Anti-KRAS / HLA antibodies and their use

Abstract

This disclosure relates to antibodies or antigen-binding fragments thereof that bind to a complex containing a KRAS peptide and an MHC molecule, as well as related antibody-drug conjugates (ADCs) derived therefrom, chimeric antigen receptors (CARs) derived therefrom, and methods of using these.

Description

Technical Field

[0001] Claim of Priority This application claims priority to PCT / CN2023 / 092478 filed on May 6, 2023, and PCT / CN2024 / 084444 filed on March 28, 2024. The entire content of the above applications is incorporated herein by reference.

[0002] Technical Field The present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to a complex comprising a KRAS peptide and an MHC molecule, and related antibody-drug conjugates (ADCs) derived therefrom, chimeric antigen receptors (CARs) derived therefrom, and methods of using these.

Background Art

[0003] Cancer is currently one of the diseases with the highest mortality rate in humans. According to the statistical data of the World Health Organization, in 2012, the number of cancer cases and deaths worldwide reached 14 million and 8.2 million respectively. In China, the number of newly diagnosed cancer cases is 3.07 million, and the number of deaths is 2.2 million.

[0004] Recently, due to the clinical and commercial success of anti-cancer antibodies, great interest has been shown in antibody-based therapies. There is a need to develop antibodies for use in various antibody-based therapies for treating cancer or autoimmune diseases.

Summary of the Invention

[0005] The present disclosure relates to anti-KRAS / HLA antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, chimeric antigen receptors (CARs) derived therefrom, and uses thereof. The present disclosure also provides multispecific antibodies (e.g., bispecific antibodies) that bind to an activated mutant KRAS peptide and a T cell-specific antigen (e.g., CD3).

[0006] In one embodiment, the disclosure relates to a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR3, and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR1, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR2, and the VL CDR3 region comprises a selected VL With respect to an antibody or antigen-binding fragment thereof that binds to a complex comprising a KRAS (KRAS proto-oncogene, GTPase) peptide and an MHC molecule, which comprises the above-mentioned light chain variable region having an amino acid sequence that is at least 80% identical to the amino acid sequence of CDR3, in some embodiments, the amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3 are one of the following.

[0007] (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 52, 53, and 54, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 5, and 6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 7, 8, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively; (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57 respectively; (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 14, and 15 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57 respectively; (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57 respectively; (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19, 20, and 21 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57 respectively; (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57 respectively; (9) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 26, and 27 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 58, 59, and 60 respectively; (10) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28, 29, and 30 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 52, 53, and 54 respectively; (11) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28, 29, and 30 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57 respectively; (12) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31, 32, and 33, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (13) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 34, 35, and 36, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively; (14) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 37, 38, and 39, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively; (15) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 40, 41, and 42, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively; (16) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 43, 44, and 45, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively; (17) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 46, 47, and 48, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively; (18) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 50, and 51, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 58, 59, and 60, respectively; (19) The above-selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 110, 111, and 112, respectively, and the above-selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 55, 56, and 57, respectively; and (20) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 113, 114, and 115, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 55, 56, and 57, respectively.

[0008] In some embodiments, according to Kabat's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 55, 56, and 57, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 55, 56, and 57, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 16, 17, and 18, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Kabat's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Kabat's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.In some embodiments, according to Kabat's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 58, 59, and 60, respectively. In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 52, 53, and 54, respectively. In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 37, 38, and 39, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 40, 41, and 42, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.In some embodiments, according to Chothia's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 43, 44, and 45, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 46, 47, and 48, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 49, 50, and 51, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 58, 59, and 60, respectively. In some embodiments, according to Kabat's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 110, 111, and 112, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 113, 114, and 115, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

[0009] In some embodiments, the antibody or its antigen-binding fragment specifically binds to a complex containing a KRAS peptide and an MHC molecule. In some embodiments, the KRAS peptide contains valine at the position corresponding to Gly12 of human KRAS (SEQ ID NO: 108). In some embodiments, the KRAS peptide contains or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the MHC is HLA (e.g., HLA-A3). In some embodiments, the antibody or its antigen-binding fragment is a human antibody or a humanized antibody or its antigen-binding fragment, a single-strand variable fragment (scFv), a one-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody or its antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, a human IgG2 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

[0010] In one embodiment, the disclosure relates to nucleic acids comprising polynucleotides that encode a polypeptide including the following:

[0011] (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 71; (2) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 52, 53, and 54, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 61; (3) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 72; (4) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 62; (5) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 7, 8, and 9, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 73; (6) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 63; (7) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 7, 8, and 9, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 74; (8) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 64; (9) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 75; (10) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 65; (11) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 75; (12) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 66; (13) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 72; (14) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 16, 17, and 18, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 75; (15) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 67; (16) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 76; (17) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 68; (18) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 77; (19) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 69; (20) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 78; (21) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 58, 59, and 60, respectively, wherein in some embodiments, the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 70; (22) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 71; (23) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 72; (24) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 73; (25) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 74; (26) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 75; (27) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 37, 38, and 39, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 75; (28) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 37, 38, and 39, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 72; (29) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 40, 41, and 42, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 75; (30) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 43, 44, and 45, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 76; (31) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 46, 47, and 48, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 77; (32) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 49, 50, and 51, respectively, wherein in some embodiments, the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 78; (33) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 110, 111, and 112, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 117; (34) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 113, 114, and 115, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 117; or (35) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 116.

[0012] In some embodiments, VH specifically binds to a complex containing the KRAS peptide and MHC molecules when paired with VL, or VL specifically binds to a complex containing the KRAS peptide and MHC molecules when paired with VH. In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., human IgG1 heavy chain or fragment thereof, human IgG2 heavy chain or fragment thereof, or human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof. In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv), a one-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid is cDNA.

[0013] In one embodiment, the disclosure relates to a vector comprising one or more nucleic acids described herein. In one embodiment, the disclosure relates to a vector comprising two nucleic acids described herein, wherein in some embodiments, the vectors together encode a VH region and a VL region that bind to a complex comprising a KRAS peptide and an MHC molecule. In one embodiment, the disclosure relates to a pair of vectors, wherein in some embodiments, each vector comprises one of the nucleic acids described herein, and in some embodiments, the pair of vectors together encode a VH region and a VL region that bind to a complex comprising a KRAS peptide and an MHC molecule.

[0014] In one embodiment, the disclosure relates to a cell comprising a vector or a pair of vectors described herein. In some embodiments, the cell is a CHO cell. In one embodiment, the disclosure relates to a cell comprising one or more nucleic acids described herein. In one embodiment, the disclosure relates to a cell comprising two nucleic acids described herein. In some embodiments, the two nucleic acids together encode a VH region and a VL region that bind together to a complex comprising a KRAS peptide and an MHC molecule.

[0015] In one embodiment, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing cells described herein under conditions sufficient to enable the cells to produce the antibody or the antigen-binding fragment, and (b) recovering the antibody or the antigen-binding fragment produced by the cells.

[0016] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to a complex comprising a KRAS peptide and an MHC molecule, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 61 and the selected VL sequence is sequence number 71; (2) The selected VH sequence is sequence number 62 and the selected VL sequence is sequence number 72; (3) The selected VH sequence is sequence number 63 and the selected VL sequence is sequence number 73; (4) The selected VH sequence is sequence number 64 and the selected VL sequence is sequence number 74; (5) The selected VH sequence is sequence number 65 and the selected VL sequence is sequence number 75; (6) The selected VH sequence is sequence number 66 and the selected VL sequence is sequence number 75; (7) (8) The selected VH sequence is sequence number 66 and the selected VL sequence is sequence number 72; (9) The selected VH sequence is sequence number 67 and the selected VL sequence is sequence number 75; (10) The selected VH sequence is sequence number 69 and the selected VL sequence is sequence number 77; (11) The selected VH sequence is sequence number 70 and the selected VL sequence is sequence number 78; and (12) The selected VH sequence is sequence number 116 and the selected VL sequence is sequence number 117.

[0017] In some embodiments, VH includes the sequence of sequence number 61, and VL includes the sequence of sequence number 71. In some embodiments, VH includes the sequence of sequence number 62, and VL includes the sequence of sequence number 72. In some embodiments, VH includes the sequence of sequence number 63, and VL includes the sequence of sequence number 73. In some embodiments, VH includes the sequence of sequence number 64, and VL includes the sequence of sequence number 74. In some embodiments, VH includes the sequence of sequence number 65, and VL includes the sequence of sequence number 75. In some embodiments, VH includes the sequence of sequence number 66, and VL includes the sequence of sequence number 75. In some embodiments, VH includes the sequence of sequence number 66, and VL includes the sequence of sequence number 72. In some embodiments, VH includes the sequence of sequence number 67, and VL includes the sequence of sequence number 75. In some embodiments, VH includes the sequence of sequence number 68, and VL includes the sequence of sequence number 76. In some embodiments, VH includes the sequence of sequence number 69, and VL includes the sequence of sequence number 77. In some embodiments, VH includes the sequence of sequence number 70, and VL includes the sequence of sequence number 78. In some embodiments, VH includes the sequence of sequence number 116, and VL includes the sequence of sequence number 117.

[0018] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to a complex containing a KRAS peptide and an MHC molecule, comprising a heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 identical to those of a selected VH sequence, and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3 identical to those of a selected VL sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 61 and the selected VL sequence is sequence number 71; (2) The selected VH sequence is sequence number 62 and the selected VL sequence is sequence number 72; (3) The selected VH sequence is sequence number 63 and the selected VL sequence is sequence number 73; (4) The selected VH sequence is sequence number 64 and the selected VL sequence is sequence number 74; (5) The selected VH sequence is sequence number 65 and the selected VL sequence is sequence number 75; (6) The selected VH sequence is sequence number 66 and the selected VL sequence is sequence number 75; (7) (8) The selected VH sequence is sequence number 66 and the selected VL sequence is sequence number 72; (9) The selected VH sequence is sequence number 67 and the selected VL sequence is sequence number 75; (10) The selected VH sequence is sequence number 69 and the selected VL sequence is sequence number 77; (11) The selected VH sequence is sequence number 70 and the selected VL sequence is sequence number 78; and (12) The selected VH sequence is sequence number 116 and the selected VL sequence is sequence number 117.

[0019] In some embodiments, the antibody or antigen-binding fragment specifically binds to a complex containing the KRAS peptide and an MHC molecule. In some embodiments, the KRAS peptide contains valine at the position corresponding to Gly12 of human KRAS (SEQ ID NO: 108). In some embodiments, the KRAS peptide contains or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the MHC is HLA (e.g., HLA-A3). In some embodiments, the antibody or antigen-binding fragment is a human antibody or humanized antibody or its antigen-binding fragment, a single-chain variable fragment (scFv), a one-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody or antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, a human IgG2 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

[0020] In one embodiment, this disclosure relates to an antibody or an antigen-binding fragment that cross-competes with an antibody or an antigen-binding fragment described herein.

[0021] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0022] In one embodiment, the present disclosure relates to a protein construct that binds to a complex comprising a KRAS peptide and an MHC molecule, comprising (1) a first functional moiety comprising an antibody or antigen-binding fragment thereof as described herein, and (2) a second functional moiety comprising a T cell-binding molecule. In some embodiments, the T cell-binding molecule (e.g., VHH or scFv) targets human CD3. In some embodiments, the KRAS peptide comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the MHC is HLA (e.g., HLA-A3). In some embodiments, the first and second functional moieties are linked via a linker.

[0023] In one embodiment, the present disclosure relates to a protein construct comprising: (1) a first functional part comprising an antibody or antigen-binding fragment thereof as described herein; (2) a second functional part comprising a T cell-binding molecule; and (3) a third functional part comprising a single-stranded human crystallizable fragment. In some embodiments, the T cell-binding molecule is a scFv or VHH targeting human CD3. In some embodiments, the first, second, and third functional parts are linked via one or more linkers. In some embodiments, the KRAS peptide comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the MHC is an HLA (e.g., HLA-A3).

[0024] In one embodiment, this disclosure relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, or to a protein construct described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor. In some embodiments, the drug-antibody ratio (DAR) is approximately 4.

[0025] In one embodiment, this disclosure relates to a recombinant receptor comprising an antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the recombinant receptor further comprises a transmembrane domain and an intracellular signaling domain. In some embodiments, the recombinant receptor is a chimeric antigen receptor ("CAR"). In some embodiments, the recombinant receptor further comprises a hinge region. In some embodiments, the transmembrane domain comprises the transmembrane domains of CD4, CD8, and / or CD28, or a portion thereof. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immunoeffector cell. In some embodiments, the intracellular signaling domain is or comprises a functional signaling domain of CD3 zeta. In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain.In some embodiments, the co-stimulatory signaling domain is MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CD11a / CD18, 4-1BB( CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1 , ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, The recombinant receptor includes a functional signaling domain from a protein selected from the group consisting of Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 ligands. In some embodiments, the co-stimulatory signaling domain includes an intracellular signaling domain of 4-1BB and / or CD28. In some embodiments, the recombinant receptor includes a signal peptide.

[0026] In some embodiments, the recombinant receptor is a chimeric T cell receptor (chimeric TCR or "cTCR"). In some embodiments, the cTCR comprises an α chain containing a variable α (Va) region and a β chain containing a variable β (Vb) region, wherein in some embodiments, the Va region is derived from VH of the antibody or its antigen-binding fragment, and the Vb region is derived from VL of the antibody or its antigen-binding fragment. In some embodiments, the α chain further comprises an α chain constant region, and the β chain further comprises a β chain constant region. In some embodiments, the cTCR further comprises a transmembrane region and a short cytoplasmic tail. In some embodiments, the cTCR is further associated with CD3.

[0027] In one embodiment, this disclosure relates to polynucleotides encoding recombinant receptors described herein.

[0028] In one embodiment, this disclosure relates to a vector comprising a polynucleotide as described herein. In some embodiments, the vector is a viral vector.

[0029] In one embodiment, the disclosure relates to recombinant cells expressing recombinant receptors described herein. In some embodiments, the recombinant cells are immune cells. In some embodiments, the immune cells are NK cells or T cells. In some embodiments, the recombinant cells are T cells. In some embodiments, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T (NK-T) cells, and γδT cells.

[0030] In one embodiment, this disclosure relates to a method for producing recombinant cells, which includes introducing a vector described herein into cells in vitro or in vitro. In some embodiments, the vector is a viral vector, and the introduction is carried out by transduction.

[0031] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of a composition comprising an antibody or its antigen-binding fragment, protein construct, antibody-drug conjugate, or recombinant cells as described herein. In some embodiments, the cancer comprises one or more cancer cells expressing KRAS G12V. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is pancreatic cancer, colorectal cancer, non-small cell lung cancer (NSCLC), ovarian cancer, esophageal cancer, or cholangiocarcinoma. In some embodiments, the method described herein further comprises administering to the subject a therapeutically effective dose of an anti-OX40 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

[0032] In one embodiment, the present disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, a protein construct, an antibody-drug conjugate, or recombinant cells as described herein.

[0033] In one embodiment, the present disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or its antigen-binding fragment, a protein construct, an antibody-drug conjugate, or recombinant cells as described herein.

[0034] In one embodiment, the present disclosure relates to a method for increasing an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or its antigen-binding fragment, a protein construct, an antibody-drug conjugate, or recombinant cells as described herein.

[0035] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an antibody or its antigen-binding fragment, a protein construct, an antibody-drug conjugate, or recombinant cells as described herein, and a pharmaceutically acceptable carrier.

[0036] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to a complex comprising a KRAS peptide and an MHC molecule, wherein in some embodiments the antibody or antigen-binding fragment specifically binds to an epitope in the KRAS peptide, and in some embodiments the epitope is an amino acid residue corresponding to Val6 of SEQ ID NO: 80.

[0037] As used herein, the term “cancer” means cells capable of autonomous proliferation. Examples of such cells include cells in an abnormal state or condition characterized by rapid proliferation. The term means cancerous growth, e.g., tumors, oncogenic processes, metastatic tissues, and malignant transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Malignancies of various organ systems, e.g., head and neck, respiratory, cardiovascular, renal, reproductive, hematological, nervous, liver, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, gliomas, and small intestine cancers. “Spontaneously occurring” cancers include any cancer that is not experimentally induced by transplanting cancer cells into a subject, e.g., spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from transgenic oncogene insertions or tumor suppressor gene knockouts, and cancers caused by infections, e.g., viral infections. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine glandular tissue. This term also includes carcinosarcoma, which is a malignant tumor composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma of glandular origin, or a carcinoma in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hematopoietic hyperplasia / neoplastic cells. Hematopoietic neoplastic diseases may originate from the bone marrow, lymphoid system, or erythrocyte lineage, or their progenitor cells.

[0038] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from immunoglobulin light chains or any of three CDRs derived from immunoglobulin heavy chains) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, and single-chain variable domains (V). HExamples include H) antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0039] As used herein, the term “antigen-binding fragment” means a portion of a full-length antibody, the portion of which is specifically capable of binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0040] As used herein, the term "anti-KRAS / HLA antibody" means an antibody that can specifically bind to an MHC complex containing a KRAS peptide and an HLA molecule (e.g., HLA-A3 or HLA-A11).

