In some embodiments, the disease or condition treatable by the methods disclosed herein is abnormal cell growth. In some embodiments, the disease or condition treatable 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.

[0269] The methods of the present disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers that can 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, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other malignancies.

[0270] In addition, the diseases or conditions provided herein include refractory or recurrent malignancies, the growth of which can be inhibited using the methods of treatment disclosed herein. In some embodiments, the cancer treated by the methods of treatment disclosed herein is selected from carcinoma, squamous carcinoma, adenocarcinoma, sarcoma, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, anorectal squamous cell carcinoma, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, head and neck squamous cell carcinoma, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancers, leukemias, lymphomas, neuromas, and combinations thereof. In some embodiments, cancers to be treated by the methods of the present disclosure include, for example, carcinomas, squamous carcinomas (e.g., cancers of the cervix, eyelids, conjunctiva, vagina, lung, oral cavity, skin, bladder, tongue, larynx, and esophagus) and adenocarcinomas (e.g., cancers of the 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 also include sarcomas (e.g., myogenic sarcomas), leukemias, neuromas, melanomas, and lymphomas. In some embodiments, the cancer to be treated by the methods of the present disclosure is breast cancer. In some embodiments, the cancer to be treated by the methods of the present disclosure is triple-negative breast cancer (TNBC). In some embodiments, the cancer to be treated by the methods of the present disclosure is ovarian cancer. In some embodiments, the cancer to be treated by the methods of the present disclosure is colorectal cancer.

[0271] In some embodiments, the patient or patient colony treated with a pharmaceutical composition of the present invention suffers from solid tumors. In some embodiments, solid tumors are 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 colony treated with a pharmaceutical composition of the present invention suffers from hematologic cancer. In some embodiments, the patient suffers from hematologic cancer, such as diffuse large B-cell lymphoma ("DLBCL"), Hodgkin lymphoma ("HL"), non-Hodgkin lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML") or multiple myeloma ("MM"). In some embodiments, the cancer of the patient to be treated or patient colony is selected from ovarian cancer, lung cancer and melanoma.

[0272] Specific examples of cancers that can be prevented and / or treated according to the present disclosure include, but are not limited to, the following: renal cancer, renal cell carcinoma, glioblastoma multiforme, metastatic breast cancer; breast cancer; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; epidermal cancer; leukemias, such as, but not limited to, acute leukemias, acute lymphocytic leukemias, acute myeloid leukemias, such as myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemias, and myelodysplastic syndromes; chronic leukemias, such as, but not limited to, chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease, Non-Hodgkin's disease; multiple myeloma, such as, 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 sarcomas, such as, but not limited to, skeletal sarcoma, 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 (hemangioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, sarcoma and synovial sarcoma; brain tumors, such as, but not limited to, gliomas, astrocytomas, brainstem gliomas, ependymomas, oligodendrogliomas, non-gliomas, acoustic neurothecatheters, craniopharyngiomas, medulloblastomas, meningiomas, pineal cell tumors, pineoblastomas, and primary brain lymphomas; breast cancer, such as, but not limited to, adenocarcinoma, 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 cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, such as, but not limited to, limited to insulinomas, gastrinomas, glucagonomas, VIPomas, somatostatin-secreting tumors, and carcinoids or islet cell tumors; pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, such as, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers, such as, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; ovarian cancers, such as, but not limited to, epithelial ovarian cancer, borderline tumors, germ cell tumors, and stromal tumors; cervical cancer;Esophageal cancer, such as 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, such as but not limited to adenocarcinoma, mycosis fungoides (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; colon cancer; colorectal cancer, RAS mutant colorectal cancer; colon cancer; rectal cancer; liver cancer, such as but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, such as adenocarcinoma; bile duct cancer, such as but not limited to papillary, nodular and diffuse; lung cancer, such as RAS mutant non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; lung cancer; testicular cancer, such as but not limited to germ cell tumor, seminoma, anaplastic, classic (typical), spermatoma, non seminoma, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor); prostate cancer, such as but not limited to androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma and rhabdomyosarcoma; pineal cancer; oral cancer, such as but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as but not limited to adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; pharyngeal cancer, such as but not limited to squamous cell carcinoma and verrucous; skin cancer, such as but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, nevus malignant melanoma, acral lentiginous melanoma; kidney cancer, such as but not limited to renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); renal cancer; Wilms' tumor; bladder cancer, such as but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma.

