Antigen-binding proteins and uses thereof

Abstract

The present invention discloses antigen-binding proteins, multispecific antibodies, nucleic acids encoding the antigen-binding proteins or multispecific antibodies, vectors comprising said nucleic acids, cells comprising the antigen-binding proteins, multispecific antibodies, nucleic acids or vectors, and methods for producing the cells. Furthermore, the present invention also discloses conjugates or compositions comprising the antigen-binding proteins or multispecific antibodies, methods for preventing and / or treating diseases using the antigen-binding proteins, multispecific antibodies or cells, and methods for detecting the presence of diseases in a subject.

Description

[Technical Field]

[0001] This application claims the benefit of priority from Chinese Patent Application No. 202310065643.3 filed on January 13, 2023, Chinese Patent Application No. 202310067716.2 filed on January 13, 2023, Chinese Patent Application No. 2023101045526 filed on January 29, 2023, Chinese Patent Application No. 202310131538.5 filed on February 17, 2023, and Chinese Patent Application No. 202310131539.X filed on February 17, 2023, which are incorporated herein by reference in their entireties and for all purposes.

[0002] The present invention relates to the field of immunology, and in particular to T cell receptors (TCRs) and their uses, especially in the prevention, treatment or detection of KRAS mutation-positive diseases and / or conditions. [Background technology]

[0003] The RAS gene family includes three genes, NRAS, HRAS, and KRAS, which encode small GTPase membrane-bound proteins and play a key role in signal transduction involved in the regulation of cell growth. RAS plays a central role in cell signal transduction, regulating processes such as transcription and translation at the molecular level and cell proliferation, differentiation, senescence, and apoptosis at the cellular level.

[0004] In 1982, a mutant RAS gene was discovered in human bladder cancer cells, making RAS the first known oncogene in humans. Among these, the KRAS gene has the greatest impact on human cancers. In its activated state, KRAS transmits upstream signals from cell surface receptors to downstream pathways, regulating normal cell function and proliferation. The most common mode of KRAS activation is by point mutation, which is one of the most frequent mutations in solid tumors. Most of these mutations are concentrated in codon 12 of exon 2, with the most common types being G12D, G12V, and G12C.

[0005] Although it is widely recognized that KRAS mutations play an important role in tumorigenesis, no drugs targeting KRAS have been approved to date. In recent years, novel drugs targeting the KRAS G12C mutation have shown some progress, and small molecule drugs that directly inhibit KRAS protein activity have been developed. However, KRAS is widely expressed, and indiscriminate inhibition of both wild-type and mutant KRAS may result in unacceptable toxicity. Another approach to directly inhibiting KRAS activity is to block the binding of KRAS to surrounding lipid membranes. However, this approach also presents challenges in distinguishing between mutant and wild-type proteins, leading to potential toxicity issues. KRAS regulates numerous downstream effector molecules, primarily the RAF / MEK / ERK and PI3K / AKT / mTOR pathways. However, due to numerous feedback mechanisms, the clinical efficacy of these downstream molecule inhibitors has been limited.

[0006] Many research results have shown that KRAS mutant antigen epitopes are targets that can be recognized with high efficiency by TCRs. KRAS mutant antigen-specific TCRs are considered to be promising means for the treatment of solid tumors with KRAS mutations, and the development of KRAS mutant antigen-specific TCR-T products is of great clinical significance. Summary of the Invention

[0007] An object of the present invention is to provide a novel antigen-binding protein that specifically binds to a KRAS mutant antigen, particularly a KRAS G12 mutant epitope, or a complex of said epitope with an MHC molecule, such as a complex of a KRAS G12 mutant epitope with HLA-A*11. The antigen-binding protein of the present disclosure may be in the form of a TCR or an antigen-binding fragment thereof. The antigen-binding protein of the present disclosure binds to a target antigen peptide with high affinity, has good expression stability and high membrane stability, mediates specific cell-killing activity against antigen-positive target cells by effector cells, and does not show alloreactivity against different HLA types.

[0008] In this regard, in one aspect, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a chain variable region (Vβ); the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1); X1 is A or V, X2 is E or V, X3 is R, P, N or a gap, X4 is D, G, S, L or a gap, X5 is I, G, D or a gap, X6 is E, T or a gap, X7 is G or a gap, X8 is A, G, T or a gap, X9 is G, S, A or a gap, X10 is N, Y or G, X11 is N, G, A or a gap, X12 is R or a gap, X13 is K or R, and X15 is I, T or M; or the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2); X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; or the Vα is a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3); X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; or the Vα comprises a CDR3 as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), X3 is S or T, X4 is E, H, L, F or S, X5 is G, W, V, S or I, X6 is F, G, D or V, X7 is Y, A or S, X8 is T, Q, P, Y or S, X9 is E, D, N or G, X10 is Y, S, R or a gap, X11 is G or a gap, X12 is T, E, P or a gap, X13 is A, Q or L, and X14 is F, Y or H; or the Vβ is a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11); X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), X3 is S or T, X4 is F or S, X5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; or the Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12 epitope.

[0009] In some embodiments, the Vα comprises a CDR1 having the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 4), wherein X1 is V, N or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S.

[0010] In some embodiments, the Vα comprises a CDR1 having X1SX3X4X5X6 (SEQ ID NO: 5), where X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S.

[0011] In some embodiments, the Vα comprises a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), where X1 is N or D, X2 is S or R, and X3 is G or A.

[0012] In some embodiments, the Vα comprises a CDR1 as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0013] In some embodiments, the Vα comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, R, or Y, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, G, or a gap, X8 is V, D, or a gap, and X9 is K or a gap.

[0014] In some embodiments, the Vα comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, or R, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, or a gap, X8 is V, D, or a gap, and X9 is K or a gap.

[0015] In some embodiments, the Vα comprises a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), where X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap.

[0016] In some embodiments, the Vα comprises a CDR2 as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0017] In some embodiments, the Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), where X1 is S, L or M, X2 is G or N, X4 is N, D, A or E, and X5 is S, T or Y.

[0018] In some embodiments, the Vβ comprises a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), where X1 is S or L, X4 is N, D or A, and X5 is S or T.

[0019] In some embodiments, the Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), where X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y.

[0020] In some embodiments, the Vβ does not comprise a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0021] In some embodiments, the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), where X1 is F, Y or S, X2 is N, Q or M, X4 is N, K or V, X5 is V, E or G, and X6 is P, L or V.

[0022] In some embodiments, the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), where X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L or V.

[0023] In some embodiments, the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), where X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G.

[0024] In some embodiments, the Vβ comprises a CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0025] In some embodiments, the present disclosure provides a T cell receptor (TCR) or antigen-binding fragment thereof comprising an α chain variable region and a Vβ of the TCR, the Vα comprises a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1), wherein X1 is A or V, X2 is E or V, X3 is R, P, N or a gap, X4 is D, G, S, L or a gap, X5 is I, G, D or a gap, X6 is E, T or a gap, X7 is G or a gap, X8 is A, G, T or a gap, X9 is G, S, A or a gap, X10 is N, Y or G, X11 is N, G, A or a gap, X12 is R or a gap, X13 is K or R, and X15 is I, T or M; and The Vα comprises a CDR1 having the following amino acid sequence: X1X2X3X4X5X6 (SEQ ID NO: 4), wherein X1 is V, N, or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q, or D, and X6 is Y or S; and the Vα comprises a CDR2 having the following amino acid sequence: X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, R, or Y, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, G, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; and / or The Vβ comprises a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), wherein X3 is S or T, X4 is E, H, L, F or S, X5 is G, W, V, S or I, X6 is F, G, D or V, X7 is Y, A or S, X8 is T, Q, P, Y or S, X9 is E, D, N or G, X10 is Y, S, R or a gap, X11 is G or a gap, X12 is T, E, P or a gap, X13 is A, Q or L, and X14 is F, Y or H; and the Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), wherein X1 is S, L, or M, X2 is G or N, X4 is N, D, A, or E, and X5 is S, T, or Y; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), wherein X1 is F, Y, or S, X2 is N, Q, or M, X4 is N, K, or V, X5 is V, E, or G, and X6 is P, L, or V; The antigen-binding domain or a fragment thereof provides a TCR or an antigen-binding fragment thereof having antigen specificity for the mutant KRAS G12 epitope.

[0026] In some embodiments, the present disclosure provides a T cell receptor (TCR) or antigen-binding fragment thereof comprising an α chain variable region and a Vβ of the TCR, the Va comprises a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2), wherein X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; or the Va comprises a CDR3 as set forth in SEQ ID NO: 22, 23 or 24, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; and The Vα comprises a CDR1 having the following amino acid sequence: X1SX3X4X5X6 (SEQ ID NO: 5), wherein X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; or the Vα comprises a CDR1 as set forth in SEQ ID NO: 27, 28 or 29, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; and said Va comprises a CDR2 having the following amino acid sequence: X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V or a gap, X2 is I or Y, X3 is T, S or R, X4 is G or S, X5 is D, G or a gap, X7 is L, M or a gap, X8 is V, D or a gap, and X9 is K or a gap; or said Va comprises a CDR2 as set forth in SEQ ID NO: 32, 33 or 34, or in a functional variant formed by insertion, deletion or substitution of one or more amino acids; and / or The Vβ comprises a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11), wherein X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; or the Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38 or 39, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; and The Vβ comprises a CDR1 having the following amino acid sequence: X1GHX4X5 (SEQ ID NO: 14), wherein X1 is S or L, X4 is N, D or A, and X5 is S or T; or the Vβ comprises a CDR1 as set forth in SEQ ID NO: 42, 43, or 44, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Vβ comprises a CDR2 having the following amino acid sequence: X1X2NX4X5X6 (SEQ ID NO: 17), wherein X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L or V; or the Vβ comprises a CDR2 as set forth in SEQ ID NO: 47, 48 or 49, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof provides a TCR or antigen-binding fragment thereof having antigen specificity for the mutant KRAS G12V epitope.

[0027] In some embodiments, the Va comprises a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3), where X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; or the Va comprises a CDR3 set forth in SEQ ID NO: 25 or 26, or in a functional variant formed by insertion, deletion, or substitution of one or more amino acids; and The Vα comprises a CDR1 having the following amino acid sequence: X1X2X3SQS (SEQ ID NO: 6), wherein X1 is N or D, X2 is S or R, and X3 is G or A; or the Vα comprises a CDR1 as set forth in SEQ ID NO: 30 or 31, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Va comprises a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), wherein X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap; or the Va comprises a CDR2 as set forth in SEQ ID NO: 35 or 36, or in a functional variant formed by insertion, deletion, or substitution of one or more amino acids; and / or The Vβ comprises a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), wherein X3 is S or T, X4 is F or S, X5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; or the Vβ comprises a CDR3 as set forth in SEQ ID NO: 40 or 41, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; and The Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), where X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y, or the Vβ comprises a CDR1 as set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Vβ comprises a CDR2 having the amino acid sequence: X1X2NX4X5V (SEQ ID NO: 18), wherein X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G; or the Vβ comprises a CDR2 as set forth in SEQ ID NO: 50 or 51, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12D epitope.

[0028] In yet another aspect, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a TCR beta chain variable region (Vβ), wherein said Vα comprises a CDR3 as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; and / or the Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12 epitope.

[0029] In some embodiments, the Vα comprises a CDR1 as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0030] In some embodiments, the Vα comprises a CDR2 as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0031] In some embodiments, the Vβ comprises a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0032] In some embodiments, the Vβ comprises a CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0033] In yet another aspect, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, said antigen binding domain or fragment thereof comprising a T cell receptor (TCR) alpha chain variable region (Vα) and a TCR beta chain variable region (Vβ), said Vα having a CDR1 sequence as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; and a CDR2 sequence as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant thereof formed by the insertion, deletion or substitution of one or more amino acids; and / or a CDR3 sequence as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant thereof formed by the insertion, deletion or substitution of one or more amino acids; The Vβ comprises a CDR1 sequence as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; a CDR2 sequence as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant thereof formed by the insertion, deletion or substitution of one or more amino acids; and a CDR3 sequence as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0034] In yet another aspect, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a beta chain variable region (Vβ); The Vα comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 27, 32 and 22, respectively; and the Vβ comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 42, 47 and 37, respectively; The Vα comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 28, 33 and 23, respectively; and the Vβ comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 43, 48 and 38, respectively; The Vα comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 29, 34 and 24, respectively, and the Vβ comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 44, 49 and 39, respectively; the Vα comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 30, 35 and 25, respectively, and the Vβ comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 45, 50 and 40, respectively; or The Vα comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 31, 36 and 26, respectively, and the Vβ comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 46, 51 and 41, respectively.

[0035] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 52, 53, 54, 55, or 56, or / and the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 57, 58, 59, 60, or 61. In some embodiments, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 52, 53, 54, 55, or 56, or / and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 57, 58, 59, 60, or 61.

[0036] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 52, or / and the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 57. Preferably, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 52, or / and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 57.

[0037] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 53, or / and the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58. Preferably, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 53, or / and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 58.

[0038] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 54, or / and the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 59. Preferably, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 54, or / and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 59.

[0039] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 55, or / and the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 60. Preferably, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 55, or / and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0040] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 56, or / and the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 61. Preferably, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 56, or / and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0041] In some embodiments, the antigen binding protein binds to a KRAS G12 mutation epitope or a complex of the epitope with an MHC molecule. In some embodiments, the KRAS mutation epitope comprises at least one of G12V and G12D.

[0042] In some embodiments, the MHC molecule is of the HLA-A*11 type, for example HLA-A*11:01.

[0043] In some embodiments, the antigen binding protein has the following properties: 1) Approximately 1.0×10 -7 It is capable of binding to the target antigen peptide with an EC50 of M or less, 2) High film stability; 3) have specific cell-killing activity against antigen-positive tumor cells; 4) No alloreactivity to different HLA types.

[0044] In some embodiments, the Vα is comprised in a first polypeptide and the Vβ is comprised in a different second polypeptide.

[0045] In some embodiments, the Vα and Vβ are comprised in one polypeptide.

[0046] In some embodiments, the antigen binding protein is soluble or membrane-bound.

[0047] In some embodiments, the antigen binding protein is selected from a TCR, a chimeric antigen receptor (CAR), an Fc polypeptide, or an antigen-binding fragment thereof.

[0048] In some embodiments, the antigen binding protein is a TCR or an antigen-binding fragment thereof, and the antigen binding protein further comprises a TCR constant region or a fragment thereof.

[0049] In some embodiments, the antigen binding protein further comprises a transmembrane domain and / or a cytoplasmic domain.

[0050] In some embodiments, the antigen binding protein further comprises an intracellular signaling region.

[0051] In some embodiments, the TCR constant region is a murine constant region or a human constant region.

[0052] In some embodiments, the TCR constant region comprises a TCR α chain constant region and / or a TCR β chain constant region.

[0053] In some embodiments, the TCR alpha chain constant region and / or the TCR beta chain constant region comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TCR alpha chain and the TCR beta chain.

[0054] In some embodiments, the TCR alpha chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 62 to 66 and 105, and / or the TCR beta chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 67 to 76.

[0055] In some embodiments, the TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO:66, and the TCR beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO:72.

[0056] In some embodiments, the fragment of a TCR constant region is the extracellular segment of a TCR constant region.

[0057] In some embodiments, the antigen binding protein comprises a TCR α chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any of SEQ ID NOs: 77-81, and / or a TCR β chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any of SEQ ID NOs: 82-86.

[0058] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 77, or / and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 82. Preferably, the antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 77 or / and a TCR beta chain set forth in SEQ ID NO: 82.

[0059] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 78, or / and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 83. Preferably, the antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 78 or / and a TCR beta chain set forth in SEQ ID NO: 83.

[0060] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 79, or / and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 84. Preferably, the antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 79 or / and a TCR beta chain set forth in SEQ ID NO: 84.

[0061] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 80, or / and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 85. Preferably, the antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 80, or / and a TCR beta chain set forth in SEQ ID NO: 85.

[0062] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 81, or / and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 86. Preferably, the antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 81 or / and a TCR beta chain set forth in SEQ ID NO: 86.

[0063] In some embodiments, the antigen binding protein further comprises one or more antigen binding regions that bind to other antigens or epitopes.

[0064] In some embodiments, the antigen binding protein is isolated or purified.

[0065] In yet another aspect, the present disclosure provides a multispecific antibody comprising an antigen-binding protein of the present disclosure.

[0066] In some embodiments, the multispecific antibody comprises an antigen binding protein of this disclosure (e.g., a TCR of this disclosure) and an antibody (e.g., an anti-CD3 antibody) linked to the C-terminus or N-terminus of the antigen binding protein of this disclosure.

[0067] In yet another aspect, the present disclosure provides a nucleic acid encoding an antigen binding protein of this disclosure or a multispecific antibody of this disclosure.

[0068] In some embodiments, the nucleic acid comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 87-91, and / or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 92-96.

[0069] In yet another aspect, the disclosure provides a vector comprising a nucleic acid of the disclosure.

[0070] In some embodiments, the vector is selected from a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0071] In yet another aspect, the present disclosure provides a cell comprising an antigen binding protein of this disclosure, a multispecific antibody of this disclosure, a nucleic acid of this disclosure, or a vector of this disclosure.