[0041] As used herein, the term “human antibody” means an antibody encoded by endogenous nucleic acids of human origin (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies are recovered from humans or produced in human cell culture media (e.g., in human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unarranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0042] As used herein, the term “chimeric antibody” means an antibody containing sequences present in at least two different species (e.g., an antibody derived from two different mammalian species, such as a human and a mouse antibody). Non-limiting examples of chimeric antibodies include antibodies containing a variable domain sequence (e.g., all or part of the light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody, as well as a constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0043] As used herein, the term “humanized antibody” means a non-human antibody that contains minimal sequences derived from non-human (e.g., mouse) immunoglobulin and sequences derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which residues in the hypervariable (e.g., CDR) region of the recipient antibody are replaced by residues in the hypervariable (e.g., CDR) region of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having desired specificity, affinity, and capability. In some embodiments, Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the performance of the antibody. In some embodiments, the humanized antibody contains substantially all, at least one, and typically two, variable domains, with all or substantially all of the hypervariable loop (CDR) corresponding to the hypervariable loop of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework region being a human immunoglobulin sequence. The humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. The humanized antibody can be produced using molecular biological methods well known in the art. Non-limiting examples of methods for producing the humanized antibody are described herein.

[0044] As used herein, the term “single-chain antibody” means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are specifically capable of binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0045] Where used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to an animal, human, or non-human being to be treated according to the methods of the present invention. Veterinary and non-veterinary uses are conceived by this disclosure. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines, and other domestic, livestock, and zoo animals.

[0046] As used herein, when referring to an antibody, the terms “specifically bind” and “specifically bind” mean that the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) in the target molecule; in other words, the antibody interacts with its target molecule, preferably more than other molecules, in order for the reagent to recognize and bind to a molecule containing a specific structure, rather than the entire molecule. An antibody that specifically binds to a target molecule may also be called a target-specific antibody. For example, an antibody that specifically binds to a KRAS / HLA complex may be called a KRAS / HLA-specific antibody, an anti-KRAS / HLA antibody, or an anti-KRAS / HLA complex antibody. In some embodiments, an anti-KRAS / HLA antibody may also be called an anti-KRAS G12V / HLA antibody.

[0047] As used herein, the term “bispecific antibody” means an antibody that binds to two different epitopes. Epitopes may be present on the same antigen or on different antigens.

[0048] As used herein, the term "multispecific antibody" means an antibody that binds to two or more different epitopes. Epitopes can be present on the same antigen or on different antigens. A multispecific antibody may be, for example, a bispecific antibody or a tripspecific antibody. In some embodiments, a multispecific antibody binds to two, three, four, five, or six different epitopes.

[0049] As used herein, “chimeric antigen receptor” or “CAR” means a fusion protein comprising an extracellular domain capable of binding to an antigen, and an intracellular domain comprising one or more intracellular signaling domains derived from a signal transduction protein. The extracellular domain may be any protein-like molecule or a part thereof capable of specifically binding to a given antigen. In some embodiments, the extracellular domain may comprise an antibody or its antigen-binding fragment. In some embodiments, the intracellular signaling domain may be any oligopeptide or polypeptide domain known to have the function of transmitting signals that cause activation or inhibition of intracellular biological processes, such as activation of immune cells such as T cells or NK cells.

[0050] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and mean polymers of at least two amino acids of any length.

[0051] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and mean, but are not limited to, polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials for use in the present invention are described herein, but other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0053] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0054] [Figure 1-1] Figures 1A to 1D show schematic structures of exemplary anti-KRAS / CD3 bispecific antibodies. [Figure 1-2] Figures 1A to 1D show schematic structures of exemplary anti-KRAS / CD3 bispecific antibodies. [Figure 2] List of Kabat CDR sequences for anti-KRAS / HLA antibody heavy chains. [Figure 3] List the Chothia CDR sequences for anti-KRAS / HLA antibody heavy chains. [Figure 4] The Kabat and Chothia CDR sequences against anti-KRAS / HLA antibody light chains are listed below. [Figure 5-1] Figure 5 lists the amino acid sequences discussed in this disclosure. [Figure 5-2] Figure 5 lists the amino acid sequences discussed in this disclosure. [Figure 5-3] Figure 5 lists the amino acid sequences discussed in this disclosure. [Figure 5-4] Figure 5 lists the amino acid sequences discussed in this disclosure. [Figure 6-1] Figures 6A to 6D show the binding curves of anti-KRAS / HLA antibodies 1B6-SI (Figure 6A), 1C10-SI (Figure 6B), 1H2-SI (Figure 6C), and 2B12-SI (Figure 6D) to COS-7-HLA-A03 cells pulsed with KRAS G12V(7-16) peptide, KRAS G12WT(7-16) peptide, KRAS G12C(7-16) peptide, or KRAS G12D(7-16) peptide, respectively. Unpulsed COS-7-HLA-A03 cells were used as a control. [Figure 6-2] Figures 6A to 6D show the binding curves of anti-KRAS / HLA antibodies 1B6-SI (Figure 6A), 1C10-SI (Figure 6B), 1H2-SI (Figure 6C), and 2B12-SI (Figure 6D) to COS-7-HLA-A03 cells pulsed with KRAS G12V(7-16) peptide, KRAS G12WT(7-16) peptide, KRAS G12C(7-16) peptide, or KRAS G12D(7-16) peptide, respectively. Unpulsed COS-7-HLA-A03 cells were used as a control. [Figure 6-3] Figures 6E to 6F show the binding curves of the anti-KRAS / HLA antibodies P03141-SI (Figure 6E) and V2-SI (Figure 6F) to COS-7-HLA-A03 cells pulsed with KRAS G12V(7-16) peptide or KRAS G12WT(7-16) peptide, respectively. Unpulsed COS-7-HLA-A03 cells were used as a control. [Figure 7]This shows the binding affinity of anti-KRAS / HLA antibodies V2-SI, 1G10-SI, 1H2-SI, 2B12-SI, 1F3-SI, 1G1-SI, 1A7-SI, 1B6-SI, 1C10-SI, and 1E12-SI to COS-7-HLA-A03 cells pulsed with KRAS G12V peptide, or to COS-7-HLA-A1101 cells pulsed with KRAS G12V(7-16) peptide, KRAS G12V(8-16) peptide, or KRAS G12WT(8-16) peptide. ISO is the antibody isotype control. [Figure 8A] This report shows the binding affinity of anti-KRAS / HLA antibodies V2-SI, 1H2-SI, 2B12-SI, 1G1-SI, 1B6-SI, 1C10-SI, 1F3-SI, 1A7-SI, 1G10-SI, 1E12-SI, and 1F9-SI to COS-7-HLA-A03 cells incubated with MRAS peptide, ERAS peptide, Rab-7b peptide, RhoJ peptide, or mRho GTPase2 peptide. COS-7-HLA-A03 cells not pulsed with peptides were used as a control. ISO indicates the antibody isotype control. [Figure 8B] This study demonstrates the binding affinity of the anti-KRAS / HLA antibody P03141-SI to COS-7-HLA-A03 cells incubated with MRAS peptide, Rab-7b peptide, RhoJ peptide, or mRho GTPase2 peptide. COS-7-HLA-A03 cells not pulsed with peptides were used as a control. [Figure 9-1]Figures 9A to 9L show the cell lysis percentage (%) of Raji cells expressing wild-type KRAS or CFPAC-1 cells expressing KRAS G12V, co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies V2-CD3 (Figure 9A), 1A7-CD3 (Figure 9B), 1B6-CD3 (Figure 9C), 1C10-CD3 (Figure 9D), 1E12-CD3 (Figure 9E), 1F1-CD3 (Figure 9F), 1F3-CD3 (Figure 9G), 1F9-CD3 (Figure 9H), 1G1-CD3 (Figure 9I), 1G10-CD3 (Figure 9J), 1H2-CD3 (Figure 9K), and 2B12-CD3 (Figure 9L), respectively. [Figure 9-2] Figures 9A to 9L show the cell lysis percentage (%) of Raji cells expressing wild-type KRAS or CFPAC-1 cells expressing KRAS G12V, co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies V2-CD3 (Figure 9A), 1A7-CD3 (Figure 9B), 1B6-CD3 (Figure 9C), 1C10-CD3 (Figure 9D), 1E12-CD3 (Figure 9E), 1F1-CD3 (Figure 9F), 1F3-CD3 (Figure 9G), 1F9-CD3 (Figure 9H), 1G1-CD3 (Figure 9I), 1G10-CD3 (Figure 9J), 1H2-CD3 (Figure 9K), and 2B12-CD3 (Figure 9L), respectively. [Figure 9-3] Figures 9A to 9L show the cell lysis percentage (%) of Raji cells expressing wild-type KRAS or CFPAC-1 cells expressing KRAS G12V, co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies V2-CD3 (Figure 9A), 1A7-CD3 (Figure 9B), 1B6-CD3 (Figure 9C), 1C10-CD3 (Figure 9D), 1E12-CD3 (Figure 9E), 1F1-CD3 (Figure 9F), 1F3-CD3 (Figure 9G), 1F9-CD3 (Figure 9H), 1G1-CD3 (Figure 9I), 1G10-CD3 (Figure 9J), 1H2-CD3 (Figure 9K), and 2B12-CD3 (Figure 9L), respectively. [Figure 9-4]Figures 9A to 9L show the cell lysis percentage (%) of Raji cells expressing wild-type KRAS or CFPAC-1 cells expressing KRAS G12V, co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies V2-CD3 (Figure 9A), 1A7-CD3 (Figure 9B), 1B6-CD3 (Figure 9C), 1C10-CD3 (Figure 9D), 1E12-CD3 (Figure 9E), 1F1-CD3 (Figure 9F), 1F3-CD3 (Figure 9G), 1F9-CD3 (Figure 9H), 1G1-CD3 (Figure 9I), 1G10-CD3 (Figure 9J), 1H2-CD3 (Figure 9K), and 2B12-CD3 (Figure 9L), respectively. [Figure 9-5] Figures 9A to 9L show the cell lysis percentage (%) of Raji cells expressing wild-type KRAS or CFPAC-1 cells expressing KRAS G12V, co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies V2-CD3 (Figure 9A), 1A7-CD3 (Figure 9B), 1B6-CD3 (Figure 9C), 1C10-CD3 (Figure 9D), 1E12-CD3 (Figure 9E), 1F1-CD3 (Figure 9F), 1F3-CD3 (Figure 9G), 1F9-CD3 (Figure 9H), 1G1-CD3 (Figure 9I), 1G10-CD3 (Figure 9J), 1H2-CD3 (Figure 9K), and 2B12-CD3 (Figure 9L), respectively. [Figure 9-6] Figures 9A to 9L show the cell lysis percentage (%) of Raji cells expressing wild-type KRAS or CFPAC-1 cells expressing KRAS G12V, co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies V2-CD3 (Figure 9A), 1A7-CD3 (Figure 9B), 1B6-CD3 (Figure 9C), 1C10-CD3 (Figure 9D), 1E12-CD3 (Figure 9E), 1F1-CD3 (Figure 9F), 1F3-CD3 (Figure 9G), 1F9-CD3 (Figure 9H), 1G1-CD3 (Figure 9I), 1G10-CD3 (Figure 9J), 1H2-CD3 (Figure 9K), and 2B12-CD3 (Figure 9L), respectively. [Figure 10]Figures 10A to 10E show the binding of COS-7-HLA-A03 cells pulsed with alanine-substituted peptide (50 μM) to anti-KRAS / HLA antibodies, including 1B6-SI (Figure 10A), 1G1-SI (Figure 10B), 1H2-SI (Figure 10C), 2B12-SI (Figure 10D), and P03141-SI (Figure 10E), as measured by flow cytometry. [Figure 11] The cell lysis percentages (%) of RKO cells, CFPAC-1 cells, or NCI-H441 cells co-cultured with CD3+ T cells in the presence of the anti-KRAS / CD3 bispecific antibodies P03141-CD3-SRY(A), P03141-CD3(B), 1H2-CD3-SRY(C), and 1H2-CD3(D), respectively. [Figure 12] The percentage of CD137+ T cells co-cultured with RKO cells, CFPAC-1 cells, or NCI-H441 cells in the presence of the anti-KRAS / CD3 bispecific antibodies P03141-CD3-SRY(A), P03141-CD3(B), 1H2-CD3-SRY(C), and 1H2-CD3(D), respectively, as measured by flow cytometry, is shown. [Figure 13] The following shows the cytokine IFN-γ release of CD3+ T cells co-cultured with RKO cells, CFPAC-1 cells, or NCI-H441 cells at 72 hours in the presence of the anti-KRAS / CD3 bispecific antibodies P03141-CD3-SRY(A), P03141-CD3(B), 1H2-CD3-SRY(C), and 1H2-CD3(D), respectively, as measured by flow cytometry. [Figure 14] Figures 14A and 14B show the killing of SW620 tumor cells in the presence of the anti-KRAS / CD3 bispecific antibody P03141-CD3-SLE (Figure 14A) or P03141-CD3-ScDb (Figure 14B), as measured by IncuCyte. [Figure 15] The binding curve of the anti-KRAS / HLA antibody P03141-CD3-SLE to modified Jarkat cells is shown. [Figure 16]This shows the binding of COS-7-HLA-A03 cells (50 μM) pulsed with amino acid substitution peptides to the anti-KRAS / HLA antibody P03141-SI, as measured by flow cytometry. [Figure 17] This shows the binding affinity of the anti-KRAS / HLA antibody P03141-SI to COS-7-HLA-A03 cells incubated with FNDC7 peptide, 3IS57 peptide, or SMIM2 peptide. COS-7-HLA-A03 cells not pulsed with peptides were used as a control. ISO is the antibody isotype control. [Modes for carrying out the invention]

[0055] The global incidence of cancer is increasing, with the global cancer burden projected to rise by 47% from 2020 to 28.4 million cases by 2040. KRAS is one of the most frequently mutated oncogenes in all human malignancies, found in one in seven human cancers. KRAS exists and is expressed as a membrane-bound protein in all human cells.

[0056] Mutations in the Kirsten rat sarcoma virus oncogene homolog (KRAS) are one of the most common oncogenic events in endodermal carcinomas. The KRAS gene can simultaneously carry multiple mutations that may enhance tumorigenic activity. In fact, KRAS mutations have been identified in 25% of all cancers, with some cancers, such as pancreatic cancer, having extremely high mutation rates (90%), while others, such as prostate cancer, show relatively low mutation rates (7%). KRAS mutations not only promote and maintain tumorigenesis but also increase the chances of resistance and poor prognosis, ultimately contributing to one million deaths per year. Although mutations in other RAS isoforms, including neuroblastoma RAS virus (v-ras) oncogene homolog (NRAS) and Harvey rat sarcoma virus oncogene homolog (HRAS), are common in many types of cancer, mutations in KRAS account for 85% of all RAS isoform mutations. Therefore, anti-KRAS antibodies may potentially be used in cancer treatment.

[0057] This disclosure provides examples of antibodies and their antigen-binding fragments that bind to MHC complexes containing a KRAS peptide (for example, having a G12V mutation).

[0058] KRAS and its oncogenic mutations KRAS has been identified as the KRAS-1 pseudogene on the short arm of chromosome 6 and the KRAS-2 gene on the short arm of chromosome 12 (12p11.1-12p12.1). The KRAS-2 coding region spans six exons and is over 45 kB in size. Two protein isoforms of KRAS-2, KRAS-4A and KRAS-4B, are produced by alternative splicing in its fourth exon, resulting in monomeric amino acid sequences of 188 and 189, respectively. For clinical and research purposes, the term KRAS refers to KRAS-4B, which constitutes the major transcriptome product in human cells. KRAS proteins belong to the superfamily of small GTPases and exclusively bind to GTP (G protein). The KRAS protein product consists of two domains: an N-terminal catalytic (guanine-binding) domain (G domain) and a C-terminal hypervariable region (HVR). The catalytic domain is a highly conserved region with very high homology. It consists of the P-loop, switch I, and switch II regions. The G-domain facilitates GTP-GDP exchange and functions as a GTP-GDP switch. The P-loop is a phosphate-binding region that stabilizes nucleotide phosphates, while the switch region forms a binding surface for effector proteins. This G-domain switch is primarily regulated by deactivators such as guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs), which promote the GDP-GTP switch and activation.

[0059] The C domain is highly variable and responsible for the fixation of the RAS to the inner surface of the plasma membrane. It contains the CAAX box (cysteine, two aliphatic amino acids, and other residues) and is responsible for post-translational modifications such as prenylation. Prenylation is a process in which farnesyl or geranylgeranyl is added to the terminal cysteine ​​of CAAX by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). This is followed by cleavage of the AAX residue and methylation of the cysteine ​​residue by isoprenylcysteine ​​methyltransferase (ICMT). KRAS monomers require localization to the cell membrane for their activity. More recent evidence suggests that KRAS monomers undergo dimerization for downstream signaling activity.

[0060] KRAS activation can be induced by several upstream signaling pathways for growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF); receptor tyrosine kinase (RTK) activity; and cytokines. KRAS activation consists of phosphorylation of GDP-bound KRAS to its GTP-bound state with the assistance of RAS GEF.

[0061] KRAS activation leads to downstream signaling in three major pathways: the MAP kinase pathway, the PI3K-AKT-mTOR pathway, and the tumor invasion and metastasis-inducing protein 1 (TIAM1-RAC) and RAS-related protein (RAL) pathways. The MAPK pathway consists of RAS, RAF, MEK, and ERK phosphorylation and regulates the cell cycle and cell proliferation. RAS activation and dimerization result in a conformational change that binds and phosphorylates the RAF molecule. In mutant RAS, its dimerization allows for increased RAF binding and activation. This constitutes the major downstream signaling pathway of mutant RAS. ERK, the final enzyme in the MAPK pathway, translocates to the nucleus and activates various transcription factors. This promotes cell proliferation and differentiation.