[0273] In some embodiments, cancer in a subject with a mutation in a RAS gene is treated by administering a pharmaceutical composition comprising a TCR, the TCR recognizing a RAS mutant epitope complexed with an MHC protein encoded by the subject's HLA allele, the TCR having a TCR α chain variable region and a TCR β chain variable region, the variable region having an 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 a messenger RNA encoding the TCR, the TCR having a TCR α chain variable region and a TCR β chain variable region, the variable region having an amino acid sequence disclosed herein. In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid sequence encoding the TCR and capable of driving the expression of the TCR, wherein the TCR recognizes a RAS mutant epitope complexed with an MHC protein encoded by the subject's HLA allele, and wherein the TCR has a TCR α chain variable region and a TCR β chain variable region, the variable region having an amino acid sequence disclosed herein. In some embodiments, the pharmaceutical composition comprises a cell comprising a nucleic acid sequence encoding a TCR that recognizes a RAS mutant epitope in complex with an MHC protein encoded by an HLA allele of the subject, the TCR having a TCR α chain variable region and a TCR β chain variable region, the variable region having an amino acid sequence disclosed herein. In some embodiments, a pharmaceutical composition is administered to a subject having cancer, the composition comprising a TCR that recognizes a RAS mutant epitope in complex with an MHC protein encoded by an HLA allele of the subject, the TCR having a TCR α chain variable region and a TCR β chain variable region, the variable region having an amino acid sequence disclosed herein, wherein the cancer is selected from biliary tract adenocarcinoma, bladder transitional cell carcinoma, breast cancer, cervical adenocarcinoma, colon adenocarcinoma, colon adenoma, neuroblastoma (autonomic ganglion), acute myeloid leukemia, chronic myeloid leukemia, chronic Myelomonocytic leukemia, juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, Burkitt lymphoma, Hodgkin lymphoma, plasma cell myeloma, hepatocellular carcinoma, large cell carcinoma, non-small cell carcinoma, ductal carcinoma, endocrine tumors, prostate adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, angiosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, myxoma, malignant fibrous histiocytoma, pleomorphic sarcoma, germ cell tumor, seminoma, anaplastic carcinoma, follicular carcinoma, papillary carcinoma, and Hurthle cell carcinoma.

[0274] Although preferred embodiments of the present invention have been shown and described herein, it will be understood by those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present invention. It will be understood that in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be employed. The following claims are intended to define the scope of the present invention and are intended to encompass methods and structures within the scope of these claims and their equivalents.

[0275] Example

[0276] Example 1: Workflow for identification and validation of antigen-specific T cells

[0277] In this example, an exemplary workflow for generating and using antigenic peptide-specific TCRs is described. Figure 1 A graphical representation of this workflow is shown. Figure 2 An exemplary timeline for this workflow is described in detail. PBMCs are first obtained and cultured from a subject. PBMCs are then stabilized in overnight culture and incubated in the presence of peptides and cytokines. Monocytes and DC-derived antigen-presenting cells (APCs) help induce T cells to respond to peptide antigens presented by APCs. Between 10 days and 2 weeks, antigen-specific T cells expand and are sorted based on antigen specificity and activation (e.g., expression of CD8+ markers indicates the generation of cytotoxic T lymphocytes). Activated antigen-reactive T cells contain antigen-specific TCRs.

[0278] Briefly, a peptide synthesizer is used to synthesize mutant peptides containing mutant target epitopes (e.g., mutant RAS peptides). The synthesized peptides are used to load APCs to stimulate T cells from healthy donor peripheral blood mononuclear cell (PBMC) samples. PBMCs from patients containing neoantigens of interest can also be used to obtain neoantigen-specific T cells. After incubation with peptide-loaded APCs, the T cell population is analyzed by flow cytometry. Antigen-specific T cells are isolated using flow cytometry ( Figure 1 、 Figure 2 、 Figure 4A 、 Figure 5 and Figure 6 ). Single-cell TCR sequencing was performed on isolated antigen-specific T cells using the 10x Genomics Single Cell V(D)J System to analyze the sequences of isolated antigen-specific TCRs. TM The analysis pipeline analyzes the sequencing reads and selects candidate TCR sequences for further analysis and functional assays ( Figure 1 and Figure 6). If the sequence of the beta chain CDR3 sequence comprises any one of SEQ ID NOs: 46-68, the candidate TCR sequence can be removed and the candidate can then be 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: 46-68, the candidate TCR can be removed without any further analysis or functional assay, such that the final identified TCR does not comprise a beta chain CDR3 sequence of any one of SEQ ID NOs: 46-68.

[0279] For TCR functional analysis, vectors or mRNA encoding RAS peptides containing target mutant epitopes were transduced into HEK293T or A375 cells to generate antigen-expressing cell lines. Nucleic acid encoding candidate TCRs ( Figure 3A ) were used to transduce Jurkat cells, TCRβ-deficient Jurkat cells, or PBMCs from healthy donors. Figure 3B An exemplary vector is shown. Figure 4B and Figure 6 Functional assays of cells transduced with vectors expressing TCRs are shown. Cell lines expressing RAS antigens were co-cultured with TCR-transduced Jurkat cells, TCRβ-deficient Jurkat cells, or PBMCs from healthy donors for antigen recognition assays to analyze the functionality of candidate TCRs ( Figure 10 or Figure 11 ).

[0280] Example 2: Obtaining antigen-specific T cells

[0281] Antigen-specific T cells were amplified using in vitro T cell induction. PBMCs from healthy human donors were seeded in AIM V medium (Invitrogen) in each well of a 24-well plate. After 24 h of incubation, mutant RAS peptides, TNF-α, IL-1β, PGE1, and IL-7 were added to the wells. The culture medium was replaced with fresh culture medium every 2 days. Antigen-specific T cells were evaluated and isolated on days 10 to 20. Figure 2 ).

[0282] Example 3: HLA multimer staining and sorting

[0283] Neoantigen-specific T cell peptides were detected by combining HLA-multimers stained with recombinant HLA (e.g., HLA-A11.01 or HLA-C01.02) multimers conjugated with two different fluorescent dyes with neoantigen peptides. Anti-CD8, anti-CD4, anti-CD19, anti-CD16, anti-CD14, anti-CD56, and antibodies and Live / Dead IR dye (Invitrogen) were used for cell surface staining. CD8+ T cells were identified as CD8 + CD4 - CD19 - CD16 - CD14 - CD56 - IR - ( Figure 1 、 Figure 2 、 Figure 4A 、 Figure 5 and Figure 6 ). For sorting, up to 5×10 6 Each cell was incubated with 1-20 μg of polymer in 100 μL 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 in PBS + 0.5% human serum. Live / dead IR dye negative cells were gated and sorted for CD8 + / polymer + T cells were sorted.