[0072] In some embodiments, the cells are selected from lymphocytes (eg, T cells, NK cells), monocytes (eg, PBMCs), and stem cells.

[0073] In some embodiments, the stem cells are lymphoid progenitor cells or induced pluripotent stem cells (iPSCs).

[0074] In some embodiments, the cell is a T cell.

[0075] In some embodiments, the T cells do not express an endogenous TCR.

[0076] In yet another aspect, the present disclosure provides a method of producing a cell of the present disclosure, comprising transducing or transfecting a cell with a vector of the present disclosure.

[0077] In some embodiments, the method further comprises expanding and / or activating the cells before or after said transduction or transfection.

[0078] In yet another aspect, the present disclosure provides a conjugate comprising an antigen binding protein of this disclosure or a multispecific antibody of this disclosure and an active agent bound or conjugated to said antigen binding protein or multispecific antibody.

[0079] In some embodiments, the active agent is selected from a detectable marker, an immunostimulatory molecule, and a therapeutic agent.

[0080] In some embodiments, the detectable marker is selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, a fluorescent, phosphorescent, or chemiluminescent molecule.

[0081] In some embodiments, the immune stimulatory molecule is selected from a cytokine, a chemokine, a platelet factor, and a complement activator.

[0082] In some embodiments, the therapeutic agent is selected from an immunomodulatory agent, a radioactive compound (eg, a nuclide), an enzyme, a chemotherapeutic agent, and a toxin.

[0083] In yet another aspect, the present disclosure provides a composition comprising an antigen-binding protein of this disclosure, a multispecific antibody of this disclosure, a nucleic acid of this disclosure, a vector of this disclosure, or a cell of this disclosure.

[0084] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0085] In some embodiments, the composition further comprises a second therapeutic agent.

[0086] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0087] In yet another aspect, the present disclosure provides a method of treating or preventing a KRAS mutation-positive disease and / or condition in a subject, comprising administering to the subject an effective amount of at least one of an antigen binding protein of this disclosure, a multispecific antibody of this disclosure, and a cell of this disclosure.

[0088] In some embodiments, the cells are autologous or allogeneic to the subject.

[0089] In some embodiments, the method comprises the steps of: (i) isolating a sample comprising cells from the subject; (ii) transducing or transfecting the cells with a vector of the present disclosure; and (iii) administering the cells obtained in step (ii) to the subject.

[0090] In some embodiments, the method further comprises the step of knocking out an endogenous TCR in said cell after step (i) and before step (ii).

[0091] In some embodiments, the KRAS mutation is KRAS G12, including at least one of G12V and G12D, for example.

[0092] In some embodiments, the KRAS mutation-positive disease and / or condition is selected from tumors, precancerous lesions, and cancers caused by KRAS mutations. In some embodiments, the tumor is selected from solid tumors and hematological tumors. More preferably, the tumor includes at least one of pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, lung cancer, malignant melanoma, prostate cancer, bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer.

[0093] In some embodiments, the method further comprises administering a second therapeutic agent.

[0094] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0095] In some embodiments, the subject has an HLA-A*11 type, for example, HLA-A*11:01.

[0096] In yet another aspect, the present disclosure provides a method of detecting a KRAS mutation-positive disease and / or condition in a subject, the method comprising the steps of (i) contacting a sample obtained from the subject with an antigen binding protein of the present disclosure, a multispecific antibody of the present disclosure, or a conjugate of the present disclosure, and (ii) detecting the presence of a KRAS mutant antigen in the sample, wherein the presence of the KRAS mutant antigen is indicative of a KRAS mutation-positive disease and / or condition.

[0097] In yet another aspect, the present disclosure provides a method of detecting a KRAS mutation-positive disease and / or condition in a subject, said method comprising administering to said subject an effective amount of an antigen binding protein or multispecific antibody of the present disclosure conjugated to a detectable marker (e.g., a radionuclide).

[0098] In some embodiments, the KRAS mutation is KRAS G12, including at least one of G12V and G12D, for example.

[0099] In some embodiments, the KRAS mutation-positive disease and / or condition is selected from tumors, precancerous lesions, and cancers caused by KRAS mutations. In some embodiments, the cancer is selected from solid tumors and hematological tumors. More preferably, the tumor includes at least one of pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, lung cancer, malignant melanoma, prostate cancer, bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer.

[0100] In yet another aspect, the present disclosure provides a reagent kit comprising an antigen binding protein, multispecific antibody, or conjugate of the present disclosure.

[0101] In one specific embodiment, the reagent kit is used to detect (e.g., diagnose) a KRAS mutation-positive disease and / or condition in a subject and comprises an antigen binding protein, multispecific antibody, or conjugate of the present disclosure.

[0102] In some embodiments of the reagent kit of the present invention, the KRAS positive mutation is KRAS G12, and includes, for example, at least one of G12V and G12D.

[0103] In some embodiments, the KRAS mutation-positive disease and / or condition is selected from tumors, precancerous lesions, and cancers caused by KRAS mutations. In some embodiments, the cancer is selected from solid tumors and hematological tumors. More preferably, the tumor includes at least one of pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, lung cancer, malignant melanoma, prostate cancer, bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer.

[0104] In another aspect, the disclosure provides the use of an antigen binding protein, multispecific antibody, nucleic acid, vector, cell, conjugate or composition of the disclosure in the manufacture of a medicament for use in treating or preventing a KRAS mutation-positive disease and / or condition in a subject.

[0105] In yet another aspect, the present disclosure provides an antigen binding protein, multispecific antibody, nucleic acid, vector, cell, conjugate or composition of the disclosure for treating or preventing a KRAS mutation-positive disease and / or condition in a subject.

[0106] In another aspect, the present disclosure provides the use of an antigen binding protein, multispecific antibody or conjugate of the present disclosure in the manufacture of a product for detecting (e.g., diagnosing) the presence of a KRAS positive epitope in a test sample, such as the use of a reagent kit for detecting (e.g., diagnosing) a KRAS mutation-positive disease and / or condition in a subject.

[0107] In yet another aspect, the present disclosure provides an antigen binding protein, multispecific antibody or conjugate of the present disclosure for detecting (e.g., diagnosing) the presence of a KRAS positive epitope in a test sample, e.g., for detecting (e.g., diagnosing) a KRAS mutation-positive disease and / or condition in a subject.

[0108] In some embodiments of the use of the present invention, the KRAS positive mutation is KRAS G12, for example, comprising at least one of G12V and G12D.

[0109] In some embodiments of the use of the present invention, the KRAS mutation-positive disease and / or condition is selected from tumors, precancerous lesions, and cancers caused by KRAS mutations. In some embodiments, the cancer is selected from solid tumors and hematological tumors. More preferably, the tumor includes at least one of pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, lung cancer, malignant melanoma, prostate cancer, bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer.

[0110] In some embodiments of the invention, the subject has an HLA-A*11 type, for example HLA-A*11:01.

[0111] The antigen-binding proteins of the present disclosure are capable of binding to target antigen peptides with high affinity, have excellent expression stability and high membrane stability, and can mediate specific cell-killing activity of effector cells against antigen-positive target cells, and do not exhibit alloreactivity against different HLA types. [Brief explanation of the drawings]

[0112] [Figure 1] 1 shows KRAS G12-related T cell immune epitopes according to the present invention. [Figures 2A-E] 1 shows the process of obtaining an antigen-specific TCR clone according to the present invention. [Figure 3] This shows that the KDA11-N02 TCR protein molecule provided by the present invention has high affinity. [Figure 4A-E] This shows that the TCR protein molecules provided by the present invention have high membrane stability. [Figure 5] 1 shows that the KRAS G12V-specific TCR-T cells provided by the present invention have specific cell-killing activity against antigen-positive tumor cells. [Figure 6] 1 shows how the TCR-T provided by the present invention specifically recognizes a tumor antigen and then releases cytokines. [Figures 7A-E] It is shown that the TCR-T provided by the present invention has specific cell-killing activity against different antigen-positive tumor cells. [Figure 8] It shows that the TCR-T cells provided by the present invention specifically react to antigen-positive target cells. [Figure 9A-B] 1 shows that the KRAS G12D-specific TCR-T cells provided by the present invention efficiently induce proliferation of antigen-specific T cells. [Figures 10A-E] 1 shows that the KRAS G12V-specific TCR provided by the present invention does not exhibit nonspecific activation of T2KO-TAP1 cell lines expressing different HLA types. [Figure 11] This shows that T2 cells themselves express KRAS G12D. [Figure 12]This shows that KVA11-N02 does not cause non-specific activation of cells derived from different tissues. [Figure 13] 1 shows a research strategy for non-specific responses to the target antigen peptide of KVA11-N02. [Figure 14] This shows that KVA11-N02 reacts nonspecifically with polypeptides in human body. [Figure 15] This shows the research strategy for non-specific responses to the target antigen peptide of KVA11-N04. [Figure 16] This shows that KVA11-N04 reacts nonspecifically with human endogenous polypeptides. [Figure 17] The in vivo antitumor activity of KVA11-N02 TCR-T and KVA11-N04 TCR-T is shown. DETAILED DESCRIPTION OF THE INVENTION

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.For example, the terms used herein are as defined in Janeway CA Jr, Travers P, Walport M et al., "Immunobiology", 5th edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0114] It should be noted that as used herein and in the appended claims, the singular forms "a," "one," and "the / said" include the plural forms unless the context clearly dictates otherwise. Thus, the terms "one," "one kind," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprise," "contain," and "have" can be used interchangeably.

[0115] Where the term "comprising" is used in the present specification and the appended claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising." When a group is defined below as comprising or having at least a certain number of embodiments, it is also understood to disclose a group preferably consisting of only these embodiments.

[0116] The following X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, and X15 are all represented as "X" in the sequence listing.

[0117] In a first aspect, the present disclosure provides an antigen-binding protein comprising an antigen-binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen-binding domain or fragment thereof comprises a TCR alpha chain variable region (Vα) and a TCR beta chain variable region (Vβ); the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1); X1 is A or V, X2 is E or V, X3 is R, P, N or a gap, X4 is D, G, S, L or a gap, X5 is I, G, D or a gap, X6 is E, T or a gap, X7 is G or a gap, X8 is A, G, T or a gap, X9 is G, S, A or a gap, X10 is N, Y or G, X11 is N, G, A or a gap, X12 is R or a gap, X13 is K or R, and X15 is I, T or M; or the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2); X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; or the Vα is a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3); X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; the Vα comprises a CDR3 as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), X3 is S or T, X4 is E, H, L, F or S, X5 is G, W, V, S or I, X6 is F, G, D or V, X7 is Y, A or S, X8 is T, Q, P, Y or S, X9 is E, D, N or G, X10 is Y, S, R or a gap, X11 is G or a gap, X12 is T, E, P or a gap, X13 is A, Q or L, and X14 is F, Y or H; or the Vβ is a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11); X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), X3 is S or T, X4 is F or S, X5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; or the Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12 epitope.

[0118] The term "antigen binding protein" as used herein refers to a protein or peptide comprising at least one TCR alpha chain CDR3 (CDR3α) and / or at least one TCR beta chain CDR3 (CDR3β) as disclosed herein and capable of binding to the antigen target KRAS G12 (e.g., G12V, G12D, G12C) mutant peptide. Further, antigen binding proteins are designed herein to comprise at least one CDR1α, CDR2α, CDR1β, CDR2β, Vα, Vβ, alpha chain and / or beta chain, or combinations thereof, in any combination with other protein domains or moieties listed herein.

[0119] The term "functional variant" as used herein refers to a peptide that has significant sequence identity with the parent peptide and retains the biological activity of the parent peptide. Functional variants include, for example, variants of the peptides or proteins described herein that retain the ability to specifically bind to KRAS G12 mutant antigens to the same extent, the same extent, or a greater extent than the parent peptide. The amino acid sequence of a functional variant can, for example, have at least about 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity with the amino acid sequence of the parent peptide.

[0120] As used herein, "functional variants formed by the insertion, deletion or substitution of one or more amino acids" refers to functional variants formed by the insertion, deletion or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0121] The term "epitope" is typically a site on an antigen, typically a (poly)peptide, recognized by a binding domain. The term "binding domain" in its broadest sense refers to an "antigen-binding site," meaning a domain of a molecule that binds to / interacts with a specific epitope on an antigen target. An antigen target may contain a single epitope, but typically contains at least two epitopes, and may contain any number of epitopes depending on the size, conformation, and type of antigen. The term "epitope" includes sequence epitopes and structural epitopes. Sequence epitopes are continuous epitopes contained in a primary amino acid sequence, typically containing at least two or more amino acids. Structural epitopes are formed by non-contiguous amino acids that become adjacent due to folding of a target antigen, particularly a target (poly)peptide.

[0122] In the context of the present invention, the term "binding domain" refers in particular to the variable regions of the TCR alpha and / or beta chains, in particular the CDR3alpha and CDR3beta of the TCR.

[0123] As used herein, the term "T cell receptor" or "TCR" includes naturally occurring TCRs as well as variants, fragments, and constructs thereof. Thus, the term includes heterodimeric, multimeric, and single-chain constructs comprising a TCR alpha chain and a TCR beta chain. Optionally, other domains and / or portions may be included, so long as the antigen-binding protein retains the ability to recognize the antigen target (preferably in complex with HLA-A*11).

[0124] In its native form, the TCR exists as a complex of multiple proteins on the surface of T cells. The T cell receptor consists of two (independent) protein chains, called the α and β chains, generated by the separate T cell receptor α and β (TCRα and TCRβ) genes. Each chain of the TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region / domain, an Ig-constant (C) region / domain, a transmembrane region that anchors the chain to the cell membrane, and a short C-terminal cytoplasmic tail.

[0125] Antigen specificity is determined by the variable regions of the α and β chains. The two variable regions of the TCR α and β chains contain three highly variable or complementarity-determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by framework (FR) regions. CDR3 is the primary determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 primarily interact with MHC molecules that present antigenic peptides.

[0126] Natural TCRs recognize antigenic peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells ("presented / displayed on MHC molecules"). Antigenic peptides presented on MHC molecules are also referred to herein as "epitope-MHC molecule complexes," "epitope-MHC complexes," or "target antigenic peptide-MHC complexes." There are two distinct classes of MHC molecules, MHC I and MHC II, which present peptides derived from different cellular compartments. MHC class I molecules are expressed on the surface of all human nucleated cells and present peptides or protein fragments derived from intracellular compartments to cytotoxic T cells. In humans, MHC is also called human leukocyte antigens (HLA). There are three major types of MHC class I: HLA-A, HLA-B, and HLA-C. When a TCR binds to its specific epitope-MHC complex, the T cell is activated and exerts its biological effect.

[0127] As described in detail below, the TCRs provided herein can advantageously (specifically) recognize KRAS G12 mutant antigens, in particular the KRAS G12V or G12D epitope, or a complex of said epitope with an MHC molecule, for example a complex of the KRAS G12V or G12D epitope with HLA-A*11.

[0128] In some embodiments of the antigen binding proteins of the present disclosure, the MHC molecule may be of the HLA-A*11 type, for example HLA-A*11:01.

[0129] In this specification, "G12" is defined with reference to the wild-type human KRAS amino acid sequence. The wild-type human KRAS amino acid epitope sequence is generally a 9-peptide (VVGA G GVGK, SEQ ID NO: 99) or 10 peptides (VVVGA G GVGK, SEQ ID NO: 100), in which the amino acids shown in bold and underlined italics are the G12 site. Thus, in some embodiments, "KRAS G12V" herein refers to VVGAV GVGK (SEQ ID NO: 101) or VVVGA V In some embodiments, "KRAS G12D" comprises the amino acid sequence of VVGA D GVGK (SEQ ID NO: 103) or VVVGA D It contains the amino acid sequence of GVGK (SEQ ID NO: 104).

[0130] In some embodiments, the antigen binding proteins of the present disclosure do not have antigen specificity for the wild-type human KRAS amino acid sequence.

[0131] In some embodiments, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a TCR beta chain variable region (Vβ); the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1); X1 is A or V, X2 is E or V, X3 is R, P, N or a gap, X4 is D, G, S, L or a gap, X5 is I, G, D or a gap, X6 is E, T or a gap, X7 is G or a gap, X8 is A, G, T or a gap, X9 is G, S, A or a gap, X10 is N, Y or G, X11 is N, G, A or a gap, X12 is R or a gap, X13 is K or R, and X15 is I, T or M; and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), X3 is S or T, X4 is E, H, L, F or S, X5 is G, W, V, S or I, X6 is F, G, D or V, X7 is Y, A or S, X8 is T, Q, P, Y or S, X9 is E, D, N or G, X10 is Y, S, R or a gap, X11 is G or a gap, X12 is T, E, P or a gap, X13 is A, Q or L, and X14 is F, Y or H; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12 epitope.

[0132] In some embodiments, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a TCR beta chain variable region (Vβ); the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2); X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; and / or the Vβ is a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11); X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12V epitope.

[0133] In some embodiments, the present disclosure provides an antigen binding protein comprising an antigen binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a TCR beta chain variable region (Vβ); the Vα comprises a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3); X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; and / or the Vβ comprises a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12); X3 is S or T, X4 is F or S, X5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12D epitope.