[0062] Activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by the GTP-RAS complex leads to the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). Subsequently, PIP3 phosphorylates AKT, which in turn leads to the phosphorylation of downstream mTOR, FOXO, and NF-κB, resulting in cell survival and resistance to apoptosis. The PI3K pathway is frequently upregulated by RAS mutations. However, the role of RAS in PI3K activation in normal cells remains unclear.

[0063] Therefore, in a normal state, KRAS acts as a key link between multiple cell cycle pathways, and activation of KRAS mutations leads to tumorigenesis via multiple downstream activation pathways.

[0064] KRAS G12C is the most common KRAS mutation in NSCLC, found in approximately 14% of all lung adenocarcinomas, followed by G12V. The spectrum of KRAS mutations in lung cancer is heterogeneous. G12C is the most common KRAS mutation among smokers (44%), followed by G12V (19%), while G12D is the most frequent mutation among non-smokers (56%). Interestingly, G12C was the most commonly mutated KRAS mutation in women, despite less exposure to tobacco than men. This suggests that women are more susceptible to acquiring the smoking-dependent G12C mutation. In another retrospective analysis of 2327 patients with KRAS variant NSCLC, KRAS G12D mutations were elevated in non-smokers (22%) or those with a low pack-year smoking history (median: 22.5 pack-years). In pancreatic cancer, KRAS G12D and G12V were dominant mutations, accounting for 40% and 32% of all KRAS mutations, respectively. However, G12R mutations accounted for approximately 17% of all KRAS mutations in pancreatic cancer. Right-sided colorectal tumors have a high incidence of KRAS mutations, with G12 mutations accounting for approximately 65% ​​of all KRAS mutations. Similar to pancreatic cancer, G12D and G12V are the most frequently occurring mutations.

[0065] Detailed descriptions of KRAS and its function can be found, for example, in Mustachio, LM, et al. “Targeting KRAS in cancer: promising therapeutic strategies.” Cancers 13.6(2021):1204; Parikh, K., et al. “Drugging KRAS: current perspectives and state-of-art review.” Journal of Hematology & Oncology 15.1(2022):152; and Huang, L., et al. “KRAS mutation: from undruggable to druggable in cancer.” Signal Transduction and Targeted Therapy 6.1(2021):386, each of which is incorporated in its entirety by reference.

[0066] This disclosure provides “TCR-like” antibodies that target the KRAS peptide-MHC complex. The development of these TCR-like antibodies based on therapeutic agents can improve therapeutic efficacy. Accordingly, in one embodiment, this disclosure provides a method for treating disorders associated with KRAS oncogenic mutations. In some embodiments, the disorder is cancer.

[0067] Among these peptides presented by cells, the KRAS G12V(7-16) peptide (SEQ ID NO: 80) is a peptide that can be presented by HLA-A3 (e.g., HLA-A0301 (HLA-A*03:01), HLA-A0302 (HLA-A*03:02)) or HLA-A11 (e.g., HLA-A1101 (HLA-A*11:01)). In some embodiments, the antibody or antigen-binding fragment described herein binds specifically to the KRAS / HLA-A3 complex.

[0068] Anti-KRAS / HLA antibodies and antigen-binding fragments This disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to MHC complexes containing KRAS peptides. In some embodiments, the MHC molecule is an HLA (e.g., HLA-A3 or HLA-A11 as described herein). The antibodies and antigen-binding fragments described herein are capable of binding to MHC complexes containing KRAS peptides. In some embodiments, the KRAS peptide described herein contains a hydrophobic amino acid residue (e.g., valine) at the position corresponding to Gly12(G12) in SEQ ID NO: 108. In some embodiments, the KRAS peptide described herein contains cysteine ​​at the position corresponding to Gly12(G12) in SEQ ID NO: 108. In some embodiments, the KRAS peptide described herein contains a negatively charged amino acid (e.g., aspartic acid) at the position corresponding to Gly12(G12) in SEQ ID NO: 108. In some embodiments, the KRAS peptides described herein include mutations to valine (V), cysteine ​​(C), or aspartic acid (D) at the position corresponding to Gly12 (G12) in SEQ ID NO: 108.

[0069] In some embodiments, the KRAS peptide contains valine at the position corresponding to Gly12 in human KRAS (SEQ ID NO: 108). In some embodiments, these antibodies can enhance the immune response.

[0070] This disclosure provides, for example, anti-KRAS / HLA antibodies 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, 2B12, and P03141, their chimeric antibodies, and their human antibodies or humanized antibodies.

[0071] CDR sequences for 1A7 and antibodies derived from 1A7 (e.g., humanized antibodies) include the heavy chain variable domain CDRs, SEQ ID NOs: 1, 2, 3, and the light chain variable domain CDRs, SEQ ID NOs: 52, 53, 54, as defined by Kabat. CDRs can also be defined by the Chothia system. Under the Chothia definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 28, 29, 30, and the light chain variable domain CDR sequences are shown in SEQ ID NOs: 52, 53, 54.

[0072] The CDR sequences for 1F3 and antibodies derived from 1F3 include the heavy chain variable domain CDRs, SEQ ID NOs: 4, 5, and 6, and the light chain variable domain CDRs, SEQ ID NOs: 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 28, 29, and 30, and the light chain variable domain CDRs are shown in SEQ ID NOs: 55, 56, and 57.

[0073] The CDR sequences for 1B6 and antibodies derived from 1B6 include the heavy chain variable domain CDRs, SEQ ID NOs. 7, 8, and 9, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 31, 32, and 33, and the light chain variable domain CDRs are shown in SEQ ID NOs. 55, 56, and 57.

[0074] CDR sequences for 1C10 and antibodies derived from 1C10 include the heavy chain variable domain CDRs, SEQ ID NOs. 7, 8, and 9, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 31, 32, and 33, and the light chain variable domain CDRs are shown in SEQ ID NOs. 55, 56, and 57.

[0075] The CDR sequences for 1E12 and antibodies derived from 1E12 include the heavy chain variable domain CDRs, SEQ ID NOs: 10, 11, and 12, and the light chain variable domain CDRs, SEQ ID NOs: 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 34, 35, and 36, and the light chain variable domain CDRs are shown in SEQ ID NOs: 55, 56, and 57.

[0076] The CDR sequences for 1F1 and antibodies derived from 1F1 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 14, and 15, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 37, 38, and 39, and the light chain variable domain CDRs are shown in SEQ ID NOs. 55, 56, and 57.

[0077] The CDR sequences for 1F9 and antibodies derived from 1F9 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 14, and 15, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 37, 38, and 39, and the light chain variable domain CDRs are shown in SEQ ID NOs. 55, 56, and 57.

[0078] The CDR sequences for 1G1 and antibodies derived from 1G1 include the heavy chain variable domain CDRs, SEQ ID NOs. 16, 17, and 18, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 40, 41, and 42, and the light chain variable domain CDRs are shown in SEQ ID NOs. 55, 56, and 57.

[0079] The CDR sequences for 1G10 and antibodies derived from 1G10 include the heavy chain variable domain CDRs, SEQ ID NOs: 19, 20, and 21, and the light chain variable domain CDRs, SEQ ID NOs: 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 43, 44, and 45, and the light chain variable domain CDRs are shown in SEQ ID NOs: 55, 56, and 57.

[0080] CDR sequences for 1H2 and antibodies derived from 1H2 include the heavy chain variable domain CDRs, SEQ ID NOs. 22, 23, and 24, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 46, 47, and 48, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 55, 56, and 57.

[0081] CDR sequences for 2B12 and antibodies derived from 2B12 include the heavy chain variable domain CDRs, SEQ ID NOs. 25, 26, and 27, and the light chain variable domain CDRs, SEQ ID NOs. 58, 59, and 60, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 49, 50, and 51, and the light chain variable domain CDRs are shown in SEQ ID NOs. 58, 59, and 60.

[0082] The CDR sequences for P03141 and antibodies derived from P03141 include the heavy chain variable domain CDRs, SEQ ID NOs. 110, 111, and 112, and the light chain variable domain CDRs, SEQ ID NOs. 55, 56, and 57, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 113, 114, and 115, and the light chain variable domain CDRs are shown in SEQ ID NOs. 55, 56, and 57.

[0083] The amino acid sequence of the heavy chain variable region of the 1A7 antibody is shown in SEQ ID NO: 61. The amino acid sequence of the light chain variable region of the 1A7 antibody is shown in SEQ ID NO: 71.

[0084] The amino acid sequence of the heavy chain variable region of the 1F3 antibody is shown in SEQ ID NO: 62. The amino acid sequence of the light chain variable region of the 1F3 antibody is shown in SEQ ID NO: 72.

[0085] The amino acid sequence of the heavy chain variable region of the 1B6 antibody is shown in SEQ ID NO: 63. The amino acid sequence of the light chain variable region of the 1B6 antibody is shown in SEQ ID NO: 73.

[0086] The amino acid sequence of the heavy chain variable region of the 1C10 antibody is shown in SEQ ID NO: 64. The amino acid sequence of the light chain variable region of the 1C10 antibody is shown in SEQ ID NO: 74.

[0087] The amino acid sequence of the heavy chain variable region of the 1E12 antibody is shown in SEQ ID NO: 65. The amino acid sequence of the light chain variable region of the 1E12 antibody is shown in SEQ ID NO: 75.

[0088] The amino acid sequence of the heavy chain variable region of the 1F1 antibody is shown in SEQ ID NO: 66. The amino acid sequence of the light chain variable region of the 1F1 antibody is shown in SEQ ID NO: 75.

[0089] The amino acid sequence of the heavy chain variable region of the 1F9 antibody is shown in SEQ ID NO: 66. The amino acid sequence of the light chain variable region of the 1F9 antibody is shown in SEQ ID NO: 72.

[0090] The amino acid sequence of the heavy chain variable region of the 1G1 antibody is shown in SEQ ID NO: 67. The amino acid sequence of the light chain variable region of the 1G1 antibody is shown in SEQ ID NO: 75.

[0091] The amino acid sequence of the heavy chain variable region of the 1G10 antibody is shown in SEQ ID NO: 68. The amino acid sequence of the light chain variable region of the 1G10 antibody is shown in SEQ ID NO: 76.

[0092] The amino acid sequence of the heavy chain variable region of the 1H2 antibody is shown in SEQ ID NO: 69. The amino acid sequence of the light chain variable region of the 1H2 antibody is shown in SEQ ID NO: 77.

[0093] The amino acid sequence of the heavy chain variable region of the 2B12 antibody is shown in SEQ ID NO: 70. The amino acid sequence of the light chain variable region of the 2B12 antibody is shown in SEQ ID NO: 78.

[0094] The amino acid sequence of the heavy chain variable region of the P03141 antibody is shown in SEQ ID NO: 116. The amino acid sequence of the light chain variable region of the P03141 antibody is shown in SEQ ID NO: 117.

[0095] The amino acid sequences for the heavy chain variable region and light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 71, 72, 73, 74, 75, 76, 77, 78, or 117. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, which together bind to the KRAS / HLA complex.

[0096] In some embodiments, the variable region is entirely human, derived from, for example, human heavy chain immunoglobulin loci (e.g., a combination of human IGHV, IGHD, and IGHJ genes) and / or human kappa chain immunoglobulin loci (e.g., a combination of human IGKV and IGKJ genes).

[0097] Furthermore, in some embodiments, the antibodies or antigen-binding fragments described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3, SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, SEQ ID NOs: 22-24, SEQ ID NOs: 25-27, SEQ ID NOs: 28-30, SEQ ID NOs: 31-33, SEQ ID NOs: 34-36, SEQ ID NOs: 37-39, SEQ ID NOs: 40-42, SEQ ID NOs: 43-45, SEQ ID NOs: 46-48, SEQ ID NOs: 49-51, SEQ ID NOs: 110-112, and SEQ ID NOs: 113-115, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 52-54, SEQ ID NOs: 55-57, and SEQ ID NOs: 58-60.

[0098] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in Figure 2 (CDR under Kabat's definition) and Figure 3 (CDR under Chothia's definition). The amino acid sequences of the selected VL CDR1, 2, and 3 are shown in Figure 4 (CDR under Kabat / Chothia's definition).

[0099] In some embodiments, the antibody or antigen-binding fragment described herein may contain one, two, or three heavy chain variable domains of the CDRs of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3

[0100] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0101] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9

[0102] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three heavy chain variable domains of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0103] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15

[0104] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18

[0105] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0106] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0107] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0108] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0109] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 33, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0110] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0111] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0112] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 40 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 41 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 42 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0113] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0114] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 48, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0115] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 49 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 50 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 51 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0116] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 112, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0117] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0118] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0119] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 57, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0120] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0121] Insertions, deletions, and substitutions can be located within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on a Kabat-defined scheme. In some embodiments, the CDR is determined based on a Chothia-defined scheme. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia-defined schemes. In some embodiments, the CDR is determined based on the IMGT definition. In some embodiments, the CDR is determined based on the contact definition.

[0122] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to the KRAS / HLA complex. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 61, and the selected VL sequence is SEQ ID NO: 71. In some embodiments, the selected VH sequence is SEQ ID NO: 62, and the selected VL sequence is SEQ ID NO: 72. In some embodiments, the selected VH sequence is SEQ ID NO: 63, and the selected VL sequence is SEQ ID NO: 73. In some embodiments, the selected VH sequence is SEQ ID NO: 64, and the selected VL sequence is SEQ ID NO: 74. In some embodiments, the selected VH sequence is SEQ ID NO: 65, and the selected VL sequence is SEQ ID NO: 75. In some embodiments, the selected VH sequence is sequence number 66, and the selected VL sequence is sequence number 75. In some embodiments, the selected VH sequence is sequence number 66, and the selected VL sequence is sequence number 72. In some embodiments, the selected VH sequence is sequence number 67, and the selected VL sequence is sequence number 75. In some embodiments, the selected VH sequence is sequence number 68, and the selected VL sequence is sequence number 76. In some embodiments, the selected VH sequence is sequence number 69, and the selected VL sequence is sequence number 77. In some embodiments, the selected VH sequence is sequence number 70, and the selected VL sequence is sequence number 78. In some embodiments, the selected VH sequence is sequence number 116, and the selected VL sequence is sequence number 117.

[0123] To measure the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to optimally align them for comparison, and non-homologous sequences may be ignored). The length of the reference sequence aligned for comparison is at least 80% of the reference sequence length, and in some embodiments, at least 90%, 95%, or 100%. Subsequently, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and measurement of identity percentage between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0124] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides containing immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chains or immunoglobulin light chains contain CDRs as shown in Figure 2, Figure 3, or Figure 4, or have sequences as shown in Figure 5. When a polypeptide forms a pair with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to the KRAS / HLA complex.

[0125] Anti-KRAS / HLA antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multimer, multispecific (e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0126] Antibody fragments are suitable for use in the provided manner, insofar as they retain the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment that binds to KRAS / HLA retains its ability to bind to the KRAS / HLA complex. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are closely associated, which can essentially be covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs, or subsets thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, though usually with lower affinity than the entire binding site. A single-stranded Fv or (scFv) antibody fragment contains the VH and VL domains (or regions) of the antibody, and these domains are located within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a desirable structure for antigen binding.

[0127] This disclosure also provides an antibody or an antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., “Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.” Journal of Virology 70.3(1996):1863-1872, which is incorporated herein by reference in its entirety. In one embodiment, this disclosure also provides an antibody or an antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol.5. No.2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0128] Antibody and antigen-binding fragments This disclosure provides various antibodies and their antigen-binding fragments derived from the anti-KRAS / HLA antibodies described herein. Generally, antibodies (also called immunoglobulins) consist of two classes of polypeptide chains: a light chain and a heavy chain. Non-limiting examples of antibodies in this disclosure may be intact four-immunoglobulin chain antibodies containing two heavy chains and two light chains. The heavy chains of the antibody may be any isotype including IgM, IgG, IgE, IgA, or IgD, or subisotypes including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chains may be κ light chains or λ light chains. The antibody may contain two identical copies of light chains and two identical copies of heavy chains, each containing one variable domain (or variable region, V H The heavy chain, which contains a variable domain (or variable region), binds to each other via disulfide bonds within its constant domains, forming the "stem" of the antibody. Each of these contains one variable domain (or variable region, V L Each light chain containing a constant domain (or constant region) is bonded to a heavy chain via a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bonded. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0129] The hypervariable regions, known as complementarity-determining regions (CDRs), form loops containing the antigen-binding surface of the antibody. The four framework regions are largely adapted to the β-sheet structure, and the CDRs form loops connecting the β-sheet structure, and in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity to the framework regions and, together with the CDRs of other chains, contribute to the formation of the antigen-binding region.

[0130] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and several definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422~439; Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al.,Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et This is described in al., Nature 342(6252):877-83(Dec.1989); and Ponomarenko and Bourne, BMC Structural Biology 7:64(2007), and the entirety of each of these is incorporated herein by reference.

[0131] CDRs are important for recognizing the epitopes of antigens. As used herein, an “epitope” is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as epitopes can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structures of the antigen.

[0132] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are well known in the art, and are described, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5(2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2(2015):171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference.

[0133] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies containing an immunoglobulin-binding domain fused to another polypeptide. The terms “antigen-binding domain” or “antigen-binding fragment” refer to any portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of an antibody that is specifically capable of binding to an epitope on the target molecule of an intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or its antigen-binding fragment may be any polypeptide containing, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and a binding domain that is an antibody-binding domain, or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0134] Antibody fragments suitable for use in the methods described herein are also provided. Fab fragments contain variable and constant domains of the light chain, as well as a variable domain and a first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments generally contain a pair of Fab fragments, commonly linked near the carboxyl terminus by a hinge cysteine ​​between them. Other chemical linkages of antibody fragments are well known in the art.