[0284] Example 4: Deep Sequencing of Antigen-Specific TCRs

[0285] Antigen-specific T cells were sorted into a mixture of PBS and 2% FBS (Hyclone Defined), and RNA from single T cells was 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 ILLUMINA's MiSeq platform. The sequencing data were then analyzed using 10xGenomics analysis software to obtain paired TCRα and β sequences. Dominant clones were identified based on the frequency of cells with common TCRα and / or TCRβ. Figure 1 and Figure 6 ).

[0286] Example 5: Antigen-specific TCR gene synthesis and cloning

[0287] Figure 3ASchematic diagram representing an exemplary vector design incorporating TCR alpha and beta chain constructs; wherein the TCR alpha and beta chain constructs comprise nucleic acid sequences encoding the variable region (V), diversity region (D) (only in the beta chain construct), joining region (J), and constant region (C) of each of the TCR alpha (α) and TCR beta (β) chains, as shown in the figure, and the elements necessary for expression of the incorporated nucleic acid sequences, such as a promoter (e.g., the EF1a region shown in the figure), are contained in a vector with upstream regulatory elements, as shown, and F2A and P2A proteolytic cleavage sites and a sequence encoding puromycin resistance (Puro) are also incorporated for puromycin-mediated selection. The lentiviral vector was constructed using pCDH-EF1-T2A-Puro (SBI system bioscience). Antigen-specific TCR lentiviral vectors were generated by inserting the TCRβ variable region, followed by the TCRβ mouse constant region, a furin cleavage site, an SGSG linker, an F2A site, a TCRα variable region, a TCRα constant region, a T2A site, and a puromycin resistance gene. Figure 3A and Figure 3B ).

[0288] Example 6: Transfection and transduction

[0289] Lentivirus encoding antigen-specific TCR was prepared by transiently transfecting 293T cells. Cells were plated at 7 × 10 6 Cells were seeded on 10 cm plates and transfected 16 hours later with 7 μg of lentiviral vector, 7 μg of a packaging plasmid mix (pPAX and pMD2.G), 28 μL of Fugene (Promega), and 1 mL of Opti-MEM (Gibco). The medium was changed one day after transfection. 72 hours after transfection, the supernatant was collected and concentrated 10-fold.

[0290] 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. 5×10 5 CD8-Jurkat cells were seeded into each well of a 96-well plate and 25 μL of concentrated lentivirus was added. The cells were centrifuged at 2400 rpm for 1 hour and incubated in a CO2 incubator. The cells were transduced again with fresh culture medium with polybrene and FBS and 25 μL of concentrated lentivirus. 24 hours after the second transduction, the culture medium was replaced with RPMI-1640 containing 10% FBS and Pen / Strep. Puromycin treatment (1 μg / ml) was started on the 4th day after transduction.

[0291] Example 7: Binding of TCR-transduced Jurkat to HLA peptides

[0292] To evaluate TCR transduction, transduced Jurkat cells or PBMCs were stained with fluorescent dye-conjugated multimers (HLA-neoantigens), anti-CD8 antibodies, anti-mTCR constant region antibodies, and live / dead IR dye. mTCR and multimer-positive cells were measured by flow cytometry ( Figure 4B and Figure 6-Figure 8 ). Figure 7 It was shown that Jurkat cells transduced with the RAS G12V peptide-specific TCR specifically expressed TCR (ie, RAS-TCR-1) relative to untransduced Jurkat cells. Figure 8 It is shown that four different PBMC lines, each transduced with a RAS G12V peptide-specific TCR, specifically express TCRs relative to untransduced PBMC lines. Figure 9 It was shown that Jurkat cells electroporated with two other RAS G12V peptide-specific TCRs (ie, RAS-TCR-2 and RAS-TCR-3) specifically expressed TCRs relative to non-electroporated Jurkat cells.

[0293] Example 8: Peptide loading of target cells and functional avidity of RAS mutant peptide-specific TCRs

[0294] For exogenous peptide pulses, 1 × 10 6 T2, 293T, or A375 cells were incubated with 10 pg / mL human β2-microglobulin (Calbiochem) and titrated (ranging from 1 × 10 -5 M to 1×10 -12 M) of RAS peptide were incubated at 37°C and 5% CO2 for 2 h. -5 M influenza peptide GIL (influenza matrix protein 58-66 GILGFVTL, Metabion)-pulsed T2 cells were used as negative controls. After washing, peptide-loaded T2 cells were used for TCR activation assays.

[0295] like Figure 10 As shown, to investigate the affinity and specificity of RAS mutant peptide-specific TCRs, Jurkat cells transduced and expressing TCRs specific for mutant RAS peptides were co-cultured with HLA-A11:01+ target cells pulsed with RAS G12V 9-mer (VVGAVGVGK (SEQ ID NO: 43); left panel), RAS G12V 10-mer (VVVGAVGVGK (SEQ ID NO: 44); right panel), or RAS WT peptide (data points on the far right of both panels). CD69 was measured as a marker of activation (at Figure 10The Y axis of the PCR reaction is shown as the percentage of CD69+ cells relative to live TCR+ and CD3+ cells. Only the mutant G12V peptide was able to induce any activation of transduced Jurkat cells, while the WT RAS peptide was not, demonstrating the specificity of the TCR for the mutant RAS peptide. In addition, the avidity of the 9-mer and 10-mer was found to be 3.8 nM and 9.6 nM, respectively.