[0134] In some embodiments, the Va of an antigen binding protein of the disclosure further comprises a CDR1 having the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 4), wherein X1 is V, N or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S.

[0135] In some embodiments, the Va of the antigen binding protein of the present disclosure further comprises a CDR1 having XSXXXX (SEQ ID NO: 5), where X is V or N, X is G or A, X is N or S, X is P, Q or D, and X is Y or S. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0136] In some embodiments, the Vα of the antigen binding protein of the present disclosure further comprises a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), where X1 is N or D, X2 is S or R, and X3 is G or A. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0137] In some embodiments, the Vα of an antigen binding protein of the disclosure comprises the CDR1 shown in SEQ ID NO: 27, 28, 29, 30, or 31, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids.

[0138] In some embodiments, the Va of an antigen binding protein of the disclosure comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), wherein Xi is Y, V, or a gap, X2 is I or Y, X3 is T, S, R, or Y, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, G, or a gap, X8 is V, D, or a gap, and X9 is K or a gap.

[0139] In some embodiments, the Va of an antigen-binding protein of the present disclosure comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), where X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, or R, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, or a gap, X8 is V, D, or a gap, and X9 is K or a gap. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0140] In some embodiments, the Va of an antigen binding protein of the present disclosure comprises a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), where X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0141] In some embodiments, the Vα of an antigen binding protein of the disclosure comprises the CDR2 set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0142] In some embodiments, the Va of an antigen binding protein of the disclosure further comprises a CDR1 having the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 4), where X1 is V, N or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; and a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), where X1 is Y, V or a gap, X2 is I or Y, X3 is T, S, R or Y, X4 is G or S, X5 is D, G or a gap, X7 is L, M, G or a gap, X8 is V, D or a gap, and X9 is K or a gap.

[0143] In some embodiments, the Va of the antigen binding protein of the present disclosure further comprises a CDR1 having X1SX3X4X5X6 (SEQ ID NO: 5), where X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S, and a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), where X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, or R, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, or a gap, X8 is V, D, or a gap, and X9 is K or a gap. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0144] In some embodiments, the Va of an antigen binding protein of the disclosure further comprises a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), where X1 is N or D, X2 is S or R, and X3 is G or A, and a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), where X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0145] In some embodiments, the Va of an antigen binding protein of the disclosure further comprises a CDR1 as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0146] In some embodiments, the Va of the antigen binding protein of the present disclosure further comprises a CDR1 as set forth in SEQ ID NO: 27, 28 or 29, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 32, 33 or 34, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0147] In some embodiments, the Va of the antigen binding protein of the present disclosure further comprises a CDR1 as set forth in SEQ ID NO: 30 or 31, or in a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 35 or 36, or in a functional variant formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0148] In some embodiments, the Vβ of an antigen binding protein of the disclosure further comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), wherein X1 is S, L, or M, X2 is G or N, X4 is N, D, A, or E, and X5 is S, T, or Y.

[0149] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), where X1 is S or L, X4 is N, D, or A, and X5 is S or T. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0150] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), where X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0151] In some embodiments, the Vβ of an antigen binding protein of the disclosure further comprises a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0152] In some embodiments, the Vβ of an antigen binding protein of the disclosure further comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), wherein X1 is F, Y or S, X2 is N, Q or M, X4 is N, K or V, X5 is V, E or G, and X6 is P, L or V.

[0153] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), where X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L, or V. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0154] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), where X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0155] In some embodiments, the Vβ of an antigen binding protein of the disclosure further comprises a CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50, or 51, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids.

[0156] In some embodiments, the Vβ of an antigen binding protein of the disclosure further comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), where X1 is S, L or M, X2 is G or N, X4 is N, D, A or E, and X5 is S, T or Y; and a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), where X1 is F, Y or S, X2 is N, Q or M, X4 is N, K or V, X5 is V, E or G, and X6 is P, L or V.

[0157] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), where X1 is S or L, X4 is N, D or A, and X5 is S or T, and a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), where X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L, or V. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0158] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), where X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y, and a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), where X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0159] In some embodiments, the Vβ of an antigen binding protein of the disclosure further comprises a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0160] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR1 as set forth in SEQ ID NO: 42, 43, or 44, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 47, 48, or 49, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0161] In some embodiments, the Vβ of the antigen binding protein of the present disclosure further comprises a CDR1 as set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the antigen binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0162] As mentioned above, CDR1 and CDR2 of the Vα and Vβ chains are thought to be primarily involved in MHC recognition. There is a limited "pool" of CDR1 and CDR2 sequences known to be involved in the recognition of HLA-A*11-restricted antigens. In principle, the CDR3 domain of the present invention can be combined with the CDR1 of the Vα chain set forth in any of SEQ ID NOS: 4-6 and 27-31, the CDR2 of the Vα chain set forth in any of SEQ ID NOS: 7-9 and 32-36, the CDR1 of the Vβ chain set forth in any of SEQ ID NOS: 13-15 and 42-46, or the CDR2 of the Vβ chain set forth in any of SEQ ID NOS: 16-18 and 47-51, provided that the antigen-binding protein retains the ability to recognize the antigen target (preferably in complex with HLA-A*11) to a degree similar, equivalent, or greater than that of the TCRs evaluated in the accompanying Examples.

[0163] In some embodiments, the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1), a CDR3 in which X1 is A or V, X2 is E or V, X3 is R, P, N or a gap, X4 is D, G, S, L or a gap, X5 is I, G, D or a gap, X6 is E, T or a gap, X7 is G or a gap, X8 is A, G, T or a gap, X9 is G, S, A or a gap, X10 is N, Y or G, X11 is N, G, A or a gap, X12 is R or a gap, X13 is K or R, and X15 is I, T or M; or a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2), a CDR3 in which X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; or a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3), a CDR3 in which X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; or a CDR3 as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and said Vα is a CDR1 having the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 4), wherein X1 is V, N or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; or a CDR1 having X1SX3X4X5X6 (SEQ ID NO: 5), wherein X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; or a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), wherein X1 is N or D, X2 is S or R, and X3 is G or A; or Comprises a CDR1 as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vα is a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, R, or Y, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, G, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; or a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, or R, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; or a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), wherein X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap; or CDR2 as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), a CDR3 in which X3 is S or T, X4 is E, H, L, F or S, X5 is G, W, V, S or I, X6 is F, G, D or V, X7 is Y, A or S, X8 is T, Q, P, Y or S, X9 is E, D, N or G, X10 is Y, S, R or a gap, X11 is G or a gap, X12 is T, E, P or a gap, X13 is A, Q or L, and X14 is F, Y or H; or a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11), a CDR3 in which X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; or a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), a CDR3 in which X3 is S or T, X4 is F or S, X5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; or a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vβ is a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), wherein X1 is S, L, or M, X2 is G or N, X4 is N, D, A, or E, and X5 is S, T, or Y; or a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), wherein X1 is S or L, X4 is N, D or A, and X5 is S or T; or a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), wherein X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y; or comprising a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vβ is a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), wherein X1 is F, Y, or S, X2 is N, Q, or M, X4 is N, K, or V, X5 is V, E, or G, and X6 is P, L, or V; or a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), wherein X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L or V; or a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), wherein X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G; or and CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0164] In some embodiments, the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1), X1 is A or V, X2 is E or V, X3 is R, P, N or a gap, X4 is D, G, S, L or a gap, X5 is I, G, D or a gap, X6 is E, T or a gap, X7 is G or a gap, X8 is A, G, T or a gap, X9 is G, S, A or a gap, X10 is N, Y or G, X11 is N, G, A or a gap, X12 is R or a gap, X13 is K or R, and X15 is I, T or M; and the Vα comprises a CDR1 having the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 4), wherein X1 is V, N or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; and the Vα comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, R, or Y, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, G, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), X3 is S or T, X4 is E, H, L, F or S, X5 is G, W, V, S or I, X6 is F, G, D or V, X7 is Y, A or S, X8 is T, Q, P, Y or S, X9 is E, D, N or G, X10 is Y, S, R or a gap, X11 is G or a gap, X12 is T, E, P or a gap, X13 is A, Q or L, and X14 is F, Y or H; and The Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), where X1 is S, L, or M, X2 is G or N, X4 is N, D, A, or E, and X5 is S, T, or Y; and The Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), where X1 is F, Y or S, X2 is N, Q or M, X4 is N, K or V, X5 is V, E or G, and X6 is P, L or V.

[0165] In some embodiments, the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2), X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; or The Vα comprises a CDR3 as set forth in SEQ ID NO: 22, 23 or 24, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and said Va comprises a CDR1 having X1SX3X4X5X6 (SEQ ID NO: 5), where X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; or said Va comprises a CDR1 as set forth in SEQ ID NO: 27, 28 or 29, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; and the Va comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V or a gap, X2 is I or Y, X3 is T, S or R, X4 is G or S, X5 is D, G or a gap, X7 is L, M or a gap, X8 is V, D or a gap, and X9 is K or a gap; or the Va comprises a CDR2 as set forth in SEQ ID NO: 32, 33 or 34, or in a functional variant formed by insertion, deletion or substitution of one or more amino acids; and / or the Vβ is a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11); X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; or The Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38 or 39, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vβ comprises a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), where X1 is S or L, X4 is N, D or A, and X5 is S or T, or the Vβ comprises a CDR1 as set forth in SEQ ID NO: 42, 43 or 44, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), wherein X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L or V; or the Vβ comprises a CDR2 as set forth in SEQ ID NO: 47, 48 or 49, or a functional variant formed by insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12V epitope.

[0166] In some embodiments, the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2), X1 is A or V, X2 is E or V, X3 is R, P or N, X4 is D, G or S, X5 is I, G or D, X6 is E, T or a gap, X8 is A, G or T, X9 is G or S, X10 is N or Y, X11 is N, G or a gap, X12 is R or a gap, and X15 is I or T; and the Vα comprises a CDR1 having the amino acid sequence XSXXXX (SEQ ID NO: 5), where X is V or N, X is G or A, X is N or S, X is P, Q or D, and X is Y or S; and the Vα comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, or R, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; and / or the Vβ is a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11); X4 is E, H or L, X5 is G, W or V, X6 is F or G, X7 is Y, A or S, X8 is T, Q or P, X9 is E or D, X10 is Y or a gap, X12 is T, E or a gap, X13 is A or Q, and X14 is F or Y; and the Vβ comprises a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), wherein X1 is S or L, X4 is N, D, or A, and X5 is S or T; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), wherein X1 is F or Y, X2 is N or Q, X4 is N or K, and X5 is V, E, or G; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12V epitope.

[0167] In some embodiments, the Vα is a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3), X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; or The Vα comprises a CDR3 as set forth in SEQ ID NO: 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vα comprises a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), where X1 is N or D, X2 is S or R, and X3 is G or A, or the Vα comprises a CDR1 as set forth in SEQ ID NO: 30 or 31, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Va comprises a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), wherein X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap; or the Va comprises a CDR2 as set forth in SEQ ID NO: 35 or 36, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), X3 is S or T, X4 is F or S5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; or The Vβ comprises a CDR3 as set forth in SEQ ID NO: 40 or 41, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and The Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), where X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y; or the Vβ comprises a CDR1 as set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), wherein X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G; or the Vβ comprises a CDR2 as set forth in SEQ ID NO: 50 or 51, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12D epitope.

[0168] In some embodiments, the Vα is a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3), X1 is A or V, X3 is N or a gap, X4 is L or a gap, X6 is T or a gap, X7 is G or a gap, X8 is A or a gap, X9 is A or a gap, X10 is N or G, X11 is N or A, X13 is K or R, and X15 is T or M; and the Vα comprises a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), where X1 is N or D, X2 is S or R, and X3 is G or A; and the Vα comprises a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), wherein X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap; and / or The Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), X3 is S or T, X4 is F or S, X5 is S or I, X6 is D or V, X8 is Y or S, X9 is N or G, X10 is S or R, X11 is G or a gap, X12 is E or P, X13 is Q or L, and X14 is F or H; and the Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), wherein X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), wherein X1 is F or S, X2 is Q or M, and X4 is N or V; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12D epitope.

[0169] In a second aspect, the present disclosure provides an antigen-binding protein comprising an antigen-binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen-binding domain or fragment thereof comprises a T cell receptor (TCR) alpha chain variable region (Vα) and a beta chain variable region (Vβ); said Vα comprises a CDR3 as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or said Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof provides an antigen-binding protein having antigen specificity for the mutant KRAS G12 epitope.

[0170] In some embodiments, the Vα comprises a CDR3 set forth in SEQ ID NO: 22, 23, or 24, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 37, 38, or 39, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0171] In some embodiments, the Vα comprises a CDR3 set forth in SEQ ID NO: 25 or 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 40 or 41, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0172] In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 22, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 37, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 22, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 37. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 22, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 37. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 22, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 37. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0173] In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 23, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 38, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 23, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 38. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 23, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 38. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 23, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 38. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0174] In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 24, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 39, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 24, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 39. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 24, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 39. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 24, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 39. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0175] In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 25, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 40, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 25, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 40. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 25, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 40. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 25, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 40. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0176] In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 41, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 26, and / or the Vβ comprises a CDR3 set forth in SEQ ID NO: 41. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 26, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 41. In some embodiments, the Va comprises a CDR3 set forth in SEQ ID NO: 26, and the Vβ comprises a CDR3 set forth in SEQ ID NO: 41. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0177] In some embodiments, the Vα comprises a CDR1 as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0178] In some embodiments, the Vα comprises a CDR2 as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0179] In some embodiments, the Vα comprises a CDR1 as set forth in SEQ ID NO: 27, 28, or 29, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 as set forth in SEQ ID NO: 32, 33, or 34, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0180] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 27, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 32, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0181] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 28, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 33, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0182] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 29, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 34, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0183] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 30 or 31, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 35 or 3, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0184] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 30, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 35, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0185] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 31, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 36, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0186] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0187] In some embodiments, the Vβ comprises a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0188] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, 43, or 44, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, 48, or 49, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0189] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0190] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 43, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 48, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0191] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 44, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 49, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0192] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 50 or 51, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0193] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 45, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 50, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0194] In some embodiments, the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 51, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0195] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0196] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 27, 28, or 29, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 32, 33, or 34, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, 43, or 44, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, 48, or 49, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0197] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 27 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 32 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0198] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 28 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 33 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 43 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 48 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0199] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 29 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 34 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 44 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 49 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0200] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 30 or 31, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 35 or 36, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 50 or 51, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0201] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 30, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 35, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 45, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 50, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0202] In some embodiments, the Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 31 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 36 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and the Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 46 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids, and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 51 or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0203] In some embodiments, the Vα comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 22, 23, 24, 25, or 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ has a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0204] In some embodiments, the Vα comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 22, 23, or 24, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 27, 28 or 29, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ has a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 37, 38 or 39, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 42, 43 or 44, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12V epitope.

[0205] In some embodiments, the Vα comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 25 or 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ has a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12D epitope.

[0206] In some embodiments, the Vα comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 22, 23, 24, 25, or 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and a CDR2 having the amino acid sequence set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ has a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0207] In some embodiments, the Vα comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 22, 23, or 24, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and a CDR2 having the amino acid sequence set forth in SEQ ID NO: 32, 33 or 34, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ has a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 37, 38 or 39, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR2 having the amino acid sequence set forth in SEQ ID NO: 47, 48 or 49, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or a fragment thereof has antigen specificity for the mutant KRAS G12V epitope.

[0208] In some embodiments, the Vα comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 25 or 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and a CDR2 having the amino acid sequence set forth in SEQ ID NO: 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ has a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR2 having the amino acid sequence set forth in SEQ ID NO: 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12D epitope.

[0209] In a third aspect, there is provided an antigen-binding protein comprising an antigen-binding domain of a T cell receptor (TCR) or a fragment thereof, wherein said antigen-binding domain or fragment thereof comprises a T cell receptor (TCR) α chain variable region (Vα) and a TCR β chain variable region (Vβ); The Vα comprises a CDR1 sequence as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vα comprises a CDR1 sequence as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vα comprises a CDR3 sequence as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ comprises a CDR1 sequence set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vβ comprises a CDR2 sequence set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and Said Vβ provides an antigen binding protein comprising the CDR3 sequence shown in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

[0210] In some embodiments, the Vα comprises a CDR3 set forth in SEQ ID NO: 22, 23, or 24, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and The Vα comprises a CDR1 as set forth in SEQ ID NO: 27, 28 or 29, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vα comprises a CDR2 as set forth in SEQ ID NO: 32, 33 or 34, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ comprises a CDR3 as set forth in SEQ ID NO: 37, 38 or 39, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vβ comprises a CDR1 as set forth in SEQ ID NO: 42, 43 or 44, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vβ comprises a CDR2 as set forth in SEQ ID NO: 47, 48 or 49, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; Preferably, the antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12V epitope.

[0211] In some embodiments, the Vα comprises a CDR3 set forth in SEQ ID NO: 25 or 26, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and The Vα comprises a CDR1 as set forth in SEQ ID NO: 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and said Vα comprises a CDR2 as set forth in SEQ ID NO: 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ comprises a CDR3 as set forth in SEQ ID NO: 40 or 41, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and The Vβ comprises a CDR1 as set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Vβ comprises a CDR2 as set forth in SEQ ID NO: 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; The antigen-binding domain or fragment thereof has antigen specificity for the mutant KRAS G12D epitope.