[0135] A diabody is a small antibody fragment containing two antigen-binding sites, and this fragment contains VH (VH and VL) attached to VL within the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domains can be paired with complementary domains on another chain, thereby generating two antigen-binding sites.

[0136] Linear antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0137] The antibodies and antibody fragments of this disclosure can be modified within the Fc region to provide a desired effector function or serum half-life.

[0138] Antibody multimerization can be achieved by the natural aggregation of antibodies or by chemical or recombinant conjugation techniques known in the art. For example, a certain proportion of purified antibody preparations (e.g., one purified IgG molecule) naturally form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0139] Alternatively, antibody homodimers can be formed by chemical bonding techniques well known in the art. For example, antibody polymers can be formed using heterobifunctional crosslinking agents, including but not limited to SMCC (4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl) and SATA (S-acetylthioacetate N-succinimidyl). Exemplary procedures for forming antibody homodimers are described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is by using the autoaffinity T15 peptide, as described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0140] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0141] Examples of bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, one antibody in a heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be produced using any convenient crosslinking method. Suitable crosslinking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0142] In some embodiments, this disclosure relates to a bispecific antibody comprising (1) a first functional part comprising an antibody or antigen-binding fragment described herein, (2) a second functional part comprising a T cell-binding molecule, and (3) a third functional part comprising a single-stranded human crystallizable fragment. In some embodiments, the T cell-binding molecule targets CD3. In some embodiments, the T cell-binding molecule is anti-CD3 VHH. In some embodiments, the antibody or antigen-binding fragment described herein is linked to a human crystallizable fragment via a hinge region, and the anti-CD3 antibody or its antigen-binding fragment is linked to the same human crystallizable fragment via a hinge region. Figure 1A shows an exemplary structure of a bispecific antibody.

[0143] In some embodiments, the bispecific antibody is an anti-KRAS / CD3 antibody, such as BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, ScDb, or tandem-scFv. In some embodiments, the anti-KRAS / CD3 antibody is VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, Diabody, crossMab, DAF(2in1), DAF(4in1), DutaMab, DT-IgG, Knob-in-hole common light chain, Knob-in-hole assembly, Charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH These include IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Mini-antibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0144] Any antibody or antigen-binding fragment described herein can be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or extend its biological activity, either in vitro (e.g., in tissue culture medium or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0145] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated with a therapeutic agent. The antibody-drug conjugate, comprising an antibody or its antigen-binding fragment, can be covalently or noncovalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinplastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, metansinoids such as DM-1 and DM-4, ​​dione, mitozantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, promycin, epirubicin, and cyclophosphamide and its analogues).

[0146] In some embodiments, the KRAS peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 80.

[0147] In some embodiments, the antibody or antigen-binding fragments described herein do not bind to MHC molecules without the KRAS peptide.

[0148] In some embodiments, the antibodies or antigen-binding fragments described herein can specifically bind to the KRAS / HLA-A3 complex. In some embodiments, the complex contains a KRAS peptide. In some embodiments, the KRAS peptide contains valine at the position corresponding to Gly12 of human KRAS (SEQ ID NO: 108). Since KRAS mutations are present in many cancer cells, antibodies or antigen-binding fragments described herein, which have high binding affinity and specificity to KRAS, can be used to form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of single-stranded variable fragments (scFv) described herein, fused to the CD3-ζ transmembrane and endodomains. In some embodiments, the chimeric antigen receptor also contains intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor contains multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, to enhance potency. Accordingly, in one embodiment, the present disclosure further provides cells (e.g., T cells) expressing the chimeric antigen receptor described herein.

[0149] In some embodiments, the scFv has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains and two light chain variable domains.

[0150] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) can be generated using the antibody or antigen-binding fragment sequence described herein (e.g., CDR or VH / VL sequence) to target the KRAS / HLA complex and additional antigens (e.g., OX40, CD3, 4-1BB, CD314, CD47, PD-1, CTLA4, CD40, or PDL1). In some embodiments, the additional antigen is a T cell-specific antigen (e.g., CD3, CD4, or CD8). For example, the multispecific or bispecific antibodies described herein can recruit and activate T cells to kill cancer cells expressing KRAS G12V.

[0151] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) having an anti-KRAS arm and an anti-CD3 arm are provided herein. In some embodiments, the anti-KRAS arm comprises a heavy chain (e.g., any heavy chain having VH as described herein) and a light chain (e.g., any light chain having VL as described herein). In some embodiments, the anti-CD3 arm comprises a heavy chain variable region (VHH) of the anti-CD3 heavy chain antibody. In some embodiments, the VHH comprises or consists of an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 103. In some embodiments, the bispecific antibodies described herein have the schematic structure shown in Figure 1A.

[0152] In some embodiments, the bispecific antibodies described herein are anti-KRAS / CD3 antibodies. In some embodiments, the bispecific antibodies in this disclosure are designed to be 1+1 (monovalent for each target) and have an IgG1 subtype structure. In some embodiments, the bispecific antibodies can crosslink target cells (e.g., cells expressing KRAS G12V) with effector cells (e.g., T cells expressing CD3), thereby enabling the effector cells to kill the target cells. In some embodiments, the anti-KRAS / CD3 antibodies described herein can specifically induce effector cells (e.g., CD3+ T cells) to kill target cells (e.g., cells expressing KRAS G12V) at E:T ratios of about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the anti-KRAS / CD3 antibodies described herein do not induce effector cells (e.g., CD3+ T cells) to kill cells that do not express KRAS G12V (e.g., cells that do not express wild-type KRAS).

[0153] In some embodiments, the anti-KRAS / CD3 antibody or its antigen-binding fragment has a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 104, and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 105 or 106.

[0154] In some embodiments, the anti-KRAS / CD3 antibody contains a knob-into-hole (KIH) mutation. In some embodiments, the anti-KRAS / CD3 antibody contains a first antigen-binding domain that specifically binds to KRAS and a second antigen-binding domain that specifically binds to CD3. In some embodiments, the first antigen-binding domain contains a heavy chain (knob heavy chain) containing one or more knob mutations, and the second antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations. In some embodiments, the first antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations, and the second antigen-binding domain contains a heavy chain (knob heavy chain) containing one or more knob mutations. In some embodiments, the anti-KRAS / CD3 antibody comprises a knob heavy chain containing a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 105. In some embodiments, the anti-KRAS / CD3 antibody comprises a hole heavy chain containing a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 106.

[0155] Antibody-drug conjugates (ADCs) The antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated with therapeutic agents (drugs). The therapeutic agent can be covalently or noncovalently bound to the antibody or antigen-binding fragment, or antigen-binding protein construct (e.g., bispecific antibodies). In some embodiments, the bispecific antibodies have a common light chain.

[0156] In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, meitansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as its analogues). Useful classes of cytotoxic, cell proliferation inhibitor, or immunomodulatory agents include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0157] In some embodiments, the therapeutic agent may be, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antibacterial agent. In some embodiments, the conjugateable therapeutic agent may be, but is not limited to, selected from MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0158] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of drastatin-10), or a derivative thereof. Auristatin can be, for example, an ester formed of auristatin E with a keto acid. For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957; International Patent Publication No. WO No. 02 / 088172, and U.S. Nos. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5, These are listed in Nos. 521,284; Nos. 5,504,191; Nos. 5,410,024; Nos. 5,138,036; Nos. 5,076,973; Nos. 4,986,988; Nos. 4,978,744; Nos. 4,879,278; Nos. 4,816,444; and Nos. 4,486,414, each of which is incorporated herein by reference for all purposes.

[0159] Auristatin has been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and has been shown to possess anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects in cancer cells. Numerous well-known assays exist in the art that can be used to measure whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation inhibitory or cytotoxic effects in desired cells.

[0160] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include thiotepa and cyclosphosphamide (CYTOXAN). TMAlkylating agents such as ); alkyl sulfonates such as busulfan, improsulfan, and biposulfan; aziridines such as benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamamine; chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, f Nitrogen mustards such as enesterine, prednimastine, trophosphamide, and uracil mustard; nitrosoureas such as carmastine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; acrasinomycin, actinomycin, anthramycin, azaserine, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophiline, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epi Antibiotics such as rubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; fludarabine, 6-mercaptopri Purine analogs such as thiamiprine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine, and 5-FU; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrine;Best Love Sil; Bisanthren; Edatrexate; Defofamine; Demecoltin; Diadiquan; Elfomitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Mitoguazone; Maitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK 7; Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxanes, e.g., Paclitaxel (TAXOL®, Bristol-Myers Examples include Squibb Oncology (Princeton, New Jersey), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives thereof. This definition includes, for example, tamoxifen, raloxifen, aromatase inhibitor 4(5)imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Furthermore, anti-hormonal agents that modulate or inhibit hormonal activity in tumors, such as anti-estrogen agents containing any of the pharmaceutically acceptable salts, acids, or derivatives mentioned above, are also included. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated in its entirety by reference.

[0161] In some embodiments, the antigen-binding construct is coupled to the drug via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is coupled to the drug via an incleavable linker, such as an MCC linker formed using SMCC or sulfo-SMCC. The selection of an appropriate linker for a given ADC can be easily made by a person skilled in the art who takes into account relevant factors such as the binding site to the antigen-binding construct, any structural limitations of the drug, and the hydrophobicity of the drug. Numerous specific linker-toxin combinations have been described and can be used in certain embodiments to prepare ADCs with the antigen-binding constructs described herein. Examples include, but are not limited to, cleavable peptide linkers with auristatins such as MMAE and MMAF, and camptothecines such as SN-38, duocalmycin, and PBD dimer; incleavable MC linkers with auristatin MMAF and MMAE; acid-unstable hydrazone linkers with calicheamycin and doxorubicin; disulfide linkers with meitansinoids such as DM1 and DM4; and bismaleimide trioxyethylene glycol (BMPEO) linkers with meitansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci.Rep.e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Publication No. US 2015 / 0374847 and U.S. Patent Publication No. US20180193477A1, which are incorporated herein by reference in their entirety.

[0162] Depending on the desired drug and the selected linker, those skilled in the art can choose a suitable method for coupling them to each other. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired drug-linker complex, which still contains a reactive group for covalently conjugating the antibody. In some embodiments, a drug-maleimide complex (i.e., a maleimide-bound drug) can be used with the payload having the reactive group in this disclosure. Maleimide is the most common reactive group that can be bound to a thiol group in ADC preparation. Furthermore, organobromids and iodides are also frequently used.

[0163] ADCs can be prepared by one of several well-known routes in the art, using organic chemical reactions, conditions, and reagents familiar to those skilled in the art (see, for example, Bioconjugate Techniques (G. Thermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) forming an antibody-linker intermediate Ab-L by covalent bonding, followed by a reaction with an activated drug site D, through the reaction of a nucleophilic or electrophilic group of the antibody with a divalent linker reagent; or (2) forming a drug-linker intermediate DL by covalent bonding, through the reaction of a nucleophilic or electrophilic group of the drug site with a linker reagent, followed by a reaction with a nucleophilic or electrophilic group of the antibody. Conjugation methods (1) and (2) can be used with various antibodies, drug moieties, and linkers to prepare the ADCs described herein. The various linkers, linker components, and toxins prepared are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G. Thermanson, 2013, Academic Press); US20210379193A1, and US20180193477A1, which are incorporated herein by reference in their entirety. Furthermore, a number of preformed drug-linkers suitable for reaction with selected antigen-binding constructs are also commercially available. For example, linker-toxins containing DM1, DM4, MMAE, MMAF, or duocalmycin SA are available from Creative BioLabs (Shirley, NY).

[0164] Several specific examples of methods for preparing ADCs are well known in the art and are described in U.S. Patent No. 8,624,003 (Pot Method), U.S. Patent No. 8,163,888 (One-Step Method), U.S. Patent No. 5,208,020 (Two-Step Method), and U.S. 20180193477A1, which are incorporated herein by reference in their entirety. Other methods are well known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0165] The drug load is expressed by the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, the drug load can be limited by the number of binding sites on the antibody. For example, when the binding is cysteinethiol, as in certain exemplary embodiments described herein, the drug load can range from 0 to 8 drug moieties per antibody. In certain embodiments, a high drug load, e.g., p ≥ 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability for certain antibody-drug conjugates. In certain embodiments, the average drug load for an antibody-drug conjugate is in the range of 1 to about 8, about 2 to about 6, or about 3 to about 5. In fact, it has been shown that for certain antibody-drug conjugates, the optimal ratio of drug moieties per antibody can be about 4. In some embodiments, the drug-antibody ratio (DAR) is about, or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is approximately 1 to approximately 2, approximately 2 to approximately 3, approximately 3 to approximately 4, approximately 3 to approximately 5, approximately 4 to approximately 5, approximately 5 to approximately 6, approximately 6 to approximately 7, or approximately 7 to approximately 8.

[0166] Antibody and ADC characteristics In some embodiments, the antibody (or its antigen-binding fragment) is 0.1s -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s-1 less than, or 0.00001 s -1 dissociates at a rate (koff) less than, and specifically binds to the KRAS / HLA complex. In some embodiments, the dissociation rate (koff) is 0.01 s -1 greater than, 0.001 s -1 greater than, 0.0001 s -1 greater than, 0.00001 s -1 greater than, or 0.000001 s -1 is greater than.

[0167] In some embodiments, the association rate (kon) is greater than 1×10 2 / M s, greater than 1×10 3 / M s, greater than 1×10 4 / M s, greater than 1×10 5 / M s, greater than, or 1×10 6 / M s is greater than. In some embodiments, the association rate (kon) is 1×10 5 less than / M s, 1×10 6 less than / M s, or 1×10 7 is less than / M s.

[0168] The affinity can be estimated from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than, or 1×10 -10 M is less than. In some embodiments, KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10 s -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M, greater than, or 1×10 -12 M is greater than.

[0169] In some embodiments, the primary target site of the anti-KRAS / HLA antibody or its antigen-binding fragment of the KRAS G12V(7-16) peptide (SEQ ID NO: 80) is different from the primary target site of the antibody V2 scFv analog.

[0170] In some embodiments, the anti-KRAS / HLA antibody binds to positions 3, 4, 5, 6, 7, and / or 8 of the KRAS peptide within the KRAS G12V(7-16) peptide / HLA-A3 complex. In some embodiments, the anti-KRAS / HLA antibody binds to positions 5, 6, and / or 7 of the KRAS peptide within the KRAS G12V(7-16) peptide / HLA complex. In some embodiments, the anti-KRAS / HLA antibody specifically recognizes position 6 of the KRAS peptide within the KRAS G12V(7-16) peptide / HLA complex.

[0171] In some embodiments, binding affinity, dissociation rate, and association rate are measured between an antibody or its antigen-binding fragment and a complex comprising a KRAS peptide (e.g., any of the KRAS peptides described herein), HLA (e.g., HLA-A*03:01), and optionally B2M.

[0172] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibodies and antigen-binding fragments described herein are conjugable to human KRAS G12V peptides (e.g., KRAS G12V(7-16) peptide (SEQ ID NO: 80)). In some embodiments, the antibodies and antigen-binding fragments described herein are not conjugable to human KRAS G12V peptides (e.g., KRAS G12V(8-16) (SEQ ID NO: 102)). In some embodiments, these antibodies are not conjugable to wild-type human KRAS peptides (e.g., KRAS G12WT(7-16) peptide (SEQ ID NO: 79) or KRAS G12WT(8-16) peptide (SEQ ID NO: 101)). In some embodiments, these antibodies are unable to bind to human KRAS peptides that include mutations in Gly12 to non-valine residues (e.g., KRAS G12C(7-16) peptide (SEQ ID NO: 81) or KRAS G12D(7-16) peptide (SEQ ID NO: 82)). In some embodiments, the antibodies and antigen-binding fragments described herein are able to bind to human KRAS proteins having the G12V mutation (human KRAS G12V protein). In some embodiments, the antibodies and antigen-binding fragments described herein do not bind to peptides derived from MRAS protein, ERAS protein, Rab-7b protein, RhoJ protein, or mRho GTPase2 protein, such as MRAS peptide (SEQ ID NO: 92), ERAS peptide (SEQ ID NO: 93), Rab-7b peptide (SEQ ID NO: 94), RhoJ peptide (SEQ ID NO: 95), mRho GTPase2 peptide (SEQ ID NO: 96), FNDC7 peptide (SEQ ID NO: 118), 3IS57 peptide (SEQ ID NO: 119), or SMIM2 peptide (SEQ ID NO: 120).

[0173] In some embodiments, the antibodies or antigen-binding fragments described herein are specifically capable of binding to human KRAS proteins containing mutations at Gly12 to a non-valine residue, e.g., cysteine ​​or aspartic acid; or to human KRAS G12V proteins rather than wild-type KRAS proteins. In some embodiments, the antibodies or antigen-binding fragments described herein have a binding affinity to human KRAS G12V proteins or fragments thereof (e.g., peptides) that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, or 1000 times greater than the binding affinity to wild-type human KRAS proteins, human KRAS proteins with a non-valine residue at the Gly12 position, or fragments thereof (e.g., peptides).