[0296] Likewise, Figure 18 As shown, Jurkat cells transduced and expressing TCRs (TCR-5, TCR-6, TCR-7) specific for mutant RAS peptides were co-cultured 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 (in Figure 18 The Y-axis shows the percentage of CD69+ cells relative to the total viable cells. The results showed that TCR was responsive.

[0297] In addition, if Figure 11 As shown, to investigate the affinity and specificity of other RAS mutant peptide-specific TCRs, Jurkat cells (each electroporated and expressing TCRs specific for mutant RAS peptides (RAS-TCR-2 and RAS-TCR-3, left and right, respectively)) were co-cultured with HLA-C01:02+ target cells pulsed with RAS G12V mutant peptide (AVGVGKSAL (SEQ ID NO: 45)) or RAS WT peptide (data points on the far right of both figures). CD69 and PD1 were measured as markers of activation (at Figure 11 The Y-axis is shown as the percentage of CD69+ and PD1+ cells relative to viable TCR+ and CD3+ cells. Only the mutant G12V peptide was able to induce any activation of transduced Jurkat cells, while the WT RAS peptide was not, demonstrating the specificity of the TCR for the mutant RAS peptide.

[0298] Figure 16 Depicted are exemplary data showing the functional avidity of RAS TCR-2 and RAS TCR-3. Transduced Jurkat NFAT luciferase reporter cells expressing RAS TCR-2 or RAS TCR-3 were co-cultured with HLA-C*01:02+ target cells pulsed with KRAS G12V 9-mer or KRAS WT peptide. 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.

[0299] Example 9: Target cells expressing neoantigens

[0300] For target cells expressing endogenous antigens, 1 × 10 cells were transduced with lentiviral vectors encoding RAS mutant peptides. 6 HEK293 or A375 cells were cultured in a 4% culture medium (DMEM with 10% FBS).

[0301] Example 10: IL-2 release assay

[0302] To investigate specificity, T cell clones (2.5 × 10 5 cells) and HEK293T, HLA-A03.01-transduced A375, or HLA-A11.01-transduced A375 cell lines (5 × 10 4 After 24 h of co-culture, culture supernatants were collected and IL-2 concentrations were assessed using a V-PLEX human IL-2 assay (MesoScale Discovery) by standard MSD.

[0303] Example 11: IFN-γ release assay

[0304] To investigate specificity, T cell clones (2 × 10 in 100 μL) were cultured. 3 cells) were incubated with cell lines expressing RAS mutant peptide neoantigens. After 24 h of co-culture, the culture supernatant was collected and analyzed using OptEIA TM IFN-γ secretion was assessed by standard ELISA using the Human IFN-γ Kit (BD Biosciences Pharmingen).

[0305] Example 12: Cytotoxicity Assay

[0306] The cytotoxic activity of T cell clones can be analyzed in a standard 4 h 51-chromium release assay. Briefly, 1 × 10 6 100 μCi Na2 51 CrO4 (ICN Biochemicals) labeling for 1-1.5h. 51 Cr-labeled target cells were cultured with T cells in RPMI 1640 with 12% FCS. To determine functional avidity, 1×10 4 T cells were added to 1×10 cells loaded with titrated amounts of RAS mutant peptide neoantigens. 3 In T2 cells pulsed with peptides, the E:T ratio was kept constant at 10:1.

[0307] After 4 h of co-culture at 37°C, 50 μL of supernatant can be collected and the radioactivity can be measured in a gamma counter. The percentage of specific 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. For the calculation of the relative percentage lysis, the maximum specific lysis percentage can be set to a reference value of 100%, and the corresponding values ​​can be calculated based on this reference. In order to determine the half-maximal lysis, the relative lysis percentage can be plotted against the peptide concentration. The peptide concentration at which the curve intersects with 50% relative lysis can be used as the value of half-maximal lysis.

[0308] Example 13: Cell Killing Assay

[0309] RAS neoantigen-specific recombinant TCRs were transduced into PBMCs and their ability to kill cancer cell lines was analyzed ( Figure 12 ). Cells expressing recombinant TCR (rTCR) showed significantly higher killing of cancer cell lines relative to the control. The SW620 cell line that 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. PBMCs transduced with rTCR were co-cultured with two different SW620 cell lines (expressing or not expressing HLA-A11:01) for 100 hours. Supernatants and cells were then collected at different time points to analyze cytokine secretion (data not shown) and the growth of SW620 cells.

[0310] The method is to co-culture cells expressing TCRs specific for mutant RAS peptides on specific HLA with target cancer cells transduced with mutant RAS peptides expressing the corresponding HLA, and to measure the relative growth of target cells and the specific measurement of the apoptosis marker Annexin V in target cancer cells. Figure 12), cytotoxic activity was assessed. Target cancer SW620 cells were engineered to express mutant peptides and appropriate MHC-I alleles. SW620 target tumor cells cultured alone or co-cultured with PBMCs transduced with irrelevant TCRs were used as negative controls. Target cells transduced with mimics (e.g., not expressing mutant peptides) can also be used as negative controls. Cells were also transduced to stably express GFP, allowing the growth of target cells to be tracked. PBMCs from healthy donors used as effector cells were transduced to express TCRs specific for mutant RAS peptides. Target cells were co-cultured with effector cells in a culture medium containing Annexin V detection reagent at a ratio of 10:1 for 100 hours. The GFP signal and Annexin-V signal that changed over time were measured using the IncuCyte S3 instrument. The AnnexinV signal from the effector cells was filtered out by size exclusion. The growth and death of target cells are expressed as the GFP and Annexin-V areas (mm) that changed over time, respectively. 2 )( Figure 12 Y-axis).