[0212] In some embodiments, the Va comprises CDRl, CDR2 and CDR3 set forth in SEQ ID NOs: 27, 32 and 22, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDRl, CDR2 and CDR3 set forth in SEQ ID NOs: 42, 47 and 37, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the Va comprises CDRl, CDR2 and CDR3 set forth in SEQ ID NOs: 27, 32 and 22, respectively, and / or the Vβ comprises CDRl, CDR2 and CDR3 set forth in SEQ ID NOs: 42, 47 and 37, respectively. In some embodiments, the Va comprises CDRl, CDR2 and CDR3 set forth in SEQ ID NOs: 27, 32 and 22, respectively, and the Vβ comprises CDRl, CDR2 and CDR3 set forth in SEQ ID NOs: 42, 47 and 37, respectively. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0213] In some embodiments, the Va comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 28, 33 and 23, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 43, 48 and 38, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the Va comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 28, 33 and 23, respectively, and / or the Vβ comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 43, 48 and 38, respectively. In some embodiments, the Vα comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 28, 33, and 23, respectively, and the Vβ comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 43, 48, and 38, respectively. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0214] In some embodiments, the Va comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 29, 34 and 24, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 44, 49 and 39, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the Va comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 29, 34 and 24, respectively, and / or the Vβ comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 44, 49 and 39, respectively. In some embodiments, the Vα comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 29, 34, and 24, respectively, and the Vβ comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 44, 49, and 39, respectively. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0215] In some embodiments, the Va comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 30, 35 and 25, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 45, 50 and 40, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the Va comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 30, 35 and 25, respectively, and / or the Vβ comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 45, 50 and 40, respectively. In some embodiments, the Vα comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 30, 35, and 25, respectively, and the Vβ comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 45, 50, or 40, respectively. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0216] In some embodiments, the Va comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 31, 36 and 26, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDRl, CDR2 and CDR3 as set forth in SEQ ID NOs: 46, 51 and 41, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids. In some embodiments, the Va comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 31, 36 and 26, respectively, and / or the Vβ comprises CDRl, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 46, 51 or 41, respectively. In some embodiments, the Vα comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 31, 36, and 26, respectively, and the Vβ comprises CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOs: 46, 51, or 41, respectively. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0217] In some embodiments, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, 53, 54, 55, or 56, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, 58, 59, 60, or 61. In some embodiments, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 52, 53, 54, 55, or 56, and / or the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 57, 58, 59, 60, or 61. In some embodiments, the Vα comprises the amino acid sequence set forth in SEQ ID NO: 52, 53, 54, 55, or 56, and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 57, 58, 59, 60, or 61.

[0218] In some embodiments, the Va comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 52, and / or the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 52, and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0219] In some embodiments, the Va comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 53, and / or the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 53, and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0220] In some embodiments, the Va comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 54, and / or the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 54, and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

[0221] In some embodiments, the Va comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 55, and / or the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 55, and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0222] In some embodiments, the Va comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 56, and / or the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the Va comprises the amino acid sequence set forth in SEQ ID NO: 56, and the Vβ comprises the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

[0223] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues in the same positions in an alignment, typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of the same sequence will have 100% identity, while sequences that are less conserved and have deletions, additions, or substitutions will have a lower degree of identity. Those skilled in the art will recognize that numerous algorithms, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197), and ClustalW, can be used to determine sequence identity using standard parameters.

[0224] Thus, the amino acid sequence of SEQ ID NO: 52, 53, 54, 55 or 56 may be, for example, a Vα "subject sequence" or "reference sequence," and the amino acid sequence of SEQ ID NO: 57, 58, 59, 60 or 61 may be, for example, a Vβ "subject sequence" or "reference sequence," and a different Vα or Vβ chain amino acid sequence may be a "query sequence."

[0225] In some embodiments of the antigen binding protein according to the above aspects of the present invention, the antigen binding protein binds a KRAS G12 mutation epitope or a complex of said epitope with an MHC molecule. In some embodiments, the KRAS mutation epitope comprises at least one of G12V and G12D. In some embodiments, the MHC molecule is of the HLA-A*11 type, for example HLA-A*11:01.

[0226] In some embodiments, the Vα chain is comprised in a first polypeptide and the Vβ is comprised in a different second polypeptide, hi some embodiments, the Vα and Vβ are comprised in a single polypeptide.

[0227] In some embodiments, the antigen binding protein is soluble. In some embodiments, the antigen binding protein is membrane-bound.

[0228] The antigen-binding proteins of the present invention may be provided in a soluble form, for example, in the form of a soluble TCR. Soluble TCRs (sTCRs) can be used as diagnostic tools and as vectors or "adapters" for specifically targeting therapeutic agents or effector cells to, for example, cancer cells expressing the antigenic target recognized by the soluble TCR. Soluble TCRs are typically fragments or constructs comprising the TCR α chain and / or β chain or their variable regions or CDRs, optionally stabilized by disulfide bonds or covalently linked by a suitable linker. Typically, soluble TCRs do not include, for example, a transmembrane region.

[0229] The antigen-binding proteins of the present invention may also be provided in a membrane-bound form, for example in the form of a membrane-bound TCR. Typically, a membrane-bound TCR comprises a transmembrane region that anchors it to the cell membrane.

[0230] In some embodiments, the antigen binding protein is selected from a TCR, a chimeric antigen receptor (CAR), an Fc polypeptide, or an antigen-binding fragment thereof.

[0231] In some embodiments, the antigen binding protein is a TCR or an antigen binding fragment thereof, and the antigen binding protein further comprises a TCR constant region or a fragment thereof.

[0232] The term "constant region" as used herein may be a human constant region or may be derived from other species, resulting in a "chimeric" TCR. For example, the human α and / or β chains may be replaced by their murine counterparts ("murine-derived"), which have been found to enhance surface expression of the human TCR and enhance binding stability of the human TCR to the CD3 co-receptor by favoring preferential pairing of the TCR α and β chains.

[0233] In some embodiments, the TCR constant region is a murine constant region or a human constant region.

[0234] It has been reported that the addition of disulfide bonds to the constant region can promote correct pairing of the TCR α and β chains (Kuball J et al. Blood. 2007 Mar 15;109(6):2331-8). Therefore, the present specification contemplates the addition of one or more cysteine ​​modifications to the constant region to form disulfide bonds between the TCR α and TCR β chains.

[0235] In some embodiments, the TCR constant region comprises a TCR alpha chain constant region and / or a TCR beta chain constant region, and preferably the TCR alpha chain constant region and / or the TCR beta chain constant region comprises at least one cysteine ​​mutation compared to the wild-type sequence to form a disulfide bond between the TCR alpha chain and the TCR beta chain.

[0236] In some embodiments, the cysteine ​​mutations are at one or more of positions 48 of the wild-type human TCR alpha chain constant region, 48 of the wild-type mouse TCR alpha chain constant region, 57 of the wild-type human TCR beta chain constant region, and 57 of the wild-type mouse TCR beta chain constant region.

[0237] The sequences of wild-type TCR constant regions can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the sequence of the TCR α chain constant domain is "TRAC*01," and the sequence of the TCR β chain constant domain is "TRBC1*01" or "TRBC2*01."

[0238] To facilitate the description of the positions of cysteine ​​mutations, the positions of the amino acid sequence of the wild-type TCR constant region in the present invention are numbered according to the numbering scheme of the International Immunogenetics Information System (IMGT). For example, if an amino acid in the TCR α chain constant region (TRAC) is designated as position number 48 in IMGT, it is described herein as the 48th amino acid of the TCR α chain constant region (TRAC); if an amino acid in the TCR β chain constant region (TRBC) is designated as position number 57 in IMGT, it is described herein as the 57th amino acid of the TCR β chain constant region (TRBC), and so on. Herein, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are based on the position numbering listed in IMGT. For example, if an amino acid in TRAV is designated as position number 46 in IMGT, it is described herein as the 46th amino acid of TRAV, and so on. In the present invention, when the sequence position numbering of other amino acids is specifically described, they are numbered as specifically described.

[0239] In some embodiments, the TCR α chain constant region comprises a TCR α chain whose constant region (and / or transmembrane region) has the amino acid sequence L L VI VL For example, when the TCR α chain comprises a human constant region, the human constant region can be one in which the constant region (and / or transmembrane region) has the amino acid sequence L L VI VL If the TCR α chain comprises a murine constant region, the murine constant region may comprise an LVL mutation such that the constant region (and / or transmembrane region) has the amino acid sequence L L V IVIt may contain LIV mutations to include LRIL.

[0240] In some embodiments, the TCR alpha chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 62-66, and / or the TCR beta chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 67-76.

[0241] In some embodiments, the TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO:66, and the TCR beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO:72.

[0242] In some embodiments, the fragment of a TCR constant region is the extracellular segment of a TCR constant region.

[0243] In some embodiments, the antigen binding protein is a TCR comprising an alpha chain and a beta chain. In some embodiments, the TCR alpha chain and / or the beta chain may comprise a leader sequence. For example, the leader sequence of the TCR alpha chain may have the amino acid sequence set forth in SEQ ID NO: 19, 21, 107, or 109. The leader sequence of the TCR beta chain may have the amino acid sequence set forth in SEQ ID NO: 20, 106, 108, or 110. In some embodiments, the leader sequence of the TCR alpha chain may have the amino acid sequence set forth in SEQ ID NO: 19, and the leader sequence of the TCR beta chain may have the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the leader sequence of the TCR alpha chain may have the amino acid sequence set forth in SEQ ID NO: 21, and the leader sequence of the TCR beta chain may have the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the leader sequence of the TCR alpha chain may have the amino acid sequence set forth in SEQ ID NO: 107, and the leader sequence of the TCR beta chain may have the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the leader sequence of the TCR alpha chain may have the amino acid sequence set forth in SEQ ID NO: 109, and the leader sequence of the TCR beta chain may have the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the leader sequence of the TCR alpha chain may have the amino acid sequence set forth in SEQ ID NO: 21, and the leader sequence of the TCR beta chain may have the amino acid sequence set forth in SEQ ID NO: 110.

[0244] In some embodiments, the antigen binding protein further comprises a transmembrane domain and / or a cytoplasmic domain.

[0245] In some embodiments, the antigen binding protein comprises a TCR α chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences selected from SEQ ID NOs: 77-81, and / or a TCR β chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences selected from SEQ ID NOs: 82-86.

[0246] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 77, and / or a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82. In some embodiments, the antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 77 and a TCR beta chain set forth in SEQ ID NO: 82.

[0247] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 78, and / or a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83. The antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 78 and a TCR beta chain set forth in SEQ ID NO: 83.

[0248] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 79, and / or a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84. The antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 79 and a TCR beta chain set forth in SEQ ID NO: 84.

[0249] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 80, and / or a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85. The antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 80 and a TCR beta chain set forth in SEQ ID NO: 85.

[0250] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 81, and / or a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86. The antigen binding protein comprises a TCR alpha chain set forth in SEQ ID NO: 81 and a TCR beta chain set forth in SEQ ID NO:86.

[0251] In some embodiments, the antigen binding protein further comprises an intracellular signaling region. In some embodiments, the antigen binding protein further comprises one or more antigen binding sites that bind to other antigens or epitopes.

[0252] In some embodiments, the antigen binding protein is isolated or purified.

[0253] As used herein, the term "isolated or purified" means identified, separated, and / or recovered from components of its production environment, such that the "isolated or purified" antigen binding protein is free or substantially free from other contaminant components from its production environment that may interfere with its therapeutic or diagnostic use. Contaminant components may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Thus, an "isolated or purified" antigen binding protein may be prepared by at least one purification step that removes or substantially removes these contaminant components.

[0254] In some embodiments, the antigen binding protein of the present disclosure comprises: 1) Approximately 1.0×10 -7 It is capable of binding to the target antigen peptide with an EC50 of M or less, 2) High film stability; 3) have specific cell-killing activity against antigen-positive tumor cells; 4) No alloreactivity to different HLA types.

[0255] Effector cells expressing the antigen-binding proteins (e.g., TCRs) of the present invention bind to their antigen targets (preferably, antigen epitopes presented by antigen-presenting cells on HLA-A*11) with high affinity. The term "affinity" or "binding affinity" refers to the ability of T cells expressing the TCRs of the present invention to respond to a given concentration of ligand, which is believed to be related to the functional capacity of the TCR-expressing cells in vivo. By definition, TCR-expressing cells with high binding affinity respond to very low amounts of antigen in in vitro tests, while such cells with low affinity require a higher amount of antigen to achieve a similar immune response. Therefore, binding affinity can be considered a quantitative determinant of the activation threshold of TCR-expressing cells, which is measured by exposing such cells to different amounts of cognate antigen in vitro. Cells with high affinity TCRs respond to low amounts of antigen. For example, if the EC50 of TCR-expressing cells to activate target cells when co-cultured with target cells expressing the target antigen peptide-HLA-A*11 is 10. -5 M or less (e.g., 10 -5 M or less, e.g. 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 M), the TCR-expressing cells are generally considered to bind to their antigen target with "high" binding affinity. In some embodiments, the antigen binding protein has an affinity of about 1.0 x 10 for the target antigen peptide. -7 In some embodiments, the EC50 is determined by introducing the TCR of the present invention into Jurkat cells carrying an NFAT-GFP reporter gene, co-culturing the cells with T2 cells loaded with different concentrations of the target antigen peptide, and measuring the reporter gene activation level of the Jurkat cells.

[0256] In some embodiments, the antigen-binding proteins of the present invention have high membrane stability on the cell membrane. In the present invention, the term "high membrane stability" refers to the property of the antigen-binding protein (e.g., TCR) being stably expressed on the cell membrane. In some embodiments, "high membrane stability" refers to the TCR being stably expressed on the cell membrane at a high level, and is indicated by the expression rate of TCR on the cell membrane. The expression rate can be measured by the method described in the Examples of the present invention. "High membrane stability" may also mean that the TCR expression rate on the cell membrane is ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧76%, ≧77%, ≧78%, ≧79%, ≧80%, ≧81%, ≧82%, ≧83%, ≧84%, ≧85%, ≧86%, ≧87%, ≧88%, ≧89%, or ≧90%.

[0257] In some embodiments, the antigen-binding proteins of the present invention have specific cell-killing activity against antigen-positive tumor cells. In the present invention, "specific cell-killing activity" means that an effective amount of effector cells (e.g., TCR-T cells) kills antigen-positive target cells upon contact with the target cells, while having no or substantially no cell-killing activity against antigen-negative cells. In one embodiment, "specific cell-killing activity" means that TCR-T cells expressing an effective amount of a specific TCR kill KRAS G12 (e.g., KRAS G12V or KRAS G12D) mutation-positive target cells upon contact, but have no or substantially no cell-killing activity against non-KRAS G12 (e.g., KRAS G12V or KRAS G12D) mutation-positive cells. In one embodiment, "specific cell-killing activity" can be measured by the method described in the examples of the present invention. Specifically, TCR-T cells expressing the TCR of the present invention or its binding fragment are used as effector cells, and non-TCR-transduced PBMCs are expanded and cultured in parallel to serve as control effector cells. KRAS G12-positive and HLA-A*11:01-positive cells are used as positive target cells with HLA antigen peptide match, and KRAS G12-negative and HLA-A*11:01-negative cells are used as negative target cells with HLA antigen peptide mismatch. These are incubated for 16 hours at different effector to target cell ratios (E:T), and luciferase substrate is added to measure the viability of the target cells, and the proportion of killed cells from the remaining target cells is calculated.

[0258] In one embodiment, "substantially free" means that target antigen-negative cells have a response value to effector cells that is equal to or less than the background response value. In other embodiments, "substantially free" means that TCR-expressing cells (effector cells) have a lysis rate of less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.3% against KRAS G12 (e.g., KRAS G12V or KRAS G12D) mutation-negative target cells at a given effector-to-target cell ratio (e.g., 9:1). The dissolution rate can be measured by the method described in the examples of the present invention.

[0259] In some embodiments, the antigen binding proteins provided herein are not alloreactive, i.e., do not elicit an alloreaction against different HLA types.

[0260] In some embodiments, the antigen binding proteins provided by the present invention do not cause non-specific activation of cells derived from different tissues and have a high degree of antigen specificity.

[0261] In another aspect, the present invention provides multispecific antibodies comprising the antigen binding proteins of this disclosure.

[0262] As used herein, the term "multispecific antibody" refers to an antibody capable of binding to at least one target antigen; for example, the multispecific antibody comprises at least two antigen-binding proteins of the present disclosure; or, for example, the multispecific antibody may comprise at least one antigen-binding protein of the present disclosure and at least one TCR or binding fragment thereof that specifically binds to another antigen target; further, for example, the multispecific antibody may comprise at least one antigen-binding protein of the present disclosure and at least one immune effector peptide.

[0263] In some embodiments, examples of TCRs or binding fragments thereof that specifically bind to other antigen targets include, but are not limited to, TCRs or binding fragments thereof that specifically bind to HPV E6 or E7, TCRs or binding fragments thereof that specifically bind to gp100, and TCRs or binding fragments thereof that specifically bind to MAGE (e.g., MAGE-A1, MAGE-A4).