[0174] In some embodiments, the antibodies or antigen-binding fragments described herein can specifically bind to cells pulsed with human KRAS protein or its peptides (e.g., any of the KRAS peptides described herein). For example, cells can be incubated with 10 μM to 1 mM (e.g., 50 μM) of human KRAS peptide (e.g., any of the KRAS peptides described herein) for at least 10, 20, 30, 40, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In some embodiments, the cells also express HLA. The anti-KRAS / HLA antibodies described herein are fusion proteins containing HLA-A*03:01 and are produced by immunization with MHC-I VH / VL mice. Therefore, in some embodiments, the antibodies or their antigen-binding fragments described herein can specifically bind to human HLA-A3 (e.g., HLA-A*03:01 or HLA-A*03:02). In some embodiments, the antibodies or their antigen-binding fragments described herein do not bind to cells pulsed with human KRAS protein or its peptide, where the cells do not express human HLA-A3 or express HLA-A11 (e.g., HLA-A*11:01).

[0175] In some embodiments, the binding activity described herein is measured between an antibody or its antigen-binding fragment and cells expressing HLA (e.g., HLA-A*03:01) that have been pulsed with a KRAS G12V peptide (e.g., any of the KRAS G12V peptides described herein). In some embodiments, the KRAS G12V peptide is the KRAS G12V(7-16) peptide (SEQ ID NO: 80).

[0176] In some embodiments, the antibodies or antigen-binding fragments described herein have a tumor growth inhibition rate (TGI) exceeding 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TV The TGI% has a tumor growth inhibition rate of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition rate (TGI%) is calculated using the following formula: TGI(%)=[1-(T i -T0) / (V i -V0)] × 100 T i V is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. i V0 is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0177] In some embodiments, the antibodies or antigen-binding fragments described herein are antagonists of the KRAS G12V protein. In some embodiments, the antibodies or antigen-binding fragments described herein are agonists of the KRAS G12V protein.

[0178] In some embodiments, the antibodies or antigen-binding fragments described herein are non-toxic. In some embodiments, no significant difference in body weight is observed between the treatment group and the control group.

[0179] In some embodiments, antibodies or antigen-binding fragments can induce complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and kill tumor cells.

[0180] In some embodiments, the antibody or its antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the antibody or its antigen-binding fragment can induce complement-dependent cell-mediated cytotoxicity (CDC).

[0181] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody optionally having the SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody optionally having the SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation.

[0182] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, and Fv fragment. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbering) or LALA-PG mutations (L234A, L235A, and P329G mutations according to EU numbering). In some embodiments, the Fc region has FLAA mutations (F234A and L235A according to EU numbering). In some embodiments, Fc has SI mutations (S239D and I332E mutations according to EU numbering). In some embodiments, Fc has an N297A mutation according to EU numbering. In some embodiments, Fc has a YTE mutation (M252Y, S254T, and T256E according to EU numbering).

[0183] In some embodiments, Fc has SI mutations (S239D and I332E mutations in EU numbering).

[0184] Method for producing anti-KRAS / HLA antibodies Synthesized and purified KRAS / MHC complexes can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, MHC-I VH / VL mice (a detailed description of MHC-I VH / VL mice can be found in PCT / CN2022 / 081924) can be immunized with KRAS peptides (e.g., KRAS G12V(7-16) peptide, SEQ ID NO: 80) that are specifically presented by human HLA-A03.

[0185] Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of an antigen peptide or protein. In some embodiments, the antigen peptide or protein is injected with at least one adjuvant. In some embodiments, the antigen peptide or protein can be conjugated with a drug that is immunogenic in the immunized species. Animals may be injected with the antigen peptide or protein two or more times (e.g., two, three, or four times).

[0186] Immunogens are typically used for antibody preparation by immunizing a suitable target (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, recombinantly expressed or chemically synthesized polypeptides. The preparation may further contain an adjuvant, such as a Freund complete or incomplete adjuvant, or a similar immunostimulant.

[0187] In some embodiments, the animal used for immunization is an MHC-I VH / VL mouse. Details of the MHC-I VH / VL mouse can be found, for example, in PCT / CN2022 / 081924, which is incorporated herein by reference in its entirety. MHC-I VH / VL mice can produce fully human antibodies or humanized antibodies during immunization, and also express humanized MHC protein complexes. Thus, antigen peptide-MHC complexes can be used to immunize mice in order to generate a diverse collection of antibodies. In some embodiments, the components of the fusion protein can form a complex recognizable by a humanized MHC protein complex expressed in an MHC-I VH / VL mouse, such as the KRAS G12V / HLA-A0301 complex. Consequently, immunized MHC-I VH / VL mice can produce immunoglobulin light chain variable domains (e.g., any of the VLs described herein) that can pair with a rather diverse family of heavy chain variable domains (e.g., any of the VHs described herein), including affinity maturation or somatic mutation variable domains.

[0188] Full-length polypeptides or proteins can be used, or their antigen peptide fragments can be used as immunogens. The protein antigen peptide contains at least 8 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30) amino acid residues of the amino acid sequence of the KRAS protein (e.g., human KRAS G12V protein), and includes an epitope of the protein such that the antibody produced against the peptide forms a specific immune complex with the protein. For example, the KRAS G12V(7-16) peptide (SEQ ID NO: 80) can be used as an immunogen to produce an antibody against the human KRAS G12V protein.

[0189] Immunogens are typically used for antibody preparation by immunizing a suitable target (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of human KRAS). The preparation may further contain an adjuvant, such as a Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0190] Polyclonal antibodies can be prepared as described above by immunizing a suitable target with KRAS G12V protein or its antigenic peptide (e.g., a portion of KRAS G12V protein, e.g., KRAS G12V(7-16) peptide (SEQ ID NO: 80)) as an immunogen. The antibody titer in the immunized target can be monitored over time using standard techniques such as enzyme-linked immunosorbent assay (ELISA) with the immobilized KRAS protein or its peptide. If desired, the antibody molecule can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein A chromatography or protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells can be obtained from the target organism and used to prepare monoclonal antibodies using standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (generally, see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, hybridoma cells that produce monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to a target polypeptide or epitope using a standard ELISA assay.

[0191] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human antibody, a humanized antibody, or a chimeric antibody, or an antibody or antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that produces the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments may exhibit increased affinity for a target, such as the KRAS / HLA complex. Any combination of deletions, insertions, and / or combinations can be realized in antibodies or antigen-binding fragments with increased binding affinity to a target. Antibodies or antigen-binding fragments can be modified, or novel post-translational modifications can be introduced, by changing the amino acids introduced into the antibody or antigen-binding fragment, such as changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing novel glycosylation sites.

[0192] The antibodies disclosed herein may be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically modified to produce human antibodies.

[0193] Examples of human antibodies and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as) human germline immunoglobulin sequences. Examples of human antibodies include amino acid residues within the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0194] Humanized antibodies typically have a human framework (FR) into which a non-human CDR has been transplanted. Therefore, a humanized antibody has one or more amino acid sequences introduced into human from a non-human source. These non-human amino acid residues are often called “import” residues, and are typically obtained from the “import” variable domain. Humanization can essentially be carried out by substituting, for example, a rodent CDR or CDR sequence with the corresponding sequence of a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Verhoeyen et al., Science, 239:1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which a portion considerably smaller than the intact human V domain is substituted with the corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which several CDR residues and several FR residues are substituted with residues derived from similar sites within the human antibody.

[0195] Furthermore, it is important to humanize antibodies while maintaining high specificity and affinity for antigens, as well as other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by analytical processes of parental sequences and various conceptual humanization products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By observing these displays, it is possible to analyze the roles that residues can play in the functionalization of candidate immunoglobulin sequences, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0196] Typically, amino acid sequence variants of human antibodies, humanized antibodies, or chimeric anti-KRAS / HLA antibodies contain amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences present in the light or heavy chain of the original antibody.

[0197] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenMab TM Antibodies are generated using mice. A heavy chain immunoglobulin locus is a chromosomal region containing the gene for the heavy chain of an antibody. Examples of loci include the human IGHV (variable) gene, the human IGHD (variability) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a chromosomal region containing the gene encoding the light chain (κ chain) of an antibody. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. RenMab TMA detailed description of the mouse can be found in PCT / CN2020 / 075698 or US20200390073A1, which are incorporated herein by reference in their entirety.

[0198] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenLite TM Antibodies are generated using mice. A heavy chain immunoglobulin locus is a chromosomal region containing the gene for the heavy chain of an antibody. Examples of loci include the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a chromosomal region containing the gene encoding a common light chain. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. RenLite TM A detailed description of the mouse can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0199] The antibodies produced by the mouse have a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to the human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4).

[0200] Identity or homology to the original sequence is typically the percentage of amino acid residues present in a candidate sequence that is identical to a sequence present in a human, humanized, or chimeric anti-KRAS / HLA antibody or fragment, after aligning the sequences, introducing gaps where necessary, and achieving the maximum percentage of sequence identity, without considering conservative substitutions as part of the sequence identity.

[0201] Further modifications can be made to anti-KRAS / HLA antibodies or antigen-binding fragments. For example, cysteine ​​residues can be introduced into the Fc region to enable interchain disulfide bond formation within this region. Homodimeric antibodies thus produced may have some kind of extended in vitro and / or in vivo half-life. For example, homodimeric antibodies with extended in vitro and / or in vivo half-lives can also be prepared using heterobifunctional crosslinking agents described by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies with a double Fc region can be recombinant (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0202] In some embodiments, covalent modifications can be added to anti-KRAS / HLA antibodies or their antigen-binding fragments. These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues.

[0203] In some embodiments, antibody variants are provided having carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans located at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues, or position 314 in Kabat numbering), however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated mutants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further recombined, and asparagine at position 297 can be replaced with alanine (N297A).

[0204] In some embodiments, to enhance production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further recombined, replacing serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is, for example, found in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated in its entirety by reference.

[0205] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) combine many aspects of normal T cell activation into a single protein. They activate T cells upon antigen binding by linking an extracellular antigen-recognition domain to an intracellular signaling domain. CARs typically consist of four regions: an antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0206] The antigen-binding domain is exposed outside the cell at the ectodomain portion of the receptor. It is responsible for targeting CAR-T cells to any cell expressing a matching molecule, which is a potential target molecule. The antigen-binding domain is typically derived from the variable region of a monoclonal antibody, linked as a single-chain variable fragment (scFv). The scFv is a chimeric protein formed from the light chain (VL) and heavy chain (VH) of an immunoglobulin, linked by a short linker peptide. The linker between the two chains consists of hydrophilic residues, with glycine and serine present in sequence for flexibility, and glutamate and lysine present in sequence for added solubility. In some embodiments, the antigen-binding domain specifically binds to tumor-associated antigens. In some embodiments, the antigen-binding domain specifically binds to the KRAS / MHC complex described herein (e.g., the KRAS / HLA-A3 complex). In some embodiments, the antigen-binding domain does not bind to MHC molecules. In some embodiments, the antigen-binding domain is derived from any antibody described herein.

[0207] Hinge domains, also known as spacers, are small structural domains located between the antigen-binding domain and the outer membrane of the cell. Ideal hinge domains increase the flexibility of the scFv receptor head, reducing spatial constraints between the CAR and its target antigen. This facilitates antigen binding and synapse formation between CAR-T cells and target cells. Hinge sequences are often based on membrane proximal regions derived from immunomolecules, such as IgG, CD8, and CD28.

[0208] The transmembrane domain is a structural element consisting of a hydrophobic alpha-helix that spans the cell membrane. It anchors the CAR to the plasma membrane and cross-links the extracellular hinge and antigen-binding domain to the intracellular signaling region. This domain, as a whole, is essential for receptor stability. Generally, the transmembrane domain derived from the most proximal component of the endodomain is used, but different transmembrane domains result in different receptor stabilities. The CD28 transmembrane domain is known to result in a highly expressed and stable receptor.

[0209] The intracellular T cell signaling domain resides in the endodomain of the receptor within the cell. After an antigen binds to the external antigen-binding domain, CAR receptors cluster together and transmit an activation signal. The inner cytoplasmic terminal of the receptor then persists the signal within the T cell. Normal T cell activation depends on the phosphorylation of the immune receptor tyrosine-based activation motif (ITAM) present in the cytoplasmic domain of CD3 zeta. To mimic this process, the cytoplasmic domain of CD3 zeta is commonly used as the primary CAR endodomain component. In addition to CD3 signaling, T cells also require co-stimulatory molecules for persistence after activation. For this reason, the endodomain of the CAR receptor typically also contains one or more chimeric domains derived from co-stimulatory proteins. We have successfully tested the signaling domains of a wide variety of co-stimulatory molecules, including CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0210] Various CAR molecules and vectors expressing these CAR molecules can be used in the methods described herein. In some embodiments, the CAR molecules specifically bind to tumor-associated antigens, such as the KRAS / HLA-A3 complex.

[0211] Exemplary structures of antigen receptors, including hinges, transmembrane domains, and intracellular T cell signaling domains, as well as methods for recombinantly introducing such receptors into cells, are described, for example, in Chandran et al., “T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance.” Immunological reviews 290.1(2019):127-147; Cartellieri, Marc, et al., “Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.” BioMed Research International 2010(2010); and PCT Publication No. WO2017173256A1; US2002 / 131960, US2013 / 287748, US2013 / 0149337, US6,451,995, US7,446,190, US8,252,592, all of which are incorporated herein by reference.

[0212] This disclosure provides chimeric antigen receptors (CARs) or fragments thereof that specifically bind to the KRAS / HLA-A3 complex. The CARs or fragments thereof described herein are capable of binding to the KRAS / HLA-A3 complex.

[0213] This disclosure provides a CAR or fragment thereof comprising (a) an extracellular antigen-binding domain that specifically recognizes the KRAS / HLA-A3 complex, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL of the CAR or fragment thereof described herein are identical to the VH and VL of any antibody or antigen-binding fragment described herein.

[0214] In some embodiments, KRAS-targeted CARs can be generated using single-stranded variable fragments (scFv) of anti-KRAS / HLA antibodies (1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, and 2B12).

[0215] In some embodiments, the CAR has an anti-KRAS / HLA antigen-binding domain (e.g., scFv derived from anti-KRAS / HLA antibodies 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, and / or 2B12). In some embodiments, the CAR has the following elements linked in a row: (1) CD8α signal peptide, (2) scFv derived from anti-KRAS / HLA antigen-binding domain (e.g., anti-KRAS / HLA antibodies 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, and / or 2B12), (3) CD8α hinge region, (4) CD8α transmembrane domain, (5) 4-1BB intracellular domain, and (6) CD3ζ intracellular domain.

[0216] T cell receptor (TCR) In some embodiments, the recombinant receptors described herein are recombinant T cell receptors (TCRs). “T cell receptor” or “TCR” refers to a molecule containing variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) that is capable of specifically binding to an antigen peptide bound to an MHC receptor. In some embodiments, the TCR is in the αβ form. Typically, TCRs existing in the αβ and γδ forms are generally structurally similar, but the T cells expressing them may have unique anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Generally, TCRs can be found on the surface of T cells (or T lymphocytes) when they are typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. For example, in some embodiments, each chain of a TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, one transmembrane region, and one short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass its functional TCR fragment. This term also encompasses intact or full-length TCRs, including αβ or γδ morphologies.

[0217] Therefore, for the purposes of this specification, reference to TCR includes any TCR or functional fragment, such as the antigen-binding region of the TCR, which binds to a specific antigen peptide bound within an MHC molecule, i.e., an MHC-peptide complex (e.g., a KRAS / HLA-A3 complex). The antigen-binding fragments described herein are usable within a TCR. In some cases, the antigen-binding region contains variable domains of the TCR, such as variable α and variable β chains of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, for example, when each chain contains three complementarity-determining regions. In some embodiments, the variable α and variable β chains of the TCR are derived from VH and VL as described in this disclosure. In some embodiments, the TCR chain contains a constant domain. For example, as in immunoglobulins, the extracellular portion of the TCR chain (e.g., α and β chains) contains two immunoglobulin domains, i.e., a variable domain (e.g., V) at the N-terminus. α or V β Typically, Kabat's numbering system, Kabat et al., “Sequences of Proteins of Immunological Interest,” US Dept. Health and Human Services, Public Health Service, National Institutes of Health, 1991, 5 th Based on the ed., amino acids 1-116), and constant domains adjacent to the cell membrane (e.g., α-chain constant domain or C α Typically, amino acids 117-259 based on Kabat, the β-chain constant domain, or C βTypically, it may contain amino acids 117-295 based on Kabat. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two CDR-containing membrane-distal variable domains. The constant domains of the TCR domain contain short connecting sequences in which cysteine ​​residues form disulfide bonds, thereby forming a bond between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains so that the TCR contains two disulfide bonds in the constant domain.

[0218] In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain along with a transmembrane region can anchor proteins within the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0219] Generally, CD3 is a multiprotein complex that can have three distinct chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex can contain one CD3γ chain, one CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a characteristic feature that allows the three chains to associate with the positively charged T cell receptor chain. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as the immunoreceptor tyrosine-type activation motif, or ITAM, although each CD3ζ chain has three motifs. Generally, ITAM is involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR to the cell. The CD3-chain and ζ-chain, together with the TCR, form what is known as the T cell receptor complex.

[0220] In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ), or the TCR may be a single-stranded TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains, or γ and δ chains) linked by a disulfide bond or the like.

[0221] In some embodiments, KRAS-targeted chimeric TCRs can be generated using the VH and / or VL sequences of anti-KRAS / HLA antibodies described herein (1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, and 2B12). In some embodiments, the chimeric TCR is derived from the VH and / or VL sequences of anti-KRAS / HLA antigen-binding variable domains (e.g., anti-KRAS / HLA antibodies 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, and / or 2B12).α or V β ) has. In some embodiments, the antigen-binding fragments described herein replace the variable α and variable β chains of the TCR. In some embodiments, the antigen-binding fragments described herein are bound to the α chain or the β chain. In some embodiments, the scFv described herein is bound to the TCR.