[0311] Specific killing of target cells was also observed when T cells were used in killing assays: CD4+ T cells isolated from PBMC expressing TCRs specific for mutant RAS peptides on a particular HLA were co-cultured with target cancer cells transduced with mutant RAS peptides expressing the corresponding HLA and the relative growth of the target cells was determined, as well as the specific measurement of the apoptosis marker Annexin V ( Figure 13 ), cytotoxic activity was assessed. Target cancer SW620 cells were engineered to express mutant peptides and appropriate MHC-I alleles. SW620 target tumor cells cultured alone or co-cultured with CD4+ T cells isolated from PBMCs that had not been transduced with RAS mutant peptide-specific TCRs were used as negative controls. Target cells transduced with a mimic (e.g., not expressing the mutant peptide) can also be used as negative controls. Target cells and effector cells were co-cultured in culture medium containing Annexin V detection reagent at ratios of 10:1, 5:1, 3:1, and 1:1 for 100 h. Figure 13 As shown, higher numbers of CD4+ T cells relative to target cells induced progressively greater amounts of cell killing in target cells expressing mutant RAS peptides. Since target cell killing was induced by isolated CD4+ T cells co-cultured with target cancer cells, this data also indicates that this killing is independent of CD8 protein.

[0312] In some examples, after co-culture, the cells were cultured with untransduced SW620 cells (Ras mut cell lines), compared with the SW620 cell lines transduced with HLA-A11:01 or HLA-C01:02 (Ras mutSignificantly higher levels of IFNγ, IL-2, and TNFα were detected in the group transduced with HLA-A11:01 or HLA-C01:02. In addition, a significantly higher percentage of caspase-3 (a marker of apoptosis)-positive cells was observed in the SW620 cell line transduced with HLA-A11:01 or HLA-C01:02 compared to the control. Therefore, rTCR-transduced PBMCs are not only able to secrete cytokines (IFNγ, IL-2, and TNFα), but are also able to functionally and specifically kill target cells.

[0313] In some examples, cytotoxic activity is assessed before TCR cloning. T cells induced by mutant RAS peptides on specific HLA are co-cultured with mutant target cancer cells expressing corresponding HLA loaded with a certain concentration of RAS mutant peptides. The relative growth of target cancer cells and the apoptosis marker Annexin V are specifically measured. Target cells are also transduced to stably express GFP, allowing the growth of target cells to be tracked. The GFP signal and Annexin-V signal that change over time are measured using the IncuCyte S3 instrument. The Annexin V signal derived from effector cells is filtered out by size exclusion. The growth and death of target cells are expressed as the GFP and Annexin-V areas (mm2) that change over time, respectively. 2 For T cells that exhibit cytotoxicity at low peptide concentrations compared to no peptide and / or wild-type peptide, TCR sequencing can be performed as described above.

[0314] Example 14A: SNG-M cell killing assay

[0315] The tumor cell line SNG-M endogenously expresses HLA-A11:01 and low levels of KRAS G12V. Healthy donor peripheral blood mononuclear cells were transduced to express RAS TCR-1. Unrelated TCR-transduced T cells were used as negative transduced T cell controls. TCR-transduced T cells were co-cultured with SNG-M tumor cells at a 3:1 effector cell to target cell ratio. Killing was measured via impedance on the xCELLigence RTCA MP. The percentage of lysis was calculated using SNG-M tumor cells cultured in the absence of any effector T cells. Compared to unrelated TCR-transduced T cells, RAS TCR-1 transduced T cells resulted in approximately 3 times more SNG-M killing over time ( Figure 14 ).

[0316] Example 14B: Engineered SW-60 Cell Killing Assay

[0317] The colorectal cancer cell line SW-620 endogenously carries the KRAS G12V mutation. SW-620 cells were engineered to express HLA C*01:02, the cognate HLA for RAS TCR-2 and RAS TCR-3. Peripheral blood mononuclear cells from healthy donors were transduced to express either RAS TCR-2 or RAS TCR-3. Untransduced T cells were used as negative controls. TCR-transduced T cells were co-cultured with SW-620 or SW-620HLA C*01:02 cells at different effector cell to target cell ratios (3:1, 6:1, and 12:1). Killing was measured by impedance on the xCELLigence RTCA MP. Percent cytolysis was calculated based on tumor cells co-cultured with untransduced T cells. Both RAS TCR-2 and RAS TCR-3 transduced T cells were able to effectively recognize and kill SW-620HLA C*01:02 (based on endogenous antigen expression). Figure 17A ).

[0318] Example 14C: Panc 03.27 cell killing assay

[0319] The pancreatic tumor cell line Panc 03.27 endogenously expresses HLA-C01:02 and the KRAS G12V mutation. Peripheral blood mononuclear cells from healthy donors were transduced to express either RAS TCR-2 or RAS TCR-3. Untransduced T cells were used as negative controls. TCR-transduced T cells were co-cultured with Panc 03.27 tumor cells at two different effector cell to target cell ratios (6:1 and 12:1). Killing was measured by impedance on the xCELLigence platform. Percent cytolysis was calculated based on tumor cells co-cultured with untransduced T cells. Both RAS TCR-2 and RAS TCR-3 transduced T cells were able to effectively recognize and kill Panc 03.27 tumor cells based on endogenous HLA and antigen expression ( Figure 17B ).