[0264] In some embodiments, the immune effector peptide is a known molecule that directly or indirectly activates components of the humoral or cellular immune system, e.g., by activating T cells, thereby inducing or stimulating an immune response. Examples of immune effector peptides include, but are not limited to, antibodies and cytokines.

[0265] Examples of the antibody may include, but are not limited to, an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD44 antibody, an anti-CD45RA antibody, an anti-CD45RB antibody, an anti-CD45RO antibody, an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49d antibody, an anti-CD49e antibody, an anti-CD49f antibody, an anti-CD16 antibody, an anti-CD28 antibody, and an anti-IL-2R antibody. In some embodiments, the antibody may be an scFv antibody, such as an anti-CD3 scFv. Anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA031, and 12F6.

[0266] Examples of said cytokines may include, but are not limited to, IL-1, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β, IFN-γ, TNFα.

[0267] Other forms of immune effector peptides can include, but are not limited to, viral proteins and peptides, bacterial proteins or peptides.

[0268] In one preferred embodiment, the immune effector peptide is an antibody. In some embodiments, the antibody is an scFv antibody, for example, an anti-CD3 scFv. Anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA031, and 12F6. In a particularly preferred embodiment, the antibody is linked to the N-terminus or C-terminus of the antigen-binding protein. In some embodiments, the antigen-binding protein is a TCR or a binding fragment thereof. In some embodiments, the Vα and Vβ of the TCR or binding fragment thereof may be comprised in different polypeptide chains (e.g., the TCR is an αβTCR). In some embodiments, the Vα and Vβ of the TCR or binding fragment thereof may be comprised in a single polypeptide chain, for example, an scTCR.

[0269] In a further aspect, the present disclosure provides a nucleic acid encoding an antigen binding protein or a multispecific antibody of the disclosure.

[0270] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 87-91, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NOs: 92-96.

[0271] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 87 and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 87 and / or the nucleotide sequence of SEQ ID NO: 92. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 87 and the nucleotide sequence of SEQ ID NO: 92.

[0272] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 88 and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 93. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 88 and / or the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 88 and the nucleotide sequence of SEQ ID NO: 93.

[0273] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 89 and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 94. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 89 and / or the nucleotide sequence of SEQ ID NO: 94. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 89 and the nucleotide sequence of SEQ ID NO: 94.

[0274] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 90 and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 95. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 90 and / or the nucleotide sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 90 and the nucleotide sequence of SEQ ID NO: 95.

[0275] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 91 and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 96. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 91 and / or the nucleotide sequence of SEQ ID NO: 96. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 91 and the nucleotide sequence of SEQ ID NO: 96.

[0276] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:87 and SEQ ID NO:97, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:92 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:87 and SEQ ID NO:97 and / or the nucleotide sequence of SEQ ID NO:92 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:87 and SEQ ID NO:97, and the nucleotide sequence of SEQ ID NO:92 and SEQ ID NO:98.

[0277] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:88 and SEQ ID NO:97, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:93 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:88 and SEQ ID NO:97 and / or the nucleotide sequence of SEQ ID NO:93 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:88 and SEQ ID NO:97, and the nucleotide sequence of SEQ ID NO:93 and SEQ ID NO:98.

[0278] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:89 and SEQ ID NO:97, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:94 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:89 and SEQ ID NO:97 and / or the nucleotide sequence of SEQ ID NO:94 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:89 and SEQ ID NO:97, and the nucleotide sequence of SEQ ID NO:94 and SEQ ID NO:98.

[0279] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:90 and SEQ ID NO:97, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:95 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:90 and SEQ ID NO:97, and / or the nucleotide sequence of SEQ ID NO:95 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:90 and SEQ ID NO:97, and the nucleotide sequence of SEQ ID NO:95 and SEQ ID NO:98.

[0280] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:91 and SEQ ID NO:97, and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:96 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:91 and SEQ ID NO:97 and / or the nucleotide sequence of SEQ ID NO:96 and SEQ ID NO:98. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:91 and SEQ ID NO:97, and the nucleotide sequence of SEQ ID NO:96 and SEQ ID NO:98.

[0281] In another aspect, the disclosure provides a vector comprising a nucleic acid of the disclosure.

[0282] As used herein, the term "vector" refers to a nucleic acid molecule that functions as a vehicle for transferring exogenous genetic material into a host cell, where the nucleic acid molecule can, for example, replicate and / or express. The term "vector" includes, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adeno-associated virus vectors (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically comprising a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors typically contain an origin for autonomous replication in a host cell (if stable expression of the polynucleotide is desired), a selectable marker, and restriction enzyme cleavage sites (e.g., polyclonal site, MCS). A vector may further comprise a promoter, a genetic marker, a reporter gene, a targeting sequence, and / or a protein purification tag. As known to those skilled in the art, many suitable vectors are known to those skilled in the art, and many are commercially available.Examples of suitable vectors are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.

[0283] In some embodiments, the vector is preferably selected from a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0284] In yet another aspect, the present disclosure provides a cell comprising an antigen binding protein, a multispecific antibody, a nucleic acid, or a vector according to the present disclosure.

[0285] As used herein, the term "cell" refers to any type of cell capable of expressing the antigen-binding protein of the present disclosure. The cell may be a eukaryotic cell, such as a plant cell (not having the potential to develop into a plant), an animal cell, a fungal cell, or an algae cell, or a prokaryotic cell, such as a bacterial cell or a protozoan cell. The cell may be a cultured cell or a primary cell, i.e., a cell isolated directly from an organism, e.g., a human. The cell may be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are well known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For purposes of producing the antigen-binding protein of the present disclosure, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

[0286] In some embodiments, the cells are selected from lymphocytes (e.g., T cells, NK cells), monocytes (e.g., PBMCs), and stem cells. As used herein, the term "stem cells" refers to stem cells used to express the antigen-binding proteins (particularly TCRs) of the present disclosure. For example, stem cells may be lymphoid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells (HSCs). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos and / or totipotent stem cells used to develop and form individual animals. Because stem cells do not express CD3 molecules on their surface, transgenic stem cells typically do not express TCRs on their cell surface. However, when stem cells differentiate into lymphoid progenitor cells that migrate to the thymus, expression of CD3 molecules initiates expression of the introduced TCR molecules on the surface of thymocytes.

[0287] In some embodiments, the stem cells are lymphoid progenitor cells or induced pluripotent stem cells (iPSCs).

[0288] In some embodiments, the cells are T cells. The T cells can be any T cells, such as cultured T cells, e.g., primary T cells or T cells from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. If obtained from a mammal, the T cells can be obtained from a number of sources, such as blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are T cells isolated from a human. The T cells can be any type of T cell and can be at any stage of development, such as CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. In some embodiments, the T cells do not express an endogenous TCR.

[0289] In another aspect, the present disclosure provides a method of producing a cell of the present disclosure, comprising transducing or transfecting a cell with a vector of the present disclosure.

[0290] As used herein, the term "transfection" refers to the process of intentionally introducing a nucleic acid molecule or polynucleotide (including a vector) into a target cell. One example is RNA transfection, i.e., the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. The term is primarily used to describe non-viral methods in eukaryotic cells. The term "transduction" is commonly used to describe virus-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves creating transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection can be performed using calcium phosphate, electroporation, cell extrusion, or by mixing the material with cationic lipids to generate liposomes that fuse with the cell membrane and release their cargo into the cell. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation.

[0291] In some embodiments, the method further comprises expanding and / or activating the cells before or after transduction or transfection.

[0292] In another aspect, the present disclosure provides a conjugate comprising an antigen binding protein and / or multispecific antibody of this disclosure and an active agent bound or conjugated to said antigen binding protein.

[0293] In some embodiments, the active agent is selected from a detectable marker, an immune stimulatory molecule, and a therapeutic agent. Preferably, the detectable marker is selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, a fluorescent, phosphorescent, or chemiluminescent molecule. Preferably, the immune stimulatory molecule is selected from a cytokine (e.g., IL-2 and IFN-γ), a chemokine (e.g., IL-8), a platelet factor (e.g., platelet factor 4), and a complement activator. Preferably, the therapeutic agent is selected from an immunomodulatory agent, a radioactive compound, an enzyme, a chemotherapeutic drug, and a toxin. Other suitable therapeutic agents include small molecule cell killers, i.e., compounds with a molecular weight of less than 700 daltons and the ability to kill mammalian cells. Such compounds may also include toxic metals that have a cell-killing effect. It should also be understood that these small molecule cell killers include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cell-killing agent.Examples of such agents are cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, isocyclic phosphorylamines, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, and the like. glucuronate), auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof capable of killing mammalian cells; for example, ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase; radionuclides, i.e., unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays upon decay, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and bismuth-213, actinium-225, and astatine-213; chelating agents that can be used to facilitate binding of these radionuclides to molecules or multimers thereof; or heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides.

[0294] In another aspect, the present disclosure provides a composition comprising an antigen binding protein, nucleic acid, vector, or cell of the disclosure, preferably said composition further comprising a pharmaceutically acceptable carrier or excipient.

[0295] The term "composition" specifically refers to a composition suitable for administration to humans. However, the term also generally encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e., active agent and optionally carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect without causing any undesired local or systemic effects in the recipient. Pharmaceutically acceptable compositions of the present invention may, for example, be sterile. Specifically, the term "pharmaceutically acceptable" may refer to approval by a regulatory agency or other recognized pharmacopoeia for use in animals, and also in humans.

[0296] The term "excipient" includes fillers, adhesives, disintegrants, coating agents, adsorbents, anti-adhesives, flow aids, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tonicity adjusters.Selecting a suitable excipient for preparing the composition of the present invention is well known to those skilled in the art.Exemplary carriers for use in the composition of the present invention include saline, buffered saline, glucose and water.Usually, the selection of a suitable excipient depends particularly on the active agent used, the disease to be treated and the desired dosage form of the composition.

[0297] The compositions of the present disclosure can be formulated into various forms, such as solid, liquid, gaseous, or lyophilized forms, depending on the active agent (e.g., soluble TCR) employed, including, in particular, ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or liquid extracts, or any form particularly suitable for the desired method of administration. Pharmaceutical production processes well known to the present invention are set forth in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co., Easton, Pa., 2012) and include, for example, conventional mixing, dissolving, granulating, sugar-coating, pulverizing, emulsifying, encapsulating, embedding, or lyophilizing. For example, compositions comprising the host cells or soluble TCRs described herein are typically provided in liquid form and preferably contain a pharmaceutically acceptable buffer.

[0298] In some embodiments, the compositions of the present disclosure further comprise a second therapeutic agent, preferably said second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0299] Examples of preferred second therapeutic agents include well-known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, isocyclic phosphorylamines, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, and the like. E), vincristine and doxorubicin; and peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase; radionuclides such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and -213, actinium-225 and astatine-213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as IL-2, chemokines such as IL-8, platelet factor 4; antibodies or fragments thereof such as anti-CD3 antibodies or fragments thereof; complement activators; heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides.

[0300] In another aspect, the present disclosure provides a method of treating or preventing a KRAS mutation-positive disease and / or condition in a subject comprising administering to the subject an effective amount of an antigen binding protein of the present disclosure.

[0301] In yet another aspect, the present disclosure provides a method of treating or preventing a KRAS mutation-positive disease and / or condition in a subject, comprising administering to the subject an effective amount of a cell of the present disclosure.

[0302] As used herein, the term "treatment" includes therapeutic or prophylactic treatment in a subject in need thereof. The term "therapeutic or prophylactic treatment" includes prophylactic treatment intended to completely prevent clinical and / or pathological findings, or therapeutic treatment intended to ameliorate or alleviate clinical and / or pathological findings. Thus, the term "treatment" also includes amelioration or prevention of disease.

[0303] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that, when administered to a subject for treating or preventing a disease, is sufficient to achieve such treatment or prevention. The "effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. A "therapeutically effective amount" refers to an amount effective for therapeutic treatment. A "prophylactically effective amount" refers to an amount effective for prophylactic treatment.

[0304] Therapeutic efficacy and toxicity can be determined by standard procedures, such as ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), in cell cultures or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio ED50 / LD50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

[0305] The exact dose of antigen-binding protein or cells to be administered can be determined by one of skill in the art using well-known techniques. A suitable dose provides a sufficient amount of the active agent of the invention and is preferably therapeutically effective, i.e., sufficient to elicit a therapeutic or prophylactic response in a subject or animal within a reasonable period of time. For example, a dose of an antigen-binding protein of the invention, e.g., a TCR, should be sufficient to bind to a cancer antigen or detect, treat, or prevent cancer for a period of about 2 hours or more, e.g., 12 to 24 hours or more, or even longer (e.g., 1 month, 2 months, 3 months, 6 months, 12 months, 24 months, etc.) from the start of administration. In some embodiments, the period may be longer. As is well known in the art, adjustments may be necessary depending on the goal of treatment (e.g., alleviation of acute disease onset), route, timing, and frequency of administration, timing and frequency of administration of the formulation, age, body weight, general health, sex, diet, severity of the disease state, drug combinations, response sensitivity, and tolerance / response to treatment.

[0306] Numerous assays for determining the dose to be administered are well known in the art. For purposes of the present invention, an assay can be used to determine the starting dose to be administered to a mammal, involving comparing the degree of target cell lysis or IFN-γ secretion from T cells after administering a particular dose of T cells expressing an antigen binding protein (e.g., TCR) of the present disclosure to a group of mammals (each receiving a different dose of T cells). The degree of target cell lysis or IFN-γ secretion achieved after administration of a dose can be determined by methods well known in the art. The dose of the antigen binding protein or cells of the present disclosure will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of the antigen binding protein or cells of the present disclosure. Typically, the dose of the antigen binding protein or cells of the present disclosure to be administered to an individual patient will be determined by the attending physician, taking into account various factors such as age, weight, general health, diet, sex, the active agent being administered, the route of administration, and the severity of the condition being treated. In some embodiments of the therapeutic methods of the present disclosure, the number of cells administered in each infusion will be, for example, about 1 x 10 cells.6 From about 1 x 10 12 In some embodiments, the number of cells may vary from 1 x 10 to 1 x 10. 6 Fewer than one may be administered.

[0307] It should be appreciated that treatment may require single or multiple administrations of a therapeutically effective amount of an active agent of the invention. For example, depending on the formulation, half-life, and clearance of the particular composition, some compositions may be administered every 3 to 4 days, every week, or every two weeks, or once a month.

[0308] The compositions of the present disclosure may be suitable for administration by various routes. Typically, administration is achieved parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0309] The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein and refer to any subject, particularly a mammalian subject, in need of treatment. Generally, mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc. However, it is readily understood that the TCRs, nucleic acids, vectors, host cells and pharmaceutical compositions provided herein are particularly anticipated to be used to treat human subjects, particularly human subjects who are HLA-A*11 positive, e.g., HLA-A*11:01 positive.

[0310] In some embodiments of the therapeutic methods of the present disclosure, the cells are autologous or allogeneic to the subject.

[0311] In some embodiments, the method includes the steps of (i) isolating a sample containing cells from the subject, (ii) transducing or transfecting the cells with a vector of the present disclosure, and (iii) administering the cells obtained in step (ii) to the subject. In some embodiments, the method further includes the step of knocking out endogenous TCR in the cells after step (i) and before step (ii). In some embodiments, the method further includes administering a second therapeutic agent. Preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug. Preferred examples of second therapeutic agents are described above.

[0312] In another aspect, the present disclosure provides a method of detecting (e.g., diagnosing) a KRAS mutation-positive disease and / or condition in a subject, the method comprising the steps of (i) contacting a sample obtained from the subject with an antigen binding protein, cell, or conjugate of the present disclosure; and (ii) detecting the presence of a KRAS mutant antigen in the sample, wherein the presence of the KRAS mutant antigen is indicative of the KRAS mutation-positive disease and / or condition.

[0313] In some examples, the sample obtained from the subject can be a blood sample, a urine sample, a tissue sample, or a cell sample. In some examples, the method is performed ex vivo. In some embodiments, the method includes: (i) contacting a sample obtained from the subject with a conjugate of the present disclosure, wherein the conjugate comprises a detectable marker; and (ii) detecting the presence of a KRAS mutant antigen in the sample by detecting the detectable marker. Examples of detectable markers include, but are not limited to, biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, a fluorescent, phosphorescent, or chemiluminescent molecule. Preferably, the marker is an enzyme or catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent, or chemiluminescent molecule.

[0314] In yet another aspect, the present disclosure provides a kit comprising an antigen binding protein, multispecific antibody, or conjugate of the disclosure.

[0315] In some embodiments, the kit is used to detect the presence of a KRAS positive epitope in a test sample.

[0316] In some embodiments, the kit is for detecting (eg, diagnosing) a KRAS mutation-positive disease and / or condition in a subject, and comprises an antigen binding protein or conjugate of the present disclosure.

[0317] In some embodiments, the conjugate comprises a detectable marker. Examples of detectable markers include, but are not limited to, biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, a fluorescent, phosphorescent, or chemiluminescent molecule. Preferably, the marker is an enzyme or a catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent, or chemiluminescent molecule. In some embodiments, the kit can further comprise instructions on how to use the kit.