[0222] Recombinant cells This disclosure provides recombinant cells (e.g., immune cells, T cells, NK cells, tumor-infiltrating lymphocytes) expressing CAR and / or various proteins described herein. These recombinant cells can be used to treat various disorders or diseases described herein (e.g., KRAS-associated cancers).

[0223] In various embodiments, recombinant cells can be obtained from, for example, humans and non-human animals. In various embodiments, recombinant cells can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. Preferably, the cells are derived from humans, rats, or mice. In some embodiments, the cells are mouse lymphocytes that have been recombinant (e.g., transduced) to express CARs, or their antigen-binding fragments. In some embodiments, the cells are obtained from humans. In various embodiments, the recombinant cells are blood cells. Preferably, the cells are leukocytes (e.g., T cells), lymphocytes, or any other suitable blood cell type. In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are T cells, B cells, or NK cells. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs). In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, human PBMCs are CD8+ cells.

[0224] In some embodiments, the cells are T cells. In some embodiments, the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. The cell surface receptor can be a wild-type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing an antigenic moiety associated with the target cell. T cells can be obtained by methods well known in the art, for example, by in vitro culture of T cells isolated from a patient (e.g., tumor-infiltrating lymphocytes). Genetically modified T cells can be obtained by transduction of T cells (e.g., isolated from the peripheral blood of a patient) with a viral vector. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are T helper 1 T cells and T helper 2 T cells. In some embodiments, the T cells expressing this receptor are αβ-T cells. In an alternative embodiment, the T cells expressing this receptor are γδ-T cells. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are naive T cells.

[0225] In some embodiments, the cells are NK cells. In some embodiments, the preparation of recombinant cells includes one or more culture and / or preparation steps. Cells for introducing binding molecules, such as CARs, can be isolated from a sample, such as a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject who requires or is administered cell therapy. In some embodiments, the subject is a human being who requires a specific therapeutic intervention, such as adoptive cell therapy, from which the cells are isolated, processed, and / or recombinant.

[0226] In some embodiments, the cells are stem cells, including induced pluripotent stem cells (iPSCs), pluripotent and multipotent stem cells. The cells may be primary cells, such as those isolated directly from the subject and / or those isolated from the subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain a desired cell type (e.g., T cells).

[0227] Various cell types can be obtained from appropriate isolation methods. Isolation methods include the separation of different cell types based on the intracellular expression or presence of one or more specific molecules, such as surface markers, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known isolation method based on such markers can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some embodiments, the separation includes, for example, incubation with an antibody or binding partner that specifically binds to such markers, the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, followed generally by a washing step to separate the antibody or binding partner-bound cells from these cells that are not bound to the antibody or binding partner.

[0228] Such separation steps can be based on positive selection, where cells bound to the reagent are retained for further use, and / or negative selection, where cells not bound to the antibody or binding partner are retained. In some embodiments, both fractions are retained for further use. In some embodiments, negative selection may be particularly useful when antibodies that specifically identify cell types are unavailable in heterogeneous populations, and separation is best performed based on markers expressed by cells other than the desired population.

[0229] Methods, nucleic acids, compositions, and kits for expressing binding molecules and for producing genetically modified cells that express such binding molecules are also provided. Genetic modification generally involves introducing nucleic acids encoding therapeutic molecules, such as CARs, e.g., TCR-like CARs, polypeptides, or fusion proteins, into cells by retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is achieved by first stimulating cells, such as in combination with stimuli that induce responses such as proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers, and then transducing the activated cells and growing them in culture medium to a number sufficient for clinical use.

[0230] In some embodiments, recombinant nucleic acids are transferred to cells using recombinant infectious viral particles, such as vectors derived from Simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred to T cells using a recombinant lentiviral vector or a retroviral vector, such as a gamma retroviral vector. In some embodiments, the retroviral vector has long-terminal repeats (LTRs), such as retroviral vectors derived from Moloney's mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or spleen fociforming virus (SFFV). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, the retrovirus may be derived from any avian or mammalian cell source. Retroviruses are usually broad-hostable, meaning they can infect host cells of several species, including humans. In some embodiments, the vector is a lentiviral vector. In some embodiments, recombinant nucleic acids are transferred to T cells by electroporation. In some embodiments, recombinant nucleic acids are transferred to T cells by translocation. Other methods for introducing genetic material into immune cells and expressing it in immune cells include calcium phosphate transfection, plasmofusion, cationic liposome-mediated transfection; tungsten particle-assisted particle impaction; and strontium phosphate DNA coprecipitation. Many of these methods are described in WO2019195486, which is incorporated herein by reference in its entirety.

[0231] Also provided are a population of recombinant cells, such as T cells, CD8+, or CD4+ cells, that express a binding molecule in a certain type of composition or cell, where such a molecule constitutes at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells; a composition containing such cells; and / or a composition concentrated for such cells.

[0232] In some embodiments, recombinant cells (e.g., CAR-T cells) can be co-cultured with target cells (e.g., antigen-presenting cells) for at least, or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, or longer, to activate the recombinant cells (e.g., CAR-T cells). In some embodiments, the target cells are Jurkat cells.

[0233] In some embodiments, IL-12 and modified IL-12 can be expressed by recombinant cells. For example, a fusion protein containing modified IL-12 as described herein can be expressed on the cell surface of recombinant cells, for example, when the fusion protein is a membrane tether protein. In some cases, a fusion protein containing modified IL-12 as described herein can be expressed and secreted, for example, when the fusion protein is a soluble protein. Expression of IL-12 in recombinant cells brings several further benefits. For example, it can increase the production of IFN-γ from NK and T cells, which is the most potent mediator of IL-12 action, activate it to stimulate the proliferation and cytotoxicity of NK cells, CD8+, and CD4+ T cells, shift the differentiation of CD4+ Th0 cells to the Th1 phenotype, increase antibody-dependent cytotoxicity (ADCC) against tumor cells, and induce IgG and suppress IgE production from B cells by at least, or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 20-fold.

[0234] In some embodiments, cytokine (e.g., IFNγ) secretion of the recombinant cells can be increased by at least, or about 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10000-fold, or more compared to the cytokine secretion level of the recombinant cells without co-culture when co-cultured with target cells.

[0235] In some embodiments, the cells are human PBMCs that are recombinant (e.g., transduced) to express a CAR or an antigen-binding fragment thereof.

[0236] In some embodiments, when the recombinant cells are co-cultured with target cells (e.g., cells expressing KRAS), the recombinant cells can increase cytokine (e.g., IFNγ) expression or secretion by at least, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more. In some embodiments, when the recombinant cells are co-cultured with target cells (e.g., cells expressing KRAS), the activated T cell population can increase by at least, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more. In some embodiments, the activation state of T cells can be measured by the CD69 expression level.

[0237] Recombinant vector This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such host cells contain polynucleotides and / or the polynucleotide-containing vectors), and the production of recombinant antibody polypeptides or fragments thereof by recombinant technology.

[0238] As used herein, “vector” is any construct that, when introduced into a host cell, can deliver one or more polynucleotides of interest to the host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as encoded polypeptides within the host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotides of interest are positioned for expression within the vector by being operably bound to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the host cell genome, at, near, or adjacent to the integration site of the polynucleotides of interest, so that the polynucleotides of interest are translated within the host cell into which the expression vector has been introduced.

[0239] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0240] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., a smallpox or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (deficient) replicable virus or a non-replicable virus. In the latter case, viral replication generally occurs only in complementary viral packaging cells. Suitable systems include, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. Preferred systems are disclosed in al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692.By coating DNA onto biodegradable beads that efficiently transport it to cells, the uptake of naked DNA can be increased.

[0241] For expression, a DNA insert containing a polynucleotide encoding an antibody or polypeptide, as disclosed herein, can be operably ligated to a suitable promoter (e.g., a heterologous promoter), such as, to name a few, the phage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoter of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may further contain sites for transcription start and end, and within the transcription region, a ribosome-binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation start at the beginning and a stop codon (UAA, UGA, or UAG) located approximately at the end of the polypeptide being translated.

[0242] As shown, the expression vector may contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes for Escherichia coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0243] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene, and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0244] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV pre-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those for Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0245] In the yeast Saccharomyces cerevisiae, several vectors containing constitutional or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0246] The construct can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference.

[0247] Transcription of the antibody-encoding DNA in more eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting elements of DNA, approximately 10–300 bp in length, that enhance the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer, located behind the origin of replication at base pairs 100–270, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer.

[0248] Appropriate secretory signals can be incorporated into expressed polypeptides to induce the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. These signals can be endogenous to the polypeptide, or they can be heterologous signals.

[0249] Polypeptides (e.g., antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST fusions) or by histidine tagging, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of a polypeptide to improve stability and endurance in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to induce secretion or excretion, improve stability, and facilitate purification is a well-known and common technique, particularly in the art.

[0250] Method for preparing recombinant cells This disclosure provides methods or processes for preparing, manufacturing, and / or using recombinant cells for treating pathological diseases or conditions.

[0251] The proteins described herein, for example, cells for introducing CARs, can be isolated from samples such as biological samples, for example, those obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject who requires or is administered cell therapy. In some embodiments, the subject is a human being who requires a specific therapeutic intervention, such as adoptive cell therapy, in which the cells are isolated, processed, and / or recombinant.

[0252] Therefore, in some embodiments, the cells are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from the subject, in addition to samples obtained from one or more processing steps such as separation, centrifugation, genetic recombination (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or processed samples. Examples of biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples (including processed samples derived therefrom).

[0253] In some embodiments, the sample from which cells are derived or isolated is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs, and / or cells derived therefrom. Samples may also include autologous and allogeneic samples in the context of cell therapy, such as adoptive cell therapy.

[0254] In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are derived from a heterologous source, such as a mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.

[0255] In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma fraction, and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium, and / or many or all divalent cations. In some embodiments, the washing step is achieved by a semi-automatic "flow-through" centrifuge. In some embodiments, the washing step is achieved by tangential flow filtration (TFF). In some embodiments, after washing, the cells are resuspended in various biocompatible buffers such as Ca 2+ / Mg 2+ -free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in the culture medium. In some embodiments, methods include density-based cell separation methods such as lysing red blood cells and preparing white blood cells from peripheral blood by centrifugation through a Percoll or Ficoll gradient.

[0256] In some embodiments, the method includes, for example, the step of isolating T cells from a patient's blood; transducing the population of T cells with a viral vector containing a nucleic acid construct encoding a recombinant antigen receptor; expanding the transduced cells in vitro; and / or injecting the expanded cells into the patient, wherein the recombinant T cells seek and destroy antigen-positive tumor cells. In some embodiments, the nucleic acid construct further includes a sequence encoding an inhibitory protein. The method further includes transfection of the T cells with a viral vector containing the nucleic acid construct.

[0257] In some embodiments, the method involves introducing any vector described herein into cells in vitro or in vitro. In some embodiments, the vector is a viral vector, and the introduction is carried out by transduction. In some embodiments, the cells are transduced for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer. In some embodiments, the method further involves introducing one or more agents into the cells, each of which can independently induce genetic disruption of the T cell receptor alpha stationary (TRAC) gene and / or the T cell receptor beta stationary (TRBC) gene. In some embodiments, the one or more agents are inhibitory nucleic acids (e.g., siRNA). In some embodiments, one or more agents are a fusion protein comprising a DNA-targeting protein and a nuclease or RNA-guided nuclease (e.g., clustered, regularly spaced short palindromic nucleic acid (CRISPR)-related nuclease).

[0258] T cell transfection can be achieved using any standard method, such as calcium phosphate, electroporation, liposome-mediated transfer, microinjection, bioristic particle delivery systems, or any other method well known to those skilled in the art. In some embodiments, T cell transfection is carried out using the calcium phosphate method.

[0259] This disclosure provides a method for producing personalized anti-tumor immunotherapy. Recombinant T cells can be produced from the patient's blood cells. These recombinant T cells are then reinjected into the patient as a cell therapy product.

[0260] Treatment method The antibodies or antigen-binding fragments thereof described herein can be used for a variety of therapeutic purposes.

[0261] In one embodiment, the disclosure provides a method for treating cancer in a subject, a method for slowing the rate of increase of tumor volume in a subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of further metastasis in a subject. In some embodiments, the treatment can interrupt, slow, stop, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction of the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0262] In one embodiment, the present disclosure is characterized by a method comprising administering a therapeutically effective dose of recombinant cells expressing CAR to a subject in need (e.g., a subject with cancer, or identified or diagnosed as having cancer).

[0263] In one embodiment, the Disclosure relates to a method comprising administering a therapeutically effective dose of an antibody, an antigen-binding fragment thereof, or recombinant cells disclosed herein to a subject in need (for example, a subject having, or being identified or diagnosed with, cancer, such as breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, nerve cancer, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, skin cancer, or hematological malignancies). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastroesophageal junction cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, glioma, hematological malignancies, in particular non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or progressive solid tumors.

[0264] In some embodiments, anti-KRAS / HLA antibodies are designed to treat solid tumors, pancreatic cancer, colorectal cancer, non-small cell lung cancer (NSCLC), ovarian cancer, esophageal cancer, or bile duct cancer. In some embodiments, anti-KRAS / HLA antibodies are designed to treat pancreatic adenocarcinoma, lung adenocarcinoma, colorectal adenocarcinoma, or rectal adenocarcinoma.

[0265] In some embodiments, the cancers described herein include acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone tumors, brain tumors, breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic bone marrow cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, and gastrointestinal carcinoid tumors. The cancer may be any cancer, including any of the following: ulcer, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and urinary tract bladder cancer. Preferred cancers are pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. Preferably, lung cancer is lung adenocarcinoma, ovarian cancer is epithelial ovarian cancer, and pancreatic cancer is pancreatic cancer. In another preferred embodiment, the cancer is a cancer expressing a mutant KRAS containing the amino acid sequence of SEQ ID NO: 80.

[0266] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients at risk of cancer can be identified by various methods well known in the art.

[0267] As used herein, “effective dose” means an amount or dosage sufficient to produce a beneficial or desired outcome, including interrupting, slowing, blocking, or inhibiting the progression of a disease, such as an autoimmune disease or cancer. The effective dose varies depending on the age and weight of the person to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector containing the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the administration can be determined on an individual basis.

[0268] An effective dose can be administered in one or more doses. For example, an effective dose of antibody or antigen-binding fragment is an amount sufficient to mitigate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to mitigate, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective dose of antibody or antigen-binding fragment may vary depending on other factors, in particular, the patient's medical history, as well as the type (and / or dose) of antibody used.

[0269] The effective doses and schedules for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dose to be administered will vary depending, for example, on the mammal receiving the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein, the route of administration, the specific type of antibody, the polynucleotide encoding the antibody, the antigen-binding fragment, and / or the compositions disclosed herein used, and other agents administered to the mammal. Guidelines for selecting an appropriate dose for an antibody or antigen-binding fragment can be found in literature on the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch.22 and pp.303-357, and Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp.365-389.

[0270] The typical daily dose of an effective amount of antibody or ADC is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may be 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dose may be 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or greater than 0.01 mg / kg. In some embodiments, the dose is approximately 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0271] In any of the methods described herein, at least one antibody, its antigen-binding fragment, recombinant cells, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a target at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0272] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and the biological activity periods of at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) overlap within the subject.

[0273] In some embodiments, at least one antibody, antigen-binding antibody fragment, recombinant cell, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, recombinant cells, or pharmaceutical compositions described herein) can be administered to a subject over a long period of time (e.g., over a period of at least one week, two weeks, three weeks, one month, two months, three months, four months, twelve months, one year, two years, three years, four years, or five years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein to diagnose or follow up on the effectiveness of the treatment (e.g., observe at least one symptom of cancer). As described herein, skilled medical professionals may also change (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and may adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0274] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0275] In some embodiments, additional therapeutic agents may include one or more inhibitors selected from the group consisting of LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated Hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0276] In some embodiments, additional therapeutic agents may include one or more agents selected from the group consisting of trabectedin, nab-paclitaxel, trevananib, pazopanib, sediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolisin, alimta, jikaida, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, IMA-901, AGS-003, cabozantinib, vinflunin, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, salomib, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0277] In some embodiments, additional therapeutic agents may include one or more agents selected from the group consisting of adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and HER2 agonists.

[0278] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

[0279] In some embodiments, additional therapeutic agents include anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-ICOS antibody, anti-CD27 antibody, anti-OX40 antibody, anti-4-1BB antibody, anti-CD40 antibody, and / or anti-GITR antibody.

[0280] In one embodiment, the present disclosure provides a combination therapy. In some embodiments, an anti-KRAS / HLA antibody or its antigen-binding fragment (e.g., any antibody described herein) may be administered together with an immunomodulator.

[0281] Pharmaceutical composition and route of administration Pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein are also provided herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein may be present in any combination in the pharmaceutical composition. The pharmaceutical composition may be formulated in any form well known in the art.

[0282] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antimicrobial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphoric acid), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). The composition preparations can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. Where necessary (e.g., in injectable formulations), adequate fluidity can be maintained by coatings such as lecithin or by surfactants. The absorption of antibodies or their antigen-binding fragments can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulation delivery systems, including biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0283] Compositions containing one or more antibodies or antigen-binding fragments described herein can be formulated in unit dosage forms (i.e., physically distinct units containing a predetermined amount of the active compound to facilitate administration and ensure uniformity of dose) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0284] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be supplied in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain compounding agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form for preparation with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0285] The present invention provides a composition containing recombinant cells for administration, including a pharmaceutical composition and a preparation, such as a unit dose form composition containing a given number of cells for administration in a given dose or fraction thereof. Examples of pharmaceutical compositions and preparations include one or more pharmaceutically acceptable carriers or excipients.