[0320] Example 15: Tumor regression assay

[0321] Immunodeficient NCG (PrkdcKO, IL2RgKO and SirpaKO NOG) mice were subcutaneously inoculated with the tumor cell line A375-HLA-A11:01-KRAS G12V, which is an A375 tumor cell line engineered to express HLA-A11:01 and KRAS G12V. Healthy donor peripheral blood mononuclear cells were transduced to express RAS TCR-1. Healthy donor peripheral blood mononuclear cells were also transduced with an irrelevant chimeric antigen receptor as a negative transduced T cell control. When tumors reached an average of 80 mm^3, PBS or effector T cells were injected intravenously into the mice. Mice were treated with 6e6 antigen-specific TCR-transduced T cells, and the total cell dose was matched for the irrelevant transduced T cell group. Mice treated with RAS TCR-1 transduced T cells showed tumor regression compared to PBS and irrelevant transduced T cell controls ( Figure 15 ).

[0322] Example 16: Summary of developed recombinant TCRs

[0323] Thi...

Claims

1. A recombinant nucleic acid encoding a T cell receptor (TCR), wherein the TCR comprises a TCR β chain construct, wherein the TCR β chain construct comprises a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:

6. 2 . The recombinant nucleic acid according to claim 1 , wherein the TCR β chain construct further comprises a complementarity determining region 1 (CDR1), wherein the CDR1 has the amino acid sequence shown in SEQ ID NO:

4. 3 . The recombinant nucleic acid according to claim 1 , wherein the TCR β chain construct further comprises a complementarity determining region 2 (CDR2), wherein the CDR2 has the amino acid sequence shown in SEQ ID NO:

5. 4 . The recombinant nucleic acid according to claim 1 , wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

12. 5 . The recombinant nucleic acid according to claim 1 , wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

12.

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

12. 7 . The recombinant nucleic acid according to claim 1 , wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

12.

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

3.

9. The recombinant nucleic acid according to any one of claims 1 to 8, comprising: (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.

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

9.

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

9.

12. The recombinant nucleic acid of any one of claims 1-11, 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 shown in SEQ ID NO:

9. 13 . The recombinant nucleic acid according to claim 1 , wherein the TCR α chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

9.

14. The recombinant nucleic acid of any one of claims 1 to 13, wherein the TCR comprises: (a) a beta chain having the amino acid sequence shown in SEQ ID NO: 14 or 70, or an amino acid sequence having at least 80% identity with SEQ ID NO: 14 or 70, (b) an α chain having the amino acid sequence shown in SEQ ID NO: 13 or 69, or an amino acid sequence at least 80% identical to SEQ ID NO: 13 or 69.

15. The recombinant nucleic acid of any one of claims 1-14, wherein the TCR binds to an epitope from human RAS comprising the mutation G12V.

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

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

18. A recombinant nucleic acid encoding a T cell receptor (TCR), wherein the TCR comprises a TCR β chain construct comprising a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:

20.

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

18.

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

19.

21. The recombinant nucleic acid of any one of claims 18-20, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

26.

22. The recombinant nucleic acid of any one of claims 18-21, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

26.

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

26.

24. The recombinant nucleic acid of any one of claims 18-23, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

26.

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

17.

26. The recombinant nucleic acid according to any one of claims 18 to 25, comprising: (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.

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

23.

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

23.

29. The recombinant nucleic acid of any one of claims 18-28, 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 shown in SEQ ID NO:

23.

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

23.

31. The recombinant nucleic acid of any one of claims 18-30, wherein the TCR comprises: (a) a beta chain having the amino acid sequence shown in SEQ ID NO: 28 or 72, or an amino acid sequence having at least 80% identity with SEQ ID NO: 28 or 72, (b) an α chain having the amino acid sequence shown in SEQ ID NO: 27 or 71, or an amino acid sequence at least 80% identical to SEQ ID NO: 27 or 71.

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

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

45.

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

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

34.

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

32.

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

33.

38. The recombinant nucleic acid of any one of claims 35-37, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

40.

39. The recombinant nucleic acid of any one of claims 35-38, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

40.

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

40.

41. The recombinant nucleic acid of any one of claims 35-40, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

40.

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

31.

43. The recombinant nucleic acid according to any one of claims 35 to 42, comprising: (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.

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

37.

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

37.

46. ​​The recombinant nucleic acid of any one of claims 35-45, 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 shown in SEQ ID NO:

37.

47. The recombinant nucleic acid of any one of claims 35-46, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

37.

48. The recombinant nucleic acid of any one of claims 35-47, wherein the TCR comprises: (a) a beta chain having the amino acid sequence shown in SEQ ID NO: 41 or 74, or an amino acid sequence at least 80% identical to SEQ ID NO: 41 or 74, (b) an α chain having the amino acid sequence shown in SEQ ID NO: 42 or 73, or an amino acid sequence at least 80% identical to SEQ ID NO: 42 or 73.

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

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

45.