[0318] In another aspect, the present disclosure provides the use of an antigen binding protein, multispecific antibody, nucleic acid, vector, cell, conjugate or composition of the disclosure in the manufacture of a medicament for treating or preventing a KRAS mutation-positive disease and / or condition in a subject.

[0319] In yet another aspect, the present disclosure provides an antigen binding protein, multispecific antibody, nucleic acid, vector, cell, conjugate or composition of the disclosure for treating or preventing a KRAS mutation-positive disease and / or condition in a subject.

[0320] In another aspect, the present disclosure provides use of an antigen binding protein, multispecific antibody or conjugate of the present disclosure in the manufacture of a reagent kit for detecting the presence of a positive epitope in a test sample, wherein said epitope is a KRAS mutation positive epitope.

[0321] In some embodiments, the present disclosure provides use of an antigen binding protein, multispecific antibody, or conjugate of the present disclosure in the manufacture of a reagent kit for detecting (e.g., diagnosing) a KRAS mutation-positive disease and / or condition in a subject.

[0322] In yet another aspect, the present disclosure provides an antigen binding protein, multispecific antibody or conjugate of the present disclosure for detecting the presence of a positive epitope in a test sample, wherein said positive epitope is a KRAS mutation positive epitope, e.g., to detect (e.g., diagnose) a KRAS mutation positive disease and / or condition in a subject.

[0323] In some embodiments of the uses of the present disclosure, the subject has an HLA-A*11 type, for example, HLA-A*11:01.

[0324] In embodiments of the present disclosure, the disease and / or condition may include a KRAS mutation-positive disease and / or condition. In some embodiments, the KRAS mutation may include a G12 mutation epitope, such as KRAS G12V or KRAS G12D. In some embodiments, the KRAS mutation-positive disease may include a tumor. In some embodiments, the tumor is selected from solid tumors and hematological tumors. More preferably, the tumor includes at least one of pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, lung cancer, malignant melanoma, prostate cancer, bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer.

[0325] The present invention will be further described in detail by the following specific embodiments. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples are carried out using conventional conditions in the art, such as those described in Sambrook and Russeii et al., Molecular Cloning: a Laboratory Manual (Third Edition) (2001), CSHL Press, or those recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are commercially available. [Example]

[0326] Example 1 Design and discovery of T cell epitopes with KRAS G12 mutation To expand the market size of TCR products specific to each KRAS mutation epitope, we used large-scale HLA genotyping data to identify the most frequently occurring HLA genotypes in the target market population. Based on the preferential selection of high-frequency HLAs, we used bioinformatics and experimental methods to identify T cell epitopes of KRAS mutation antigens presented by specific HLA genotypes. Systematic bioinformatics and immunological studies were conducted on the antigen epitopes that could be formed by the KRAS G12 mutation, and 12 distinct T cell epitopes were identified. We established MHC tetramer and ELISpot detection methods to detect T cells specifically reacting with these epitopes. Furthermore, we implemented a drug development process, including TCR cloning, affinity optimization, and nonspecific exclusion for future HLA target antigen epitopes (Figure 1).

[0327] Example 2: Cloning and acquisition of TCR that specifically recognizes KRAS G12 presented by HLA-A*11:01 After obtaining ethical approval and informed consent from the patient, tumor-infiltrating lymphocytes (TILs) were cultured and expanded from postoperative tumor tissue of a KRAS G12 mutation-positive colorectal cancer patient (HLA-A*11:01). After sufficient expansion of TILs, CD8 and Tetramer (HLA-A*11:01, KRAS G12V 8-16, VVGA) were used. V GVGK, designated KV1, SEQ ID NO: 101; HLA-A*11:01, KRAS G12V 7-16, VVVGA V GVGK, designated KV2, SEQ ID NO: 102; HLA-A*11:01, KRAS G12D 8-16, VVGA D GVGK, designated KD1, SEQ ID NO: 103; HLA-A*11:01, KRAS G12D 7-16, VVVGA D Cells that strongly stained positive for GVGK and KD2 (designated GVGK and KD2, SEQ ID NO: 104) were single-cell sorted by flow cytometry. Cellular mRNA from the sorted CD8+ / Tetramer+ cells was reverse-transcribed and amplified to obtain the TCRα and β V-region genes. The obtained TCRα and β V-region genes were cloned into lentiviral vectors containing the TCR C region, respectively, to construct vectors capable of expressing the complete TCRα or β chain (Figure 2). Different candidate TCR pairs were transfected into established T cell reporter cell lines, and the antigen specificity and affinity of the TCR pairs were confirmed using antigen-specific activation assays. Using the above cloning and confirmation strategy, five TCRs presented by HLA-A*11:01 were isolated, as shown in Figure 2. The specific details are as follows: KRAS G12V 7-16 epitope-specific TCR: KVA11-N02 (Figure 2A) KRAS G12V 8-16 epitope-specific TCR: KVA11-N03 (Figure 2B) KRAS G12V 7-16 epitope-specific TCR: KVA11-N04 (Figure 2C) KRAS G12D 7-16 epitope-specific TCR: KDA11-N01 (Figure 2D) KRAS G12D 7-16 epitope-specific TCR: KDA11-N02 (Figure 2E)

[0328] The amino acid sequences of the variable regions of each TCR obtained and the nucleotide sequences encoding them are shown in Table 1, and the CDR sequences in the variable regions of the α and β chains are shown in Table 2. The constant region sequences of the recombinant TCRs are shown in Table 3, and the amino acid sequences of the α and β chains of the recombinant TCRs are shown in Table 4.

[0329] [Table 1]

[0330] [Table 2]

[0331] [Table 3]

[0332] [Table 4]

[0333] The Vα and Vβ of KVA11-N02, KVA11-N03, KVA11-N04, KDA11-N01, and KDA11-N02 were fused to mouse-derived C regions (sequence number 66 (α chain) and sequence number 72 (β chain)), respectively, linked in the order of β-T2A-α, and constructed into a lentiviral vector for subsequent use.

[0334] Example 3.1 Affinity Measurements KVA11-N02, KVA11-N03, KVA11-N04, and KDA11-N01 were transfected into Jurkat cells (Jurkat-NFAT, endogenous TCR knockout) carrying the NFAT-GFP reporter gene, respectively, and co-cultured with T2 cells loaded with different concentrations of target antigen peptide (e.g., 1.0 x 10^-5M, 1.0 x 10^-6M, 1.0 x 10^-7M, 1.0 x 10^-8M, 1.0 x 10^-9M, or 0 M). The reporter gene activation levels in Jurkat cells were measured. The EC50 values ​​for each TCR are shown in Table 5.

[0335] [Table 5]

[0336] Example 3.2 Affinity Measurements KDA11-N02 and TK412 (Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors, Qiong J. Wang, et al., Published Online First December 23, 2015; DOI: 10.1158 / 2326-6066.CIR-15-0188) were transfected into Jurkat cells (Jurkat-NFAT, endogenous TCR knockout) carrying the NFAT-GFP reporter gene, respectively, and co-cultured with T2 cells loaded with different concentrations of target antigen peptide (e.g., 1.0×10^-5M, 1.0×10^-6M, 1.0×10^-7M, 1.0×10^-8M, 1.0×10^-9M, or 0M of the target antigen peptide), and the reporter gene activation level of the Jurkat cells was measured. As shown in Figure 3, the EC50 of KDA11-N02 was 1.73 x 10^-8 M, and the EC50 of the control group, TK412, was 4.62 x 10^-8 M.

[0337] Example 4 Film Stability Equal amounts of TK34 (a TCR induced in vivo against the same target in mice as reported in the literature (TRAV3-3*01 / BV*401, hereafter referred to as "TK34"; Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors, Qiong J et al., Cancer Immunology Research)), KVA11-N02, KVA11-N03, KVA11-N04, KDA11-N01, and KDA11-N02 were transduced into PBMCs via lentivirus and cultured until day 9. Expression of TCR-T was measured by staining with anti-TCRβ-APC antibody, and the CD4 / CD8 ratio was measured by staining with human CD3-PE, CD4-PE-Cy7, and CD8-FITC antibodies. Flow cytometry analysis results are shown below.

[0338] 1) KVA11-N02 (Figure 4A) and KVA11-N04 (Figure 4C): Under identical transfection and culture conditions, TK34-, KVA11-N02-, and KVA11-N04-transfected cells all had essentially the same CD4 / CD8 ratios, and the TCR expression rates of KVA11-N02 and KVA11-N04 were significantly higher than those of TK34. This suggests that the protein molecules of KVA11-N02 and KVA11-N04 have better membrane stability and may have superior antigen reactivity and antitumor activity.

[0339] 2) KVA11-N03 (Figure 4B): The TCR expression rate of KVA11-N03 was 40% or more (Figure 4B), confirming that the KVA11-N03 protein molecule was stably expressed on the membrane.

[0340] 3) KDA11-N01 (Figure 4D): The TCR expression rate of KDA11-N01 reached 90%, and the mean fluorescence intensity of TCR expression was 25851. This suggests that the TCR protein molecule of KDA11-N01 has very high membrane stability and may have excellent antigen reactivity and antitumor activity.

[0341] 4) KDA11-N02 (Figure 4E): The TCR expression rate of KDA11-N02 was approximately 70%, and the mean fluorescence intensity of TCR expression was 6719. This suggests that the TCR protein molecule of KDA11-N02 has very high membrane stability and may have excellent antigen reactivity and antitumor activity.

[0342] Example 5: The TCR of the present disclosure specifically kills tumor antigens, and its cell killing activity shows a positive correlation with the amount of antigen expression. KVA11-N02 and KVA11-N04 specifically recognize the VVVGAVGVGK epitope peptide of KRAS G12V, while KVA11-N03 and TK34 specifically recognize the VVGAVGVGK epitope peptide of KRAS G12V. TCR-T cells expressing KVA11-N02, KVA11-N03, KVA11-N04, or TK34 were used as effector cells, and non-TCR-transduced PBMCs expanded in parallel were used as effector control cells. T2KO-A1101 (HLA-A*11:01) cells loaded with 10^-5M, 10^-6M, 10^-7M, 10^-8M, 10^-9M, 10^-10M, or 10^-11M KRAS G12V 7-16 epitope peptide or 8-16 epitope peptide were used as HLA-antigen peptide-matched positive target cells, while unloaded T2KO-A1101 cells (HLA-A*11:01) were used as HLA-matched but antigen peptide-mismatched negative target cells. All T2 cells stably express luciferase. Effector cells and different concentrations of target cells were cultured at an effector-to-target cell ratio of 9:1 for 20 hours. Luciferase substrate was added to measure surviving target cells, and the percentage of killed target cells was calculated from the remaining target cells. The EC50 value of each TCR-T was calculated from the killing rate of target cells with different concentrations of antigen peptide, and an EC50 curve was created. The results are as follows:

[0343] 1) TK34 and KVA11-N02 exhibited clear killing activity against the G12V7-16 epitope peptide presented by HLA-A*11:01. The killing activity increased with increasing antigen expression, eventually reaching a plateau and achieving nearly 100% killing. Furthermore, KVA11-N02 clearly demonstrated stronger killing activity against T2 cells with low antigen density compared to TK34. EC50 curves based on the killing percentages showed that the EC50 values ​​for TK34 and KVA11-N02 were 2.14E-9 and 1.32E-9, respectively, indicating that KVA11-N02 exhibited stronger killing activity (Figure 5A).

[0344] 2) KVA11-N03 exhibited significant killing activity against the G12V8-6 epitope peptide presented by HLA-A*11:01. The killing activity increased with increasing antigen expression, eventually reaching a plateau and achieving nearly 100% killing. The EC50 curve based on the killing rate showed that KVA11-N03 had a strong killing activity of 1.466E-9 (Figure 5B).

[0345] 3) TK34 and KVA11-N04 are G12V8-presented by HLA-A*11:01. 16 and G12V7~ 16 The killing activity of KVA11-N04 against the epitope peptide increased with increasing antigen expression, eventually reaching a plateau and achieving nearly 100% killing. Furthermore, KVA11-N04 clearly demonstrated stronger killing activity against T2 cells with low antigen density compared to TK34. The EC50 curves based on the killing percentages showed that the EC50 values ​​for TK34 and KVA11-N04 were 2.14E-9 and 9.726E-10, respectively, indicating that KVA11-N04 exhibited stronger killing activity (Figure 5C).

[0346] Example 6.1 The TCR-T of the present disclosure specifically recognizes tumor antigens and secretes cytokines, the amount of which is proportional to the amount of antigen expression KVA11-N02 and KVA11-N04 specifically recognize the KRAS G12V VVVGAVGVGK epitope peptide, KVA11-N03 specifically recognizes the KRAS G12V VVGAVGVGK epitope peptide, and TK34 specifically recognizes the KRAS G12V VVGAVGVGK epitope peptide. TCR-T cells expressing KVA11-N02, KVA11-N03, KVA11-N04, or TK34 were used as effector cells, and non-TCR-transduced PBMCs expanded in parallel were used as effector cell controls. -5 M, 10^ -6 M, 10^ -7 M, 10^ -8 M, 10^ -9 M, 10^ -10 M, 10^ -11 T2KO-A1101 (HLA-A*11:01) cells loaded with the M KRAS G12V peptide were used as HLA-antigen peptide-matched positive target cells, and unloaded T2KO-A1101 cells were used as HLA-matched but antigen peptide-mismatched negative target cells. Effector cells and different concentrations of antigen-target cells were cultured at an effector-to-effector-to-target cell ratio (E:T) of 1:1 for 20 hours, and IFN-γ secretion in the supernatant was measured using an IFN-γ ELISA kit (ExCell Bio, Cat# EH008-96). EC50 values ​​for each TCR-T were calculated based on the amount of IFN-γ secreted after effector cell activation, and EC50 curves were generated. The results are shown below.

[0347] 1) TK34 and KVA11-N02 are G12V8-presented by HLA-A*11:01 16 / 7 ~ 16The cells showed a strong specific response to the epitope peptide, and IFN-γ secretion increased with increasing antigen expression. Furthermore, compared with TK34, KVA11-N02 clearly demonstrated a stronger cytokine secretion ability against T2 cells, even at low antigen densities. The EC50 curves for IFN-γ secretion were 3.024E-8 and 2.217E-8, respectively, indicating that KVA11-N02 exhibited a stronger specific response (Figure 6A).

[0348] 2) KVA11-N03 is G12V8 presented by HLA-A*11:01 16 The cells showed a strong specific response to the epitope peptide, and IFN-γ secretion increased with increasing antigen expression. The EC50 curve for KVA11-N03 was 5.175E-8, indicating that KVA11-N03 showed a stronger specific response (Figure 6B).

[0349] 3) TK34 and KVA11-N04 are G12V8~, presented by HLA-A*11:01. 16 / 7 ~ 16 The cells showed a strong specific response to the epitope peptide, and IFN-γ secretion increased with increasing antigen expression. Furthermore, compared with TK34, KVA11-N04 clearly demonstrated a stronger cytokine secretion ability against T2 cells, even at low antigen densities. The EC50 curves for IFN-γ secretion were 3.042E-8 and 2.204E-8, respectively, indicating that KVA11-N04 exhibited a stronger specific response (Figure 6C).

[0350] Example 6.2 The TCR-T of the present disclosure specifically recognizes tumor antigens and secretes cytokines, the amount of which is proportional to the amount of antigen expression KDA11-N01 and KDA11-N02 specifically recognize the KRAS G12D 7-16 epitope peptide VVVGADGVGK presented by HLA-A*11:01. TCR-T cells expressing KDA11-N01 were used as effector cells, and non-TCR-transduced PBMCs expanded in parallel were used as effector cell controls. 10^ -5 M, 10^ -6 M, 10^ -7 M, 10^ -8 M, 10^ -9 M, 10^ -10 M, 10^ -11 T2KO-A1101 (HLA-A*11:01) cells loaded with the M KRAS G12D peptide were used as HLA-antigen peptide-matched positive target cells, and unloaded T2KO-A1101 cells were used as HLA-matched but antigen peptide-mismatched negative target cells. Effector cells and different concentrations of antigen target cells were cultured at an effector-to-target cell ratio (E:T) of 1:1 for 20 hours, and IFN-γ secretion in the supernatant was measured using an IFN-γ ELISA kit (ExCell Bio, Cat# EH008-96). EC50 values ​​for each TCR-T were calculated based on the amount of IFN-γ secreted after effector cell activation, and EC50 curves were generated. The results are shown below.

[0351] 1) KDA11-N01 is G12D7~ presented by HLA-A*11:01 16 KDA11-N01 showed a strong specific response to the epitope peptide, and IFN-γ secretion increased with increasing antigen expression. Furthermore, it was clearly observed that KDA11-N01 exhibited strong cytokine secretion ability even against T2 cells with low antigen density. The EC50 curve generated from the IFN-γ secretion yielded an EC50 of 4.96E-8, confirming the strong specific response of KDA11-N01 (Figure 6D).

[0352] 2) KDA11-N02 is G12D7~ presented by HLA-A*11:01 16KDA11-N02 showed a strong specific response to the epitope peptide, and IFN-γ secretion increased with increasing antigen expression. Furthermore, it was clearly observed that KDA11-N02 exhibited a strong cytokine secretion ability against T2 cells with low antigen density. The EC50 curve generated from the IFN-γ secretion was 1.33E-8, ​​confirming the strong specific response of KDA11-N02 (Figure 6E).