[0286] The toxicity and therapeutic effect of a composition can be determined by standard pharmaceutical procedures in cell culture media or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (effective dose for 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs exhibiting a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize the potential harm (i.e., mitigate the undesirable side effects). Toxicity and therapeutic effect can be determined by other standard pharmaceutical procedures.

[0287] Data obtained from cell culture assays and animal studies can be used in the formulation of appropriate doses of any given agent for use in subjects (e.g., humans). A therapeutically effective dose of one or more antibodies (e.g., 1, 2, 3, or 4) or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) is the amount that treats the disease (e.g., kills cancer cells) or reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who has previously had cancer but is now cured), or the amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods well known in the art, in addition to observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors may influence the dose and timing required to effectively treat the subject (e.g., severity of the disease or disability, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0288] Exemplary doses include the amount (milligrams or micrograms) of either the antibody or antigen-binding fragment described herein per kilogram of body weight of the subject (e.g., approximately 1 μg / kg to approximately 500 mg / kg; approximately 100 μg / kg to approximately 500 mg / kg; approximately 100 μg / kg to approximately 50 mg / kg; approximately 10 μg / kg to approximately 5 mg / kg; approximately 10 μg / kg to approximately 0.5 mg / kg; or approximately 1 μg / kg to approximately 50 μg / kg). Although these doses cover a wide range, those skilled in the art will understand that the efficacy and effective dose of therapeutic agents containing antibodies and their antigen-binding fragments can be determined by methods well known in the art. Typically, a relatively low dose is administered first, and the dose can be subsequently and gradually increased by the healthcare professional or veterinary professional (in the case of therapeutic use), or the researcher (if still working in the development stage), until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject depends on various factors, including the activity of the specific compound used, the subject's age, weight, overall health, sex, and diet, administration time, route of administration, excretion rate, and the half-life of the antibody or antibody fragment in the body.

[0289] The pharmaceutical composition may be included in a container, pack, or dispenser, along with instructions for administration. This disclosure also provides methods for producing antibodies or their antigen-binding fragments for various applications described herein.

[0290] Examples The present invention will be further illustrated by the following embodiments, which are not intended to limit the scope of the invention as described in the claims.

[0291] Example 1. Production of anti-KRAS / HLA antibodies MHC-I VH / VL mice (a detailed description of MHC-I VH / VL mice can be found in their entirety in PCT / CN2022 / 081924, which is incorporated herein by reference) were immunized with human KRAS G12V (HLA-A*03:01) protein (Kactus Biosystems, catalog number: MHC-HM418). MHC-I VH / VL mice can have humanized light chain immunoglobulin loci and humanized heavy chain immunoglobulin loci, and the mice also express humanized major histocompatibility complex (MHC) protein complexes.

[0292] If a desirable immune response was achieved, antigen-specific immune cells were isolated from the immunized mice to obtain further anti-KRAS / HLA antibodies, or to obtain the light and heavy chain variable region sequences of anti-KRAS / HLA antibodies. For example, plasma cells secreting antigen-specific monoclonal antibodies were screened and isolated using single-cell technology (e.g., using Beacon® Optofluidic System, Berkeley Lights Inc.), and the antibody variable region sequences were subsequently obtained by reverse transcription and PCR sequencing. The obtained variable region sequences were cloned into vectors containing sequences encoding the human IgG constant region for antibody expression. Next, the specificity of the expressed antibodies binding to the KRAS G12V / HLA-A0301 complex could be verified by FACS (fluorescence-activated cell sorting). Exemplary antibodies obtained by this method include 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, and 2B12.

[0293] In another experiment, phage display was performed to screen for and discover monoclonal antibodies specific to the KRAS G12V / HLA-A0301 complex. P03141 was cited as an exemplary antibody obtained by this method.

[0294] The heavy and light chain variable regions of 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, 2B12, and P03141 are shown in Figure 5. Figures 2, 3, and 4 show the heavy and light chain CDRs of 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, 2B12, and P03141, respectively, under the definitions of Kabat and Chothia.

[0295] Various IgG1, IgG2, and IgG4 antibodies were produced. Regarding antibody naming, when the antibody VH / VL is linked to various isotypes, the isotype is added to the name. For example, when the VH and VL of 1A7 are linked to the constant region of IgG1, the antibody is named 1A7-IgG1 (or 1A7). Examples of other isotypes include 1A7-IgG2 and 1A7-IgG4. The constant region can also contain several mutations. For example, when SI mutations (EU numbering: S239D and I332E mutations) are introduced into the Fc region of 1A7-IgG1, the resulting antibody is named 1A7-IgG1-SI (or 1A7-SI).

[0296] Example 2. Binding affinity of anti-KRAS / HLA antibody The affinity of anti-KRAS / HLA antibodies for the KRAS G12V / HLA-A0301 complex was measured using a Biacore equipped with a pre-immobilized protein A sensor chip. TM Measurements were taken using surface plasmon resonance (SPR) with an 8K biosensor (Biacore, INC, Piscataway NJ).

[0297] Dilute the His-tagged human KRAS G12V (HLA-A*03:01) complex protein (Kactus Biosystems, catalog number: MHC-HM418) to 2 μg / mL, and then inject it into Biacore at a rate of 10 μL / min for approximately 50 seconds. TMThe desired protein density was achieved by injecting into an 8K biosensor. Purified anti-KRAS G12V / HLA antibody was then injected at concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, or 3.125 nM at a rate of 30 μL / min for 180 seconds. Dissociation was monitored for 400 seconds. After the final injection of each titration, the tip was refilled with glycine (pH 1.7, 30 μL / min for 30 seconds).

[0298] Biacore TM Using 8K Evaluation software 3.0, the entire dataset was fitted to a 1:1 Langmuir coupled model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) to simultaneously obtain kon and koff. The affinity was estimated from the quotient of the velocity constant (KD = koff / kon).

[0299] The same method, with appropriately adjusted parameters (e.g., antibody concentration) as those skilled in the art would understand, was performed for each test antibody. The results for each test antibody are shown in the table below.

[0300] [Table 1]

[0301] Under the steady-state affinity model, Biacore TM Affinity (KD) was obtained using 8K Evaluation Software 3.0. The results for the test antibody are shown in the table below.

[0302] V2 scFv is a monoclonal antibody targeting the KRAS G12V / HLA-A0301 complex, as described in Douglass et al., Sci.Immunol.6, eabd5515 (2021). The VH and VL sequences of V2 scFv are shown in SEQ ID NOs. 97 and 98, respectively. V2 was obtained by conjugating a V2 scFv analog to the human IgG1 constant region. Furthermore, V2-SI was obtained by introducing the SI mutation into the IgG1 constant region.

[0303] [Table 2]

[0304] The results showed that all anti-KRAS / HLA antibodies exhibited good binding affinity to the human KRAS G12V / HLA-A0301 complex. In particular, 1A7-SI, 1B6-SI, 1C10-SI, 1E12-SI, 1F1-SI, 1G1-SI, 1G10-SI, and 1H2-SI showed better affinity to the human KRAS G12V / HLA-A0301 complex than the positive control V2-SI.

[0305] In another experiment, the affinity of P03141-SI for the KRAS G12V / HLA-A0301 complex was measured using biolayer interferometry (BLI) with a ForteBio Octet system equipped with a pre-immobilized protein A sensor chip.

[0306] Purified anti-KRAS G12V / HLA antibody was diluted to 5 μg / mL and then captured at 1000 rpm for approximately 200 seconds. A 200 nM concentration of His-tagged human KRAS G12V (HLA-A*03:01) complex protein was then injected at 1000 rpm for 180 seconds. Dissociation was monitored for 600 seconds. After the final injection of each titration, the tip was refilled with glycine (pH 1.7, 1000 rpm for 30 seconds).

[0307] The results show that the kon(1 / Ms), koff(1 / s), and KD(M) values ​​of P03141-SI are 1.36E+05, 3.87E-03, and 2.852E-08, respectively, indicating good affinity for the human KRAS G12V / HLA-A0301 complex.

[0308] Example 3. Verification of the binding activity of anti-KRAS / HLA antibody to COS-7-HLA-A03 cells pulsed with KRAS peptide. The binding activity of anti-KRAS / HLA antibodies to COS-7-HLA-A03 cells (COS-7 cells expressing HLA-A03 (SEQ ID NO: 109)) pulsed with KRAS G12WT(7-16) peptide (SEQ ID NO: 79), KRAS G12V(7-16) peptide (SEQ ID NO: 80), KRAS G12C(7-16) peptide (SEQ ID NO: 81), or KRAS G12D(7-16) peptide (SEQ ID NO: 82) was verified by flow cytometry.

[0309] Specifically, COS-7-HLA-A03 cells were incubated with peptide (50 μM) for 1 hour at 37°C and 5% CO2. After washing, the cells were suspended for 30 minutes at 4°C in cold PBS containing serially diluted anti-KRAS / HLA antibodies (100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM), and subsequently incubated for 30 minutes with Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170).

[0310] Cells were collected and the mean fluorescence intensity (MFI) was determined. A fitting curve was obtained using antibody concentration (nM) on the X axis and MFI on the Y axis. The results are shown in Figures 6A to 6D.

[0311] The results showed that the anti-KRAS / HLA antibodies 1B6-SI, 1C10-SI, 1H2-SI, and 2B12-SI specifically bound to COS-7-HLA-A03 cells pulsed with the KRAS G12V(7-16) peptide.

[0312] In another similar experiment, the binding activity of P03141-SI or V2-SI to COS-7-HLA-A03 cells pulsed with KRAS G12WT(7-16) peptide or KRAS G12V(7-16) peptide was investigated. The results are shown in Figures 6E-6F, where the binding EC50 of P03141-SI and V2-SI to KRAS G12V(7-16) peptide was 13.96 nM and 169.4 nM, respectively. This indicates that P03141-SI exhibits specific binding to COS-7-HLA-A03 cells pulsed with KRAS G12V(7-16) peptide, and its binding activity is stronger than that of V2-SI.

[0313] Example 4. Measurement of the binding site of anti-KRAS / HLA antibody by alanine scan. To investigate the binding site of anti-KRAS / HLA antibodies to the KRAS G12V(7-16) / HLA-A03 complex, the recognition epitopes of the anti-KRAS / HLA antibodies were measured using an alanine scan assay. The amino acids at positions 1, 2, 3, 4, 6, 7, 8, 9, and 10 of KRAS G12V(7-16) were individually substituted with alanine to obtain a series of peptides (p.V1A, p.V2A, p.V3A, p.G4A, p.V6A, p.G7A, p.V8A, p.G9A, and p.K10A) (as shown in the table below). The binding of anti-KRAS / HLA antibodies (antibody concentration: 10 μg / mL) to COS-7-HLA-A03 cells pulsed with these peptides (peptide concentration: 50 μM) was measured using flow cytometry. The secondary antibody was Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170). The mean fluorescence intensity (MFI) results are shown in Figures 10A-10E.

[0314] [Table 3]

[0315] Example 5. Cross-binding activity of anti-KRAS / HLA antibodies Flow cytometry was used to verify the binding activity of anti-KRAS / HLA antibodies to COS-7-HLA-A03 cells pulsed with KRAS G12V(7-16) peptide, and to COS-7-HLA-A1101 cells (COS-7 cells expressing human HLA-A1101 (SEQ ID NO: 100)) pulsed with KRAS G12WT(7-16) peptide, KRAS G12WT(8-16) peptide (SEQ ID NO: 101), KRAS G12V(7-16) peptide, or KRAS G12V(8-16) peptide (SEQ ID NO: 102).

[0316] COS-7-HLA-A03 or COS-7-HLA-A1101 cells were incubated with peptide (50 μM) for 1 hour at 37°C in 5% CO2. After washing, the cells were suspended in cold PBS containing anti-KRAS / HLA antibody (antibody concentration: 10 μg / mL) at 4°C for 30 minutes, followed by incubation with Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170) for 30 minutes. The cells were harvested and the Myoplastic Inhibitory Factor (MFI) was determined.

[0317] The results showed that all anti-KRAS / HLA antibodies did not show cross-binding to any of the peptide / HLA-A1101 complexes, while the positive control V2-SI did show cross-binding to the KRAS G12V(7-16) / HLA-A1101 complex. Some exemplary MFI results for these anti-KRAS / HLA antibodies are shown in Figure 7.

[0318] Example 6. Binding of anti-KRAS / HLA antibodies to potential off-target peptides. Flow cytometry was used to verify the binding of anti-KRAS / HLA antibodies to KRAS G12V(7-16) and potential off-target peptides (sequence-similar peptides derived from MRAS protein, ERAS protein, Rab-7b protein, RhoJ protein, and mRho GTPase2 protein).

[0319] COS-7-HLA-A03 cells were incubated with 50 μM MRAS peptide, ERAS peptide, Rab-7b peptide, RhoJ peptide, and mRho GTPase2 peptide at 37°C and 5% CO2 for 1 hour. After washing, the cells were suspended in cold PBS and incubated with 10 μg / mL anti-KRAS G12V / HLA antibody at 4°C for 30 minutes. Subsequently, the cells were incubated with Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170). The results are shown in Figure 8A.

[0320] The sequences of MRAS peptide, ERAS peptide, Rab-7b peptide, RhoJ peptide, and mRho GTPase2 peptide are shown in the table below.

[0321] [Table 4]

[0322] The results showed that the anti-KRAS / HLA antibodies 1B6-SI, 1C10-SI, 1G1-SI, 1H2-SI, and 2B12-SI had good binding affinity to KRAS G12V(7-16). Furthermore, these antibodies did not bind to MRAS peptide, ERAS peptide, Rab-7b peptide, RhoJ peptide, or mRho GTPase2 peptide. However, the positive control V2-SI also bound to the Rab-7b peptide with a high MFI.

[0323] Therefore, antibodies 1B6-SI, 1C10-SI, 1G1-SI, 1H2-SI, and 2B12-SI showed specific binding to the KRAS G12V(7-16) / HLA-A03 complex.

[0324] In another similar experiment, the binding activity of P03141-SI to KRAS G12V(7-16) or potential off-target peptides (sequence-similar peptides derived from MRAS protein, Rab-7b protein, RhoJ protein, and mRho GTPase2 peptide) was examined. The results are shown in Figure 8B, where P03141-SI was shown to bind to KRAS G12V(7-16), but not to MRAS peptide, Rab-7b peptide, RhoJ peptide, or mRho GTPase2 peptide.

[0325] Example 7. Preparation of anti-KRAS / CD3 bispecific antibody Various bispecific antibodies having heavy chain and light chain sequences can be produced from anti-KRAS / HLA antibodies (e.g., 1A7, 1B6, 1C10, 1E12, 1F1, 1F3, 1F9, 1G1, 1G10, 1H2, 2B12, and P03141) and anti-CD3 antibodies described herein. Some exemplary structures of bispecific antibodies are shown in Figures 1A-1D.

[0326] Structural form I An anti-KRAS / CD3 bispecific antibody can be generated, having an anti-KRAS arm containing heavy and light chains, and an anti-CD3 arm containing a heavy chain variable domain of an anti-CD3 heavy chain antibody (VHH) (e.g., CD3 VHH, SEQ ID NO: 103) connected to the CH2 and CH3 domains of human IgG.

[0327] Many methods can be used to reduce the chance of incorrect pairing between the two heavy chains of a bispecific antibody. In the Fc region, knob-into-hole mutations were introduced into the anti-CD3 arm heavy chain and the anti-KRAS arm heavy chain. Exemplary antibody structures are shown in Figure 1A. Exemplary bispecific antibodies obtained include 1A7-CD3, 1B6-CD3, 1C10-CD3, 1E12-CD3, 1F1-CD3, 1G1-CD3, 1F3-CD3, 1F9-CD3, 1G10-CD3, 1H2-CD3, 2B12-CD3, and P03141-CD3. For example, 1A7-CD3 is a bispecific antibody with an IgG1 heavy chain constant region, where the constant region of the anti-CD3 arm (CD3) heavy chain contains a knob mutation, and the constant region of the anti-KRAS arm (1A7) heavy chain contains a hole mutation. The sequences of the light chain constant region, the heavy chain constant region with a knob mutation, and the heavy chain constant region with a hole mutation are shown in SEQ ID NOs: 104, 105, and 106, respectively.

[0328] Furthermore, the VH of the anti-KRAS arm can be substituted with the VH of V2, resulting in the anti-KRAS / CD3 bispecific antibody V2-CD3, which acts as a reference antibody.

[0329] Structural form II KRAS-scFv (1A7-scFv, 1B6-scFv, 1C10-scFv, 1E12-scFv, 1F1-scFv, 1G1-scFv, 1F3-scFv, 1F9-scFv, 1C10-scFv, 1H2-scFv, 2B12-scFv, or P03141-scFv) and CD3-scFv (VH SEQ ID NO: 121; VL SEQ ID NO: 122) can be paired to form bispecific antibodies.

[0330] The bispecific antibody is a BITE (bispecific T cell engager) molecule linked to the IgG1 Fc region (containing the N297G mutation). The BITE molecule consists of two single-stranded variable fragments (scFv) linked in a line by a flexible fusion linker; one scFv binds to the cell surface protein CD3, and the other scFv binds to the tumor cell surface antigen KRAS. An exemplary structure is shown in Figure 1B. Exemplary bispecific antibodies obtained include 1A7-CD3-SLE, 1B6-CD3-SLE, 1C10-CD3-SLE, 1E12-CD3-SLE, 1F1-CD3-SLE, 1G1-CD3-SLE, 1F3-CD3-SLE, 1F9-CD3-SLE, 1G10-CD3-SLE, 1H2-CD3-SLE, 2B12-CD3-SLE, and P03141-CD3-SLE.