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

52. The recombinant nucleic acid of any one of claims 1-51, wherein the recombinant nucleic acid is a vector.

53. The recombinant nucleic acid of any one of claims 1-52, wherein the epitope is 8-25 amino acids in length.

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

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

56. according to the recombinant nucleic acid described in any one of claims 1-55, wherein said epitope is with the K of less than 500nM, 250nM, 150nM, 100nM, 50nM, 25nM or 10nM. D or IC 50 Binds to the human MHC.

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

58. The recombinant nucleic acid of any one of claims 1-57, wherein the TCR is expressed 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 Binds to the MHC-peptide complex.

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

60. A cell comprising the recombinant nucleic acid of any one of claims 1-59.

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

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

63. The cell of claim 60 or 61, wherein the cell is a CD8+ T cell.

64. The cell of claim 60 or 61, wherein the cell is isolated from a subject having a RAS mutation.

65. 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. The pharmaceutical composition of claim 65, further comprising an immunomodulator or adjuvant.

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

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

69. The pharmaceutical composition according to any one of claims 65-67, for use in treating immune diseases or cancer.

70. The pharmaceutical composition according to claim 68, which is used to treat immune diseases or cancer.

71. Use of the pharmaceutical composition according to any one of claims 65-67 for treating immune diseases or cancer.

72. Use of the pharmaceutical composition according to claim 68 for treating immune diseases or cancer.

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

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

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

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

77. A method of identifying a subject having cancer as a candidate for a therapeutic agent, the method comprising identifying the subject as a subject expressing a protein encoded by an HLA-A11:01 allele or an HLA C01:02 allele, wherein the therapeutic agent is a pharmaceutical composition selected from any one of claims 65-67.

78. A method of identifying a subject having cancer as a candidate for a therapeutic agent, the method comprising identifying 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 according to any one of claims 64-68.

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

80. A recombinant nucleic acid encoding a T cell receptor (TCR), comprising a TCR β chain construct and a TCR α chain construct, wherein the TCR specifically binds to a mutant epitope from human RAS complexed with a human MHC encoded by an HLA-A68:01 allele, wherein the mutant epitope from human RAS is characterized by a G12V mutation.

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

82.

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

80.

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

81.

84. The recombinant nucleic acid of any one of claims 80-83, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

84.

85. The recombinant nucleic acid of any one of claims 80-84, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

84.

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

84.

87. The recombinant nucleic acid of any one of claims 80-86, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

84.

88. The recombinant nucleic acid of any one of claims 80-87, further comprising a TCR alpha chain construct comprising CDR1, CDR2 and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 77; The CDR2 has the amino acid sequence shown in SEQ ID NO: 78; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

79.

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

83.

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

83.

91. The recombinant nucleic acid of any one of claims 80-90, 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 shown in SEQ ID NO:

83.

92. The recombinant nucleic acid of any one of claims 80-91, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

83.

93. The recombinant nucleic acid of any one of claims 80-92, wherein the epitope from human RAS comprising 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 mutant epitope from human RAS, the mutant epitope from human RAS complexed with a human MHC encoded by an HLA-C03:03 allele, wherein the mutant epitope from human RAS is characterized by a G12V mutation.

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

97. The recombinant nucleic acid of claim 96, wherein the TCR(i) has 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 binds to the mutant epitope from human RAS in complex with the human MHC encoded by the HLA-C03:03 allele; and (ii) 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 Binds to the mutant epitope from human RAS in complex with the human MHC encoded by the HLA-C03:04 allele.

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

109.

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

107.

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

108.

101. The recombinant nucleic acid of any one of claims 95-100, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

111.

102. The recombinant nucleic acid of any one of claims 95-101, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

111.

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

111.

104. The recombinant nucleic acid of any one of claims 95-103, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

111.

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

106.

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

110.

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

110.

108. The recombinant nucleic acid of any one of claims 95-107, 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 shown in SEQ ID NO:

110.

109. The recombinant nucleic acid of any one of claims 95-108, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

110.

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

113.

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

88.

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

89.

113. The recombinant nucleic acid of any one of claims 95-97 and 110-111, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

115.

114. The recombinant nucleic acid of any one of claims 95-97 and 110-113, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

115.

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

115.

116. The recombinant nucleic acid of any one of claims 95-97 and 110-115, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

115.

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

112.

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

114.

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

114.

120. The recombinant nucleic acid of any one of claims 95-97 and 110-119, 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 shown in SEQ ID NO:

114.

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

114.

122. The recombinant nucleic acid of any one of claims 95-97, wherein the TCRβ chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

117.

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

107.

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

108.

125. The recombinant nucleic acid of any one of claims 95-97 and 122-124, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

119.

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

119.

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

119.

128. The recombinant nucleic acid of any one of claims 95-97 and 122-127, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

119.

129. The recombinant nucleic acid of any one of claims 95-97 and 122-128, further comprising a TCR alpha chain construct comprising CDR1, CDR2 and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 104; The CDR2 has the amino acid sequence shown in SEQ ID NO: 105; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

116.

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

118.

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

118.

132. The recombinant nucleic acid of any one of claims 95-97 and 122-131, 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 shown in SEQ ID NO:

118.

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

118.

134. The recombinant nucleic acid of any one of claims 95-97, wherein the TCRβ chain construct comprises a complementarity determining region 3 (CDR3), wherein the CDR3 has the amino acid sequence of SEQ ID NO:

123.

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

121.

136. The recombinant nucleic acid of any one of claims 95-97 and 134-135, wherein the TCRβ chain construct comprises a complementarity determining region 2 (CDR2), wherein the CDR2 has the amino acid sequence of SEQ ID NO:

122.