[0353] Example 7.1 TCR-T of the present disclosure exhibits highly efficient and specific cell-killing activity against antigen-positive tumor cells KVA11-N02 and KVA11-N04 specifically recognize the KRAS G12V VVVGAVGVGK epitope peptide, while KVA11-N03 and TK34 both recognize the KRAS G12V8~ 16 TCR-T cells expressing KVA11-N02 or TK34 were used as effector cells, and non-TCR-transduced PBMCs expanded in parallel were used as control effector cells. SW480-B2M-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive), Hela-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive), and PANC-1-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive) were used as HLA-antigen peptide-matched positive target cells, and HUCCT-1 (KRAS G12V negative, HLA-A*11:01 positive) was used as HLA-antigen peptide-mismatched negative target cells. All target cells stably express the luciferase gene. Effector cells and different target cells were cultured at the indicated effector-to-target cell ratio (E:T) for 16 hours, and target cell survival was measured by adding luciferase substrate. The percentage of killed target cells was calculated from the remaining target cells. The results are shown below.

[0354] 1) Both TK34 and KVA11-N02 exhibited significant cell-killing activity. Compared with TK34, KVA11-N02 exhibited stronger cell-killing activity against the adherent positive target cells SW480, Hela, and PANC-1, and this activity was concentration-dependent. However, no significant killing was observed against the adherent negative target cell HUCCT-1 (Figure 7A). This indicates that KVA11-N02 has superior cell-killing activity and specificity.

[0355] 2) Both TK34 and KVA11-N03 showed clear cell-killing activity, which was concentration-dependent, but not clear killing of the negative target cell, HUCCT-1. This indicates that KVA11-N03 has superior cell-killing activity and specificity, demonstrating superior killing function compared to TK34 (Figure 7B).

[0356] 3) Compared with TK34, KVA11-N04 exhibited stronger killing activity against the adherent positive target cells SW480, Hela, and PANC-1, and this activity was concentration-dependent, but no significant killing was observed against the adherent negative target cell HUCCT-1 (Figure 7C). This indicates that KVA11-N04 has superior cell killing activity and specificity.

[0357] Example 7.2 The disclosed TCR-T exhibits highly efficient and specific cell-killing activity against antigen-positive tumor cells KDA11-N01-expressing TCR-T cells and KDA11-N02-expressing TCR-T cells were used as effector cells, respectively. Parallel cultures of non-TCR-transduced PBMCs were used as effector control cells. PANC-1-A1101 (KRAS G12D positive, HLA-A*11:01 positive) and HUCCT-1-A1101-G12D (KRAS G12D positive, HLA-A*11:01 positive) were used as HLA-antigen peptide-matched positive target cells, and Hela-A2 (KRAS G12D negative, HLA-A*11:01 negative) was used as HLA-antigen peptide-mismatched negative target cells. All target cells stably express luciferase. Effector cells and different target cells were cultured at the indicated effector-to-target cell ratio (E:T) for 16 hours, and target cell survival was measured by adding luciferase substrate. The percentage of killed target cells was calculated from the remaining target cells. The results are shown below.

[0358] 1) KDA11-N01 exhibited significant killing activity against multiple positive target cells, and this activity was concentration-dependent. In particular, KDA11-N01 exhibited stronger killing activity against the adherent positive target cell, PANC-1, which may be related to the antigen expression level of the target cell. On the other hand, no significant killing was observed against the negative target cell, HeLa (Figure 7D). This indicates that KDA11-N01 has excellent cell-killing activity and specificity.

[0359] 2) KDA11-N02 exhibited significant killing activity against multiple positive target cells, and this activity was concentration-dependent. In particular, KDA11-N02 exhibited stronger killing activity against the adherent positive target cell, PANC-1, which may be related to the antigen expression level of the target cell. On the other hand, no significant killing was observed against the negative target cell, HeLa (Figure 7E). This indicates that KDA11-N02 has excellent cell-killing activity and specificity.

[0360] Example 8.1 TCR-T cells of the present disclosure exhibit specific responses to antigen-positive target cells: IFN-γ secretion assay TCR-T cells expressing KVA11-N02, KVA11-N03, KVA11-N04, or TK34 were used as effector cells. Parallel cultures of untransduced PBMCs were used as control effector cells. SW480-B2M-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive), Hela-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive), and PANC-1-A1101-G12V (KRAS G12V positive, HLA-A*11:01 positive) were used as HLA-antigen peptide-matched target cells, and HUCCT-1 (KRAS G12V negative, HLA-A*11:01 positive) was used as HLA-antigen peptide-mismatched target cells. TCR-T positive effector cells were cultured at a ratio of 1:1 with different target cells for 20 hours, and IFN-γ secretion in the supernatant was measured using an IFN-γ ELISA kit (ExCell Bio, Cat# EH008-96). The results are as follows:

[0361] 1) KVA11-N02 was activated by the adherent cells Hela-A1101-G12V, PANC-1-A11N02-G12V, and SW480-A1101-B2M-TAP-G12V and secreted IFN-γ, but was unable to be activated by the antigen-negative adherent cell line HUCCT-1. Compared with TK34, KVA11-N02 secreted significantly more IFN-γ, and its reactivity against KRAS G12V-positive cell lines was stronger than that of TK34 (Figure 8A). These results demonstrate that KVA11-N02 has superior specificity to TK34.

[0362] 2) Both KVA11-N03 and TK34 were activated by the adherent cells Hela-A1101-G12V, PANC-1-A11N04-G12V, and SW480-A1101-B2M-TAP-G12V and secreted IFN-γ, but were unable to be activated by the antigen-negative adherent cell line HUCCT-1. This indicates that KVA11-N03 has high specificity. Furthermore, the amount of IFN-γ secreted by KVA11-N03 was significantly higher than that of TK34 (Figure 8B).

[0363] 3) KVA11-N04 was activated by the adherent cells Hela-A1101-G12V, PANC-1-A11N04-G12V, and SW480-A1101-B2M-TAP-G12V and secreted IFN-γ, but was unable to be activated by the antigen-negative adherent cell line HUCCT-1. Compared with TK34, KVA11-N04 secreted significantly more IFN-γ, and its reactivity against KRAS G12V-positive cell lines was stronger than that of TK34 (Figure 8C). These results demonstrate that KVA11-N04 has superior specificity to TK34.

[0364] Example 8.2 TCR-T cells of the present disclosure exhibit specific responses to antigen-positive target cells: IFN-γ secretion assay KDA11-N01 and KDA11-N02-expressing TCR-T cells were used as effector cells, and non-transduced PBMCs were cultured in parallel as control effector cells. PANC-1-A1101 (KRAS G12D positive, HLA-A*11:01 positive), HPAF-II-A1101 (KRAS G12D positive, HLA-A*11:01 positive), and HUCCT-1-A1101-G12D (KRAS G12D positive, HLA-A*11:01 positive) were used as HLA-peptide-matched target cells, and Hela-A2 (KRAS G12D negative, HLA-A*11:01 negative) was used as HLA-peptide-mismatched target cells. TCR-T positive effector cells were cultured at a ratio of 1:1 with different target cells for 20 hours, and IFN-γ secretion in the supernatant was measured using an IFN-γ ELISA kit (ExCell Bio, Cat# EH008-96). The results are as follows:

[0365] 1) KDA11-N01 was activated by the adherent cells PANC-1-A1101, HUCCT-1-G12D, and HPAF-II-A1101 and secreted IFN-γ, but was not activated by the antigen-negative adherent cell line Hela-A2 (Fig. 8D). These results demonstrate the high specificity of KDA11-N01.

[0366] 2) KDA11-N02 was activated by the adherent cells PANC-1-A1101, HUCCT-1-G12D, and HPAF-II-A1101 and secreted IFN-γ, but was not activated by the antigen-negative adherent cell line Hela-A2 (Fig. 8E). These results demonstrate the high specificity of KDA11-N02.

[0367] Example 9 TCRs of the present disclosure can efficiently induce antigen-specific T cell proliferation KDA11-N01 TCR-T, KDA11-N02 TCR-T, and untransduced PBMCs were incubated in IL-2-free T cell medium for 24 hours, then stained with CFSE (C34554, Invitrogen). They were then co-cultured with T2 cells loaded with the KRAS G12D 7-16 peptide for 3 days. The mixed cells were then stained with mouse TCRβ-APC and human CD8α-PE Cy7, and CD8 and mouse TCRβ-positive cells were selected for cell proliferation analysis. T2 cells loaded with an irrelevant peptide served as a peptide control, and untransduced TCR-T PBMCs served as a negative control. The experimental results showed that the KRAS G12D 7~16 peptide could specifically stimulate the proliferation of KDA11-N01 TCR-T and KDA11-N02 TCR-T, further demonstrating the antigen specificity of KDA11-N01 TCR-T and KDA11-N02 TCR-T (Figures 9A and 9B).

[0368] Example 10: TCRs of the present disclosure do not exhibit non-specific activation against T2KO-TAP1 cell lines expressing different HLA types To evaluate whether KVA11-N02, KVA11-N03, KVA11-N04, KDA11-N01, and KDA11-N02 induce alloreactivity, i.e., antigen-independent cross-reactivity with mismatched HLAs, cell lines expressing 66 different HLAs were constructed using T2KO-TAP1 cells (HLA knockout and TAP1 expression). The selected high-frequency HLA-A, HLA-B, and HLA-C genes cover over 90% of the population. T2KO-TAP1 cells transfected with different HLA genes were sorted by flow cytometry, and the HLA expression rates were all over 95%.

[0369] TCR-T cells (1E5) expressing the TCR of the present invention were used as effector cells and co-cultured with 1E5 target cells for 16 to 24 hours, and the supernatant was collected and IFN-γ was measured. T2-A1101 cells loaded with KRAS G12V7-16 peptide, KRAS G12V8-16 peptide, or KRAS G12D7-16 peptide at 10^-6 M served as a positive control.

[0370] The results showed that target cells did not significantly stimulate IFN-γ secretion by KVA11-N02, KVA11-N03, and KVA11-N04 (Figures 10A to 10C). This suggests that KVA11-N02, KVA11-N03, and KVA11-N04 of the present disclosure do not exhibit alloreactivity against different HLA types, and therefore do not generate alloreactivity.

[0371] Target cells also barely stimulated IFN-γ secretion by KDA11-N01 and KDA11-N02 (Fig. 10D-E). However, T2-TAP-A1101, T2-TAP-A1102, and T2-TAP-B5801 stimulated IFN-γ secretion by KDA11-N01 (Fig. 10D), and T2-TAP-A1101 and T2-TAP-A1102 stimulated IFN-γ secretion by KDA11-N02 (Fig. 10E). This suggests that KDA11-N01 and KDA11-N02 may respond to an antigenic peptide presented by HLA*A11:02. This antigenic peptide could be KRAS G12D or another non-target antigenic peptide. Subsequently, the expression of the target antigen in T2 cells was detected by PCR, and the genomic sequence of the amplified product was analyzed, confirming that T2 cells themselves express KRAS G12D (Figure 11).

[0372] Example 11 The TCRs of the present disclosure do not exhibit non-specific activation of cells derived from different tissues To evaluate the safety of KVA11-N02, KVA11-N03, and KVA11-N04, we first selected tumor cell lines derived from different tissues, transfected them with the HLA-A*11:01 gene, and sorted them by flow cytometry to ensure HLA-A*11:01 expression was 90% or higher. These cells were used as nonspecific antigen-presenting cells to detect whether KVA11-N02, KVA11-N03, and KVA11-N04 reacted with nonspecific antigens. TCR-T cells expressing KVA11-N02, KVA11-N03, and KVA11-N04 were used as effector cells, and non-TCR-transfected PBMCs expanded in parallel served as effector control cells. Hela-G12V-1101 (overexpressing KRAS G12V and HLA-A*11:01) was used as a positive target cell line, and a tumor cell line transfected with HLA-A*11:01 was used as a target cell line. Effector cells were cultured with different target cells at a 1:1 effector-to-target cell ratio for 24 hours. IFN-γ secretion in the supernatant was measured using an IFN-γ ELISA kit (ExCell Bio, Cat# EH008-96).

[0373] The results showed that KVA11-N02, KVA11-N03, and KVA11-N04 TCR-T cells secreted no more IFN-γ than control PBMCs when exposed to all nonspecific antigen-presenting target cells, demonstrating that KVA11 cells did not exhibit nonspecific activation responses to HLA-A*11:01-positive tumor cell lines derived from different tissues and have excellent antigen specificity (Figures 12A-C).

[0374] Example 12 Study of non-specific reaction of target antigen peptide to TCR of the present disclosure: X-SCAN, no off-target sites detected We loaded antigen-presenting cells with a mutant peptide library and either secreted interferon by TCR-T cells or activated reporter cell lines to compile peptide response profiles for candidate TCRs. Based on the compiled results, we searched for peptides that predicted responses. We evaluated the binding ability of peptides to MHC using software. We detected the activation ability of synthetic peptides with TCR-T cells. We constructed gene expression vectors and introduced them into antigen-presenting cells to detect presentation ability. We investigated whether the antigens were expressed in different tissues. Finally, we determined the nonspecific response characteristics of TCR-T cells.

[0375] To more comprehensively evaluate the nonspecific reactivity of KVA11-N02 and KVA11-N04, single-point mutations were performed on the target antigen peptide KRAS G12V 7-16 VVVGAVGVGK of KVA11-N02 and KVA11-N04. Mutation principle: Each amino acid at each position was mutated to one of 19 other amino acids while maintaining the other amino acids, resulting in 19 × 9 (number of amino acids in the peptide) mutant peptides. The peptide positions are labeled P1, P2, P3, etc., starting from the N-terminus. For example, the amino acid V at P1 in the G12V 8-16 peptide VVGAVGVGK was mutated to G, A, L, I, S, P, F, M, W, Q, T, C, N, Y, D, E, K, R, and H, respectively, resulting in 19 P1 mutant peptides. The group was named KV1-P1X (X is the generic name for the mutated amino acid), and the sequence was XVGAVGVGK (X refers to the mutated amino acid, meaning any mutated amino acid). Individual peptide names were KV1-P1G, KV1-P1A, etc. Although Adaptimmune uses 90% pure peptides, we purchased peptides with a purity of 95% from GenScript to comprehensively characterize the specificity of KVA11-N02 and KVA11-N04. A total of 191 mutant peptides were detected. T2 cells were loaded with 10-7 M mutant peptides and co-cultured overnight with KVA11-N02 TCR-T cells and KVA11-N04 TCR-T cells (1E5 each) at a 1:1 ratio. IFN-γ secretion was measured using an IFN-γ ELISA kit. To summarize the reactivity of the single-point mutant peptides, mutations that prevented TCR-T cells from producing IFN-γ were determined to be important amino acids, while other positions were determined to be non-essential amino acids (results for KVA11-N02 TCR-T are shown in Figure 13, and results for KVA11-N04 TCR-T are shown in Figure 15).

[0376] Summary of positive amino acid responses at each position where KVA11-N02 stimulates IFN-γ production in TCR-T cells: P1[A V ]-P2[I V ]-P3[AICQMSEG V ]-P4[ G ]-P5[CSTVP A ]-P6[I V]-P7[ G ]-P8[ILFG V ]-P9[ANQSD G ]-P10[ K ] (The underlined parts are the target peptide amino acids.) An amino acid pattern search of the human protein database (https: / / myhits.sib.swiss / cgi-bin / pattern_search) detected no potentially positive peptides (Figure 14). Comprehensive evaluation by X-Scan revealed that KVA11-N02 showed no nonspecific reactions with peptides in the human body and exhibited excellent specificity.

[0377] Summary of positive amino acid responses at each position where KVA11-N04 stimulates IFN-γ production in TCR-T cells: P1[T V ]-P2[I V ]-P3[AIG V ]-P4[ G ]-P5[CS A ]-P6[IL V ]-P7[AQ G ]-P8[ICT V ]-P9[ G ]-P10[ K ] (The underlined parts are the target peptide amino acids.) An amino acid pattern search of the human protein database (https: / / myhits.sib.swiss / cgi-bin / pattern_search) detected no potentially positive peptides (Figure 16). Comprehensive evaluation by X-Scan revealed that KVA11-N04 showed no nonspecific reactions with peptides in the human body and exhibited excellent specificity.

[0378] Example 13: In vivo antitumor activity A tumor model was established using the pancreatic cancer cell line PANC-1 (KRAS G12V, A1101) in B-NDG B2M KO immunodeficient mice (5-6 weeks old, female), and 5 × 10 6 KVA11-N02 TCR-T cells, 5 x 10 6 5 × 10 KVA11-N04 TCR-T cells were administered via the tail vein. 6 PBMCs were administered as a negative control, and 5 x 106 TK34 TCR-T cells were administered as a control, and the same amount of saline was used as a blank control. The growth of subcutaneous tumors in each group of mice was monitored, and the results are shown in Figure 17. Figure 17 demonstrates that the TCR-T cells of the present invention can more effectively suppress tumor cell growth than TK34.