[0331] Structural form III As shown in Figure 1C, it is possible to generate an anti-KRAS / CD3 bispecific antibody having a 2+1 IgG CrossFab inversion structure (changed to a CD3 binding domain, VH SEQ ID NO: 121; VL SEQ ID NO: 122) with a change in charge in the KRAS binding domain. The 2+1 IgG CrossFab inversion structure includes a first Fab molecule, a second Fab molecule, and a third Fab molecule. The first and third Fab molecules are identical and specifically bind to KRAS / HLA-A03, while the second Fab molecule specifically binds to CD3. The first Fab molecule fuses at the C-terminus of its heavy chain to the N-terminus of the heavy chain of the second Fab molecule. On the other hand, the second Fab molecule fuses at the C-terminus to the N-terminus of the first subunit of the Fc domain. The third Fab molecule fuses at the C-terminus to the N-terminus of the second subunit of this Fc domain.

[0332] The sequences of KRAS CH1, KRAS CL, KRAS CH1-Fc, and CD3 CH1-Fc are shown in sequence numbers 123 to 126, respectively.

[0333] Exemplary bispecific antibodies obtained include 1A7-CD3-SRY, 1B6-CD3-SRY, 1C10-CD3-SRY, 1E12-CD3-SRY, 1F1-CD3-SRY, 1G1-CD3-SRY, 1F3-CD3-SRY, 1F9-CD3-SRY, 1G10-CD3-SRY, 1H2-CD3-SRY, 2B12-CD3-SRY, and P03141-CD3-SRY.

[0334] Structural form IV As shown in Figure 1D, the anti-KRAS / CD3 bispecific antibody has an ScDb structure in which the VL domain and VH domain are arranged in the following order: VL(KRAS)-SL-VH(CD3, e.g., SEQ ID NO: 121)-LL-VL(CD3, e.g., SEQ ID NO: 122)-SL-VH(KRAS), where SL is a short linker (GGGGS; SEQ ID NO: 127); LL is a long linker (GGGGS) 3 (SEQ ID NO: 128).

[0335] Exemplary bispecific antibodies obtained included 1A7-CD3-ScDb, 1B6-CD3-ScDb, 1C10-CD3-ScDb, 1E12-CD3-ScDb, 1F1-CD3-ScDb, 1G1-CD3-ScDb, 1F3-CD3-ScDb, 1F9-CD3-ScDb, 1G10-CD3-ScDb, 1H2-CD3-ScDb, 2B12-CD3-ScDb, and P03141-CD3-ScDb. Chinese hamster ovary (CHO) cells were transfected with an anti-KRAS / CD3 bispecific antibody expression vector to express the bispecific antibodies. The CHO cell supernatant containing the anti-KRAS / CD3 bispecific antibodies was collected and purified by protein A affinity chromatography to obtain the anti-KRAS / CD3 bispecific antibodies.

[0336] Example 8. Cytotoxicity of anti-KRAS / CD3 bispecific antibody Anti-KRAS / CD3 bispecific antibodies (1A7-CD3, 1B6-CD3, 1C10-CD3, 1E12-CD3, 1F1-CD3, 1G1-CD3, 1F3-CD3, 1F9-CD3, 1G10-CD3, 1H2-CD3, 2B12-CD3, and V2-CD3) were serially diluted (3-fold) to the maximum concentration of 100 nM. Then, to test cell killing, purified effector cells (CD3+ T cells) and target cells (Raji cells expressing wild-type KRAS (ATCC, catalog number: CCL-86)) or KRAS-driven pancreatic cancer cells (CFPAC-1 cells expressing endogenous KRAS G12V (ATCC, catalog number: CRL-1918)) were co-incubated for 72 hours in a 5:1 ratio (E:T). Lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells was measured by a non-radioactive colorimetric assay using a cytotoxicity detection kit (ROCHE, catalog number: 04744926001). The results are shown in Figures 9A-9L.

[0337] The results showed that V2-CD3 had a limited killing effect (less than 10% cell lysis) in CFPAC-1. However, the anti-KRAS / CD3 bispecific antibodies 1A7-CD3, 1B6-CD3, 1C10-CD3, 1F1-CD3, 1G1-CD3, 1F3-CD3, 1F9-CD3, 1G10-CD3, 1H2-CD3, and 2B12-CD3 were able to kill CFPAC-1 cells but generally did not kill Raji cells expressing wild-type KRAS.

[0338] Example 9. In vitro killing activity of anti-KRAS / CD3 bispecific antibody. Detection of in vitro toxic activity by flow cytometry Anti-KRAS / CD3 bispecific antibodies (P03141-CD3, P03141-CD3-SRY, 1H2-CD3, or 1H2-CD3-SRY) were serially diluted (10-fold) to the maximum concentration of 100 nM, and then co-incubated with purified effector cells (CD3+ T cells) and target cells (RKO cells, CFPAC-1 cells, or NCI-H441 cells) in a 10:1 ratio (E:T) for 72 hours. Negative controls were designated as NC1 and NC2, respectively, either without antibody or with target cells only. RKO cells expressed wild-type KRAS, while NCI-H441 and CFPAC-1 cells expressed KRAS G12V. Killing activity was verified by flow cytometry. As shown in Figure 11, P03141-CD3, P03141-CD3-SRY, 1H2-CD3, and 1H2-CD3-SRY induced varying levels of tumor cell lysis in the CFPAC-1 and NCI-H441 groups, but they did not kill RKO cells even at the maximum concentration.

[0339] Furthermore, the percentage of CD137+ T cells was determined by flow cytometry analysis, and BioLegend LEGEND MAX TM Using the Human IFN-γ ELISA Kit (BioLegend, catalog number: 430107), T cell activation, including the secretion level of the cytokine IFN-γ, was determined. As shown in Figures 12 and 13, all bispecific antibody treatment groups significantly increased CD137+ T cells and stimulated IFN-γ secretion in a dose-dependent manner.

[0340] Detection of in vitro lethal activity by incucyte Anti-KRAS / CD3 bispecific antibodies (P03141-CD3-SLE or P03141-CD3-ScDb) were serially diluted (10-fold) to a maximum concentration of 100 nM, and then co-incubated with purified effector cells (CD3+ T cells) and target cells (CFPAC-1 cells or SW620-HLA-A03 cells (recombined to express HLA-A03 and KRAS G12V) in a 10:1 ratio (E:T) for 7 days. Cytotoxic activity was detected using IncuCyte (Sartorius AG, IncuCyte® S3). The results are shown in Figures 14A-14B. Here, P03141-CD3-SLE and P03141-CD3-ScDb showed good cytotoxicity at concentrations of 1 nM, 10 nM, and / or 100 nM.

[0341] Example 10. Binding activity of anti-KRAS / CD3 bispecific antibody The binding activity of anti-KRAS / HLA antibodies to modified Jarkat cells (Promega, catalog number: J1601) was investigated by flow cytometry.

[0342] Specifically, Jurkat-Luc-OX40 cells (transfected Jurkat-Luc cells expressing human OX40 protein) were transferred to a 96-well plate. Serially diluted anti-KRAS / CD3 antibody P03141-CD3-SLE was added to the 96-well plate and incubated at 4°C for 30 minutes. The same procedure was performed for each test antibody, with parameters (e.g., antibody concentration) appropriately adjusted as those skilled in the art would understand. The cells were then treated with the secondary antibody R-filicoerythrin AffiniPure. TMGoat anti-human IgG with F(ab')2 fragment, specifically targeting the Fcγ fragment (Jackson Immuno Research Laboratories, Inc., catalog number: 109-116-098), was incubated at 4°C in the dark for 30 minutes, followed by flow cytometry analysis. Human IgG1 protein was used as an ISO control. Cells were harvested, and the mean fluorescence intensity (MFI) was determined. A fitting curve was obtained using antibody concentration (nM) on the X axis and MFI on the Y axis. The results are shown in Figure 15. Here, P03141-CD3-SLE showed good binding activity to modified Jarkat cells.

[0343] Example 11. Antitumor activity in the NCI-H441 xenograft model The effect of the anti-KRAS / CD3 bispecific antibody P03141-CD3 on tumor growth in a lung cancer model was investigated. (Approximately 5 × 10⁻⁶) 6 Human PBMCs were injected (by IV injection) into B-NDG mice (Biocytogen, catalog number: B-CM-001). On the same day, 5 × 10 6 NCI-H441 cells were simultaneously injected into mice. Tumor volume was 50-150 mm². 3 When the tumor volume reached a certain level, the mice were randomly divided into different groups (6 mice per group) based on tumor volume. The mice were then administered intravenously (iv) with either P03141-CD3 or an equal volume of phosphate-buffered saline (PBS). The administration frequency was twice a week (a total of 6 doses).

[0344] Tumor volume was measured twice a week, and the body weight of the mice was also recorded. When the tumor volume of the mice reached 2000 mm³... 3 Euthanasia was performed when the condition was reached.

[0345] Measure the length of the long axis and short axis of the tumor, and calculate the tumor volume as 0.5 × (long axis) × (short axis). 2The following formula was used to calculate tumor growth inhibition (TGI): TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0. A t-test was performed for statistical analysis. P < 0.05 is the threshold for showing a statistically significant difference.

[0346] The results showed that the anti-KRAS / CD3 bispecific antibody P03141-CD3 exhibited good antitumor activity with a TGI% of 51.3% 28 days after grouping patients into lung cancer models.

[0347] Example 12. Antitumor activity The tumor growth effects of anti-KRAS / CD3 bispecific antibodies 1B6-CD3, 1G1-CD3, 2B12-CD3, and P03141-CD3 were tested in colorectal models or lung cancer models. Approximately 5 × 10⁻⁶ 6 Human PBMCs were injected into B-NDG mice (by IV injection), and on the same day, 5 × 10 6 Individual CFPAC-1 cells or SW620 cells were simultaneously injected into mice. The tumor volume in the mice was 200-250 mm². 3 When the tumor volume reached a certain level, the mice were randomly divided into different groups (6 mice per group) based on tumor volume. The mice were then administered intravenously (iv) either with an antibody or an equal volume of phosphate-buffered saline (PBS). The administration frequency was twice a week.

[0348] The results showed that all four anti-KRAS / CD3 bispecific antibodies exhibited good antitumor activity in colorectal cancer or lung cancer.

[0349] Example 13. Binding of anti-KRAS / HLA antibodies to potential off-target peptides. Potential off-target peptides were discovered using an X-amino acid scan (X-scan) assay when the anti-KRAS / HLA antibody P03141-SI recognizes the KRAS G12V protein. In the X-scan experiment, a series of peptides were obtained by individually substituting the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of KRAS G12V(7-16) with various other amino acids. Then, the binding of COS-7-HLA-A03 cells pulsed with these peptides (peptide concentration: 50 μM) to the anti-KRAS / HLA antibody (antibody concentration: 10 μg / mL) was measured using flow cytometry. The secondary antibody was Alexa Fluor® 647 anti-human IgG Fcγ. Cells were harvested and the mean fluorescence intensity (MFI) was determined. The results are shown in Figure 16.

[0350] Next, a set of mutant peptides exhibiting higher binding MFI than the original peptide was selected. These peptides were then selected based on several factors, including human expression, HLA-presented ability, and / or high affinity to pMHC using tools such as ScanProsite, MetMHCpan, and / or MetCTLpan. Subsequently, the binding of anti-KRAS / HLA antibodies to these peptides (potential off-target peptides) was verified.

[0351] Figure 17 shows the results of P03141-SI binding to KRAS G12V(7-16) and to the three potential off-target peptides mentioned above (FNDC7 peptide, 3IS57 peptide, and SMIM2 peptide in Table 5), as measured by flow cytometry.

[0352] [Table 5]

[0353] The results showed that P03141-SI specifically binds to KRAS G12V(7-16), but does not bind to KRAS G12WT(7-16), FNDC7 peptide, 3IS57 peptide, or SMIM2 peptide.

[0354] Other Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to a complex containing a KRAS (KRAS proto-oncogene, GTPase) peptide and an MHC molecule, A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR1, the VH CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR2, and the VH CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR3, and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR1, the VL CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR2, and the VL CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3, are one of the following: an antibody or its antigen-binding fragment: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 52, 53, and 54, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 5, and 6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 7, 8, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 14, and 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19, 20, and 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (9) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 26, and 27, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 58, 59, and 60, respectively; (10) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28, 29, and 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 52, 53, and 54, respectively; (11) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28, 29, and 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (12) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31, 32, and 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (13) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 34, 35, and 36, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (14) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 37, 38, and 39, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (15) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 40, 41, and 42, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (16) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 43, 44, and 45, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (17) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 46, 47, and 48, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; (18) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 50, and 51, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 58, 59, and 60, respectively; (19) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 110, 111, and 112, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively; and (20) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 113, 114, and 115, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55, 56, and 57, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 55, 56, and 57, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 7, 8, and 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 16, 17, and 18, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 58, 59, and 60, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 52, 53, and 54, respectively.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 37, 38, and 39, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

16. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 40, 41, and 42, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

17. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 43, 44, and 45, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

18. The antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 46, 47, and 48, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

19. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 49, 50, and 51, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 58, 59, and 60, respectively.

20. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 110, 111, and 112, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

21. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 113, 114, and 115, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof specifically binds to a complex containing a KRAS peptide and an MHC molecule.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the KRAS peptide contains valine at the position corresponding to Gly12 of human KRAS (SEQ ID NO: 108).

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the KRAS peptide comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

80.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, wherein the MHC is HLA (e.g., HLA-A3).

26. The antibody or antigen-binding fragment according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

27. The antibody or antigen-binding fragment according to any one of claims 1 to 26, wherein the antibody or antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, a human IgG2 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

28. Nucleic acids containing polynucleotides that encode polypeptides including the following: (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 71; (2) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, which comprises the amino acid sequences shown in SEQ ID NOs. 52, 53, and 54, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 61; (3) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO: 72; (4) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 62; (5) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO: 73; (6) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 63; (7) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO: 74; (8) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 64; (9) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 75; (10) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 65; (11) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 75; (12) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 66; (13) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 72; (14) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 16, 17, and 18, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 75; (15) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 67; (16) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 76; (17) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 68; (18) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 77; (19) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 69; (20) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 78; (21) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 58, 59, and 60, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 70; (22) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 71; (23) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 72; (24) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 73; (25) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 74; (26) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 75; (27) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 37, 38, and 39, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 75; (28) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 37, 38, and 39, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 72; (29) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 40, 41, and 42, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 75; (30) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 43, 44, and 45, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 76; (31) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 46, 47, and 48, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 77; (32) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 49, 50, and 51, respectively, wherein VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with VL comprising the amino acid sequence shown in SEQ ID NO. 78; (33) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 110, 111, and 112, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VL comprising the amino acid sequence shown in SEQ ID NO. 117; (34) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 113, 114, and 115, respectively, wherein the VH binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VL comprising the amino acid sequence shown in SEQ ID NO. 117; or (35) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively, wherein the VL binds to a complex comprising a KRAS peptide and an MHC molecule when paired with a VH comprising the amino acid sequence shown in SEQ ID NO.

116.

29. The nucleic acid according to claim 28, wherein when VH forms a pair with VL, it specifically binds to a complex containing the KRAS peptide and the MHC molecule, or when VL forms a pair with VH, it specifically binds to a complex containing the KRAS peptide and the MHC molecule.

30. The nucleic acid according to claim 28 or 29, wherein the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof, a human IgG2 heavy chain or fragment thereof, or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

31. The nucleic acid according to any one of claims 28 to 30, wherein the nucleic acid encodes a single-stranded variable fragment (scFv), a single-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

32. The nucleic acid according to any one of claims 28 to 31, wherein the nucleic acid is cDNA.

33. A vector comprising one or more nucleic acids as described in any one of claims 28 to 32.

34. The vector comprises two nucleic acids according to any one of claims 28 to 32, each encoding the VH region and the VL region that bind to a complex comprising a KRAS peptide and an MHC molecule.

35. A pair of vectors, each comprising one of the nucleic acids described in any one of claims 28 to 32, wherein the pair of vectors both encode the VH region and the VL region that bind to a complex comprising a KRAS peptide and an MHC molecule.

36. A cell comprising the vector according to claim 33 or 34, or the pair of vectors according to claim 35.

37. The cell according to claim 36, wherein the cell is a CHO cell.

38. A cell comprising one or more nucleic acids according to any one of claims 28 to 32.

39. A cell comprising two nucleic acids according to any one of claims 28 to 32.

40. The cell according to claim 39, wherein the two nucleic acids both encode the VH region and the VL region, and bind together to a complex comprising the KRAS peptide and the MHC molecule.

41. A method for producing an antibody or an antigen-binding fragment thereof, wherein the method is: (a) Culturing the cells according to any one of claims 36 to 40 under conditions sufficient to produce the antibody or the antigen-binding fragment, (b) recovering the antibody or antigen-binding fragment produced by the cells, method.

42. An antibody or antigen-binding fragment thereof that binds to a complex containing a KRAS peptide and an MHC molecule, An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) containing an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 61, and the selected VL sequence is sequence number 71; (2) The selected VH sequence is sequence number 62, and the selected VL sequence is sequence number 72; (3) The selected VH sequence is sequence number 63, and the selected VL sequence is sequence number 73; (4) The selected VH sequence is sequence