137. The recombinant nucleic acid of any one of claims 95-97 and 134-136, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

125.

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

125.

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

125.

140. The recombinant nucleic acid of any one of claims 95-97 and 134-139, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

125.

141. The recombinant nucleic acid of any one of claims 95-97 and 134-140, further comprising a TCR alpha chain construct comprising CDR1, CDR2 and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 104; The CDR2 has the amino acid sequence shown in SEQ ID NO: 105; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

120.

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

124.

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

124.

144. The recombinant nucleic acid of any one of claims 95-97 and 134-143, 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 shown in SEQ ID NO:

124.

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

124.

146. The recombinant nucleic acid of any one of claims 95-145, wherein the epitope from human RAS comprising the mutation G12V is SEQ ID NO: 133 or 134.

147. A recombinant nucleic acid encoding a T cell receptor (TCR), wherein the TCR comprises a TCR β chain construct, wherein the TCR β chain construct comprises a complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:

129.

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

127.

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

128.

150. The recombinant nucleic acid of any one of claims 147-149, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

131.

151. The recombinant nucleic acid of any one of claims 147-150, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

131.

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

131.

153. The recombinant nucleic acid of any one of claims 147-152, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

131.

154. The recombinant nucleic acid of any one of claims 147-153, further comprising a TCR alpha chain construct comprising CDR1, CDR2, and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 29; The CDR2 has the amino acid sequence shown in SEQ ID NO: 30; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

126.

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

130.

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

130.

157. The recombinant nucleic acid of any one of claims 147-156, 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 shown in SEQ ID NO:

130.

158. The recombinant nucleic acid of any one of claims 147-157, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

130.

159. The recombinant nucleic acid of any one of claims 147-158, wherein the TCR binds to an epitope from human RAS comprising mutation G12D.

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

135.

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

162. A recombinant nucleic acid encoding a T cell receptor (TCR), said TCR comprising a TCR β chain construct and a TCR α chain construct, wherein said TCR has a K of at most 1000 nM. D Specifically binds to a mutant epitope from human RAS in complex with human MHC encoded by an HLA-C05:01 allele, wherein the mutant epitope from human RAS is characterized by a G12D mutation.

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

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

90.

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

88.

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

89.

167. The recombinant nucleic acid of any one of claims 162-166, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

92.

168. The recombinant nucleic acid of any one of claims 162-167, wherein the TCR β chain construct comprises a variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

92.

169. The recombinant nucleic acid of any one of claims 162-168, wherein the TCRβ chain construct comprises a variable region having at least 95%, 96%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:

92.

170. The recombinant nucleic acid of any one of claims 162-169, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

92.

171. The recombinant nucleic acid of any one of claims 162-170, further comprising a TCR alpha chain construct comprising CDR1, CDR2, and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 85; The CDR2 has the amino acid sequence shown in SEQ ID NO: 86; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

87.

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

91.

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

91.

174. The recombinant nucleic acid of any one of claims 162-173, 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 shown in SEQ ID NO:

91.

175. The recombinant nucleic acid of any one of claims 162-174, wherein the TCR alpha chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

91.

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

95.

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

94.

178. The recombinant nucleic acid of any one of claims 162-163 and 176-177, wherein the TCRβ chain construct comprises a complementarity determining region 2 (CDR2), wherein the CDR2 has the amino acid sequence of SEQ ID NO:

81.

179. The recombinant nucleic acid of any one of claims 162-163 and 176-178, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

97.

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

97.

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

97.

182. The recombinant nucleic acid of any one of claims 162-163 and 176-181, wherein the TCR β chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

97.

183. The recombinant nucleic acid of any one of claims 162-163 and 176-182, further comprising a TCR alpha chain construct comprising CDR1, CDR2 and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 15; The CDR2 has the amino acid sequence shown in SEQ ID NO: 16; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

93.

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

96.

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

96.

186. The recombinant nucleic acid of any one of claims 162-163 and 176-185, 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 shown in SEQ ID NO:

96.

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

96.

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

101.

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

94.

190. The recombinant nucleic acid of any one of claims 162-163 and 188-189, wherein the TCRβ chain construct comprises a complementarity determining region 2 (CDR2), wherein the CDR2 has the amino acid sequence of SEQ ID NO:

81.

191. The recombinant nucleic acid of any one of claims 162-163 and 188-190, wherein the TCR β chain construct comprises a variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

103.

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

103.

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

103.

194. The recombinant nucleic acid of any one of claims 162-163 and 188-193, wherein the TCRβ chain construct comprises a variable region having the amino acid sequence shown in SEQ ID NO:

103.

195. The recombinant nucleic acid of any one of claims 162-163 and 188-194, further comprising a TCR alpha chain construct comprising CDR1, CDR2 and CDR3, in, The CDR1 has the amino acid sequence shown in SEQ ID NO: 98; The CDR2 has the amino acid sequence shown in SEQ ID NO: 99; and The CDR3 has the amino acid sequence shown in SEQ ID NO:

100.

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

102.

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

102.

198. The recombinant nucleic acid of any one of claims 162-163 and 188-197, 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 shown in SEQ ID NO:

102.

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

102.

200. The recombinant nucleic acid of any one of claims 162-199, wherein the epitope from human RAS comprising the mutation G12D is SEQ ID NO:

132.

201. The recombinant nucleic acid of any one of claims 162-200, wherein the TCR has an EC of at least 1000 nM. 50 specifically binds to the mutant epitope.

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