[0379] Although the above has described in detail preferred embodiments of the present invention, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including combining each specific technical feature in any appropriate manner. To avoid unnecessary repetition, the present invention does not separately describe various possible combinations. However, these simple modifications and combinations are also part of the content disclosed by the present invention and fall within the scope of protection of the present invention.

Claims

1. An antigen-binding protein comprising an antigen-binding domain of a T cell receptor (TCR) or a fragment thereof, wherein the antigen-binding domain or fragment thereof comprises a T cell receptor (TCR) α chain variable region (Vα) and a TCR β chain variable region (Vβ); the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13LX15 (SEQ ID NO: 1); X1 is A or V; X2 is E or V; X3 is R, P, N or a gap; X4 is D, G, S, L or a gap; X5 is I, G, D or a gap; X6 is E, T or a gap; X7 is G or a gap; X8 is A, G, T or a gap; X9 is G, S, A or a gap; X10 is N, Y or G; X11 is N, G, A or a gap; X12 is R or a gap; X13 is K or R; X15 is a CDR3 that is I, T, or M; or a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2), X1 is A or V; X2 is E or V; X3 is R, P or N; X4 is D, G or S; X5 is I, G or D; X6 is E, T or a gap; X8 is A, G or T; X9 is G or S; X10 is N or Y; X11 is N, G or a gap; X12 is R or a gap; X15 is a CDR3 that is I or T; or a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3), X1 is A or V; X3 is N or a gap; X4 is L or a gap; X6 is T or a gap; X7 is G or a gap; X8 is A or a gap; X9 is A or a gap; X10 is N or G; X11 is N or A; X13 is K or R; X15 is a CDR3 that is T or M; or a CDR3 as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or the Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10); X3 is S or T; X4 is E, H, L, F or S; X5 is G, W, V, S or I; X6 is F, G, D or V; X7 is Y, A or S; X8 is T, Q, P, Y or S; X9 is E, D, N or G; X10 is Y, S, R or a gap; X11 is G or a gap; X12 is T, E, P or a gap; X13 is A, Q or L; CDR3 where X14 is F, Y, or H; or a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11), X4 is E, H or L; X5 is G, W or V; X6 is F or G; X7 is Y, A or S; X8 is T, Q or P; X9 is E or D; X10 is Y or a gap; X12 is T, E or a gap; X13 is A or Q; CDR3 where X14 is F or Y; or a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12), X3 is S or T; X4 is F or S; X5 is S or I; X6 is D or V; X8 is Y or S; X9 is N or G; X10 is S or R; X11 is G or a gap; X12 is E or P; X13 is Q or L; CDR3, wherein X14 is F or H; or comprising a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and wherein the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12 epitope.

2. said Vα is a CDR1 having the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 4), wherein X1 is V, N or D, X2 is S or R, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; or a CDR1 having X1SX3X4X5X6 (SEQ ID NO: 5), wherein X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S; or a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), wherein X1 is N or D, X2 is S or R, and X3 is G or A; or comprising a CDR1 as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or the Vα is a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 7), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, R, or Y, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, G, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; or a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V, or a gap, X2 is I or Y, X3 is T, S, or R, X4 is G or S, X5 is D, G, or a gap, X7 is L, M, or a gap, X8 is V, D, or a gap, and X9 is K or a gap; or a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), wherein X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap; or 2. The antigen binding protein of claim 1, comprising a CDR2 as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

3. the Vβ is a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 13), wherein X1 is S, L, or M, X2 is G or N, X4 is N, D, A, or E, and X5 is S, T, or Y; or a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), wherein X1 is S or L, X4 is N, D, or A, and X5 is S or T; or a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), wherein X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y; or comprising a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or the Vβ is a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 16), wherein X1 is F, Y, or S, X2 is N, Q, or M, X4 is N, K, or V, X5 is V, E, or G, and X6 is P, L, or V; or a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), wherein X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E, or G, and X6 is P, L, or V; or a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), wherein X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G; or 3. An antigen-binding protein according to claim 1 or 2, comprising a CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

4. the Vα is a CDR3 having the following amino acid sequence: X1X2X3X4X5X6GX8X9X10X11X12KLX15 (SEQ ID NO: 2); X1 is A or V; X2 is E or V; X3 is R, P or N; X4 is D, G or S; X5 is I, G or D; X6 is E, T or a gap; X8 is A, G or T; X9 is G or S; X10 is N or Y; X11 is N, G or a gap; X12 is R or a gap; X15 is a CDR3 that is I or T; or comprising a CDR3 as set forth in SEQ ID NO: 22, 23 or 24, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vα comprises a CDR1 having X1SX3X4X5X6 (SEQ ID NO: 5), where X1 is V or N, X3 is G or A, X4 is N or S, X5 is P, Q or D, and X6 is Y or S, or comprises a CDR1 as set forth in SEQ ID NO: 27, 28, or 29, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; and said Vα comprises a CDR2 having the amino acid sequence X1X2X3X4X5NX7X8X9 (SEQ ID NO: 8), wherein X1 is Y, V or a gap, X2 is I or Y, X3 is T, S or R, X4 is G or S, X5 is D, G or a gap, X7 is L, M or a gap, X8 is V, D or a gap, and X9 is K or a gap; or comprises a CDR2 as set forth in SEQ ID NO: 32, 33 or 34, or in a functional variant formed by insertion, deletion or substitution of one or more amino acids; and / or the Vβ is a CDR3 having the following amino acid sequence: ASSX4X5X6X7X8X9X10X12X13X14 (SEQ ID NO: 11); X4 is E, H or L; X5 is G, W or V; X6 is F or G; X7 is Y, A or S; X8 is T, Q or P; X9 is E or D; X10 is Y or a gap; X12 is T, E or a gap; X13 is A or Q; CDR3 where X14 is F or Y; or a CDR3 as set forth in SEQ ID NO: 37, 38 or 39, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vβ comprises a CDR1 having the amino acid sequence X1GHX4X5 (SEQ ID NO: 14), where X1 is S or L, X4 is N, D or A, and X5 is S or T, or a CDR1 as set forth in SEQ ID NO: 42, 43, or 44, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5X6 (SEQ ID NO: 17), wherein X1 is F or Y, X2 is N or Q, X4 is N or K, X5 is V, E or G, and X6 is P, L or V; or a CDR2 as set forth in SEQ ID NO: 47, 48, or 49, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; The antigen-binding protein according to any one of claims 1 to 3, wherein the antigen-binding domain or a fragment thereof has antigen specificity for a mutant KRAS G12V epitope.

5. the Vα is a CDR3 having the following amino acid sequence: X1VX3X4X6X7X8X9X10X11X13LX15 (SEQ ID NO: 3); X1 is A or V; X3 is N or a gap; X4 is L or a gap; X6 is T or a gap; X7 is G or a gap; X8 is A or a gap; X9 is A or a gap; X10 is N or G; X11 is N or A; X13 is K or R; X15 is T or M, or a CDR3 as set forth in SEQ ID NO: 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and The Vα comprises a CDR1 having the amino acid sequence X1X2X3SQS (SEQ ID NO: 6), wherein X1 is N or D, X2 is S or R, and X3 is G or A, or a CDR1 as shown in a functional variant of SEQ ID NO: 30 or 31; and the Vα comprises a CDR2 having the amino acid sequence X1X2X3SX5NX7X8 (SEQ ID NO: 9), wherein X1 is V or a gap, X2 is I or Y, X3 is S or Y, X5 is G or a gap, X7 is G or a gap, and X8 is D or a gap, or a CDR2 as set forth in SEQ ID NO: 35 or 36, or in a functional variant formed by insertion, deletion, or substitution of one or more amino acids; and / or the Vβ is a CDR3 having the following amino acid sequence: ASX3X4X5X6SX8X9X10X11X12X13X14 (SEQ ID NO: 12); X3 is S or T; X4 is F or S; X5 is S or I; X6 is D or V; X8 is Y or S; X9 is N or G; X10 is S or R; X11 is G or a gap; X12 is E or P; X13 is Q or L; CDR3, wherein X14 is F or H; or a CDR3 as set forth in SEQ ID NO: 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and the Vβ comprises a CDR1 having the amino acid sequence X1X2HX4X5 (SEQ ID NO: 15), where X1 is S or M, X2 is G or N, X4 is A or E, and X5 is T or Y, or a CDR1 as set forth in SEQ ID NO: 45 or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and the Vβ comprises a CDR2 having the amino acid sequence X1X2NX4X5V (SEQ ID NO: 18), wherein X1 is F or S, X2 is Q or M, X4 is N or V, and X5 is E or G, or a CDR12 as set forth in SEQ ID NO: 50 or 51, or a functional variant formed by insertion, deletion, or substitution of one or more amino acids; The antigen-binding protein according to any one of claims 1 to 3, wherein the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12D epitope.

6. An antigen-binding protein comprising an antigen-binding domain of a T cell receptor (TCR) or a fragment thereof, wherein the antigen-binding domain or fragment thereof comprises a T cell receptor (TCR) α chain variable region (Vα) and a TCR β chain variable region (Vβ); said Vα comprises a CDR3 whose amino acid sequence is set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ comprises a CDR3 having the amino acid sequence set forth in SEQ ID NO: 37, 38, 39, 40, or 41, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; and wherein the antigen-binding domain or fragment thereof has antigen specificity for a mutant KRAS G12 epitope.

7. said Vα comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or 7. The antigen binding protein of claim 6, wherein the Vα comprises a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

8. said Vβ comprises a CDR1 whose amino acid sequence is set forth in SEQ ID NO: 42, 43, 44, 45 or 46, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or 8. The antigen binding protein of claim 6 or 7, wherein the Vβ comprises a CDR2 whose amino acid sequence is set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

9. An antigen-binding protein comprising an antigen-binding domain of a T cell receptor (TCR) or a fragment thereof, wherein the antigen-binding domain or fragment thereof comprises a T cell receptor (TCR) α chain variable region (Vα) and a TCR β chain variable region (Vβ); The Vα has a CDR1 sequence as set forth in SEQ ID NO: 27, 28, 29, 30 or 31, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; a CDR2 sequence as set forth in SEQ ID NO: 32, 33, 34, 35 or 36, or a functional variant thereof formed by the insertion, deletion or substitution of one or more amino acids; and a CDR3 sequence as set forth in SEQ ID NO: 22, 23, 24, 25 or 26, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and / or The Vβ comprises a CDR1 as set forth in SEQ ID NO: 42, 43, 44, 45, or 46, or a functional variant formed by the insertion, deletion, or substitution of one or more amino acids; CDR2 as set forth in SEQ ID NO: 47, 48, 49, 50 or 51, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids; and a CDR3 as set forth in SEQ ID NO: 37, 38, 39, 40 or 41, or a functional variant formed by the insertion, deletion or substitution of one or more amino acids.

10. said Vα comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 27, 32 and 22, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids; and / or said Vβ comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 42, 47 and 37, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids; The Vα comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 28, 33 and 23, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 43, 48 and 38, respectively, or functional variants formed by the insertion, deletion or substitution of one or more amino acids, The Vα comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 29, 34 and 24, respectively, or in functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or the Vβ comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 44, 49 and 39, respectively, or in functional variants formed by the insertion, deletion or substitution of one or more amino acids, said Vα comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 30, 35 and 25, respectively, or in functional variants formed by the insertion, deletion or substitution of one or more amino acids; and / or said Vβ comprises CDR1, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 45, 50 and 40, respectively, or in functional variants formed by the insertion, deletion or substitution of one or more amino acids; or 10. The antigen binding protein of claim 9, wherein said Va comprises CDRl, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 31, 36 and 26, respectively, or in functional variants formed by the insertion, deletion or substitution of one or more amino acids, and / or said Vβ comprises CDRl, CDR2 and CDR3 as set forth in the amino acid sequences of SEQ ID NOs: 46, 51 and 41, respectively, or in functional variants formed by the insertion, deletion or substitution of one or more amino acids.

11. the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, 53, 54, 55, or 56, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, 58, 59, 60, or 61; Preferably, the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 52, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 57; the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58; the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59; the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60, or 11. The antigen-binding protein of any one of claims 1 to 10, wherein the Vα comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56, and / or the Vβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

61.

12. the antigen binding protein binds to a KRAS G12 mutation epitope or a complex comprising said epitope and an MHC molecule, and said KRAS mutation epitope comprises at least one of G12V and G12D; Preferably, the MHC molecule is of the HLA-A*11 type, such as HLA-A*11:

01.

13. The antigen binding protein has the following properties: 1) Approximately 1.0×10 -7 EC below M 50 capable of binding to a target antigen peptide; 2) High film stability; 3) have specific cell-killing activity against antigen-positive tumor cells; 4) the absence of alloreactivity against different HLA types.

14. the Vα is comprised in a first polypeptide and the Vβ is comprised in a different second polypeptide; or 14. The antigen-binding protein of any one of claims 1 to 13, wherein said Vα and Vβ are comprised in a single polypeptide.

15. The antigen-binding protein of any one of claims 1 to 14, wherein the antigen-binding protein is soluble or membrane-bound.

16. the antigen binding protein is selected from a TCR, a chimeric antigen receptor (CAR), an Fc polypeptide, or an antigen-binding fragment thereof; Preferably, the antigen-binding protein according to any one of claims 1 to 15, wherein the antigen-binding protein is a TCR or an antigen-binding fragment thereof, and further comprises a TCR constant region or a fragment thereof, and / or the antigen-binding protein further comprises a transmembrane region and / or a cytoplasmic region, and / or the antigen-binding protein further comprises an intracellular signalling region.

17. the TCR constant region is a murine or human constant region; and / or the TCR constant region comprises a TCR alpha chain constant region and / or a TCR beta chain constant region; 17. The antigen binding protein of claim 16, wherein the TCR alpha and / or beta chain constant regions comprise at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TCR alpha and beta chains.

18. the TCR alpha chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 62-66 and 105, and / or the TCR beta chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 67-76; 18. The antigen binding protein of claim 17, wherein the TCR alpha chain constant region preferably comprises the amino acid sequence set forth in SEQ ID NO: 66 and the TCR beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO:

72.

19. 17. The antigen binding protein of claim 16, wherein the fragment of the TCR constant region is the extracellular segment of the TCR constant region.

20. the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any of SEQ ID NOs: 77-81, and / or a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any of SEQ ID NOs: 82-86, Preferably, the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 77, and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 82; the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 78, and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 83; the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 79, and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 84; the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 80, and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 85; or 20. The antigen binding protein of any one of claims 16 to 19, wherein the antigen binding protein comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 81, and a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:

86.

21. 21. The antigen-binding protein of any one of claims 1 to 20, wherein the antigen-binding protein further comprises one or more antigen-binding sites that bind to other antigens or epitopes.

22. 22. The antigen-binding protein of any one of claims 1 to 21, wherein the antigen-binding protein is isolated or purified.

23. A multispecific antibody comprising the antigen-binding protein of any one of claims 1 to 20.

24. A nucleic acid encoding the antigen-binding protein of any one of claims 1 to 22 or the multispecific antibody of claim 23.

25. 25. The nucleic acid of claim 24, wherein the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any of SEQ ID NOs: 87-91 and / or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any of SEQ ID NOs: 92-96.

26. A vector comprising the nucleic acid of claim 24 or 25.

27. 27. The vector of claim 26, wherein the vector is selected from a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, or an artificial chromosome.

28. A cell containing or expressing an antigen-binding protein according to any one of claims 1 to 22, a multispecific antibody according to claim 23, a nucleic acid according to claim 24 or 25, or a vector according to claim 26 or 27.

29. the cell is selected from a lymphocyte (e.g., a T cell, an NK cell), a monocyte (e.g., a PBMC), or a stem cell; Preferably, the stem cells are lymphoid progenitor cells or induced pluripotent stem cells (iPSCs); More preferably, the cell is a T cell.

30. 30. The cell of claim 28 or 29, wherein the T cell does not express an endogenous TCR.

31. A method for producing a cell according to any one of claims 28 to 30, comprising the step of transducing or transfecting the cell with a vector according to claim 26 or 27.

32. 32. The method of claim 31, further comprising the step of amplifying and / or activating the cells before or after said transduction or transfection.

33. 24. A conjugate comprising the antigen-binding protein of any one of claims 1 to 22 or the multispecific antibody of claim 23 and an active agent bound or conjugated to said antigen-binding protein or said multispecific antibody.

34. the active agent is selected from a detectable marker, an immunostimulatory molecule, or a therapeutic agent; Preferably, said detectable marker is selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, a fluorescent, phosphorescent or chemiluminescent molecule, Preferably, the immune stimulatory molecule is selected from a cytokine, a chemokine, a platelet factor and a complement activator; 34. The conjugate of claim 33, wherein the therapeutic agent is selected from an immunomodulator, a radioactive compound (e.g., a nuclide), an enzyme, a chemotherapeutic agent, and a toxin.

35. 20. The method of claim 10, further comprising administering to said subject an antigen-binding protein according to any one of claims 1 to 22, a multispecific antibody according to claim 23, a nucleic acid according to claim 24 or 25, a vector according to claim 26 or 27, or a cell according to any one of claims 28 to 30, Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

36. 36. The composition of claim 35, wherein the composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, or a small molecule drug.

37. A kit comprising an antigen-binding protein according to any one of claims 1 to 22, a multispecific antibody according to claim 23, or a conjugate according to claim 33 or 34.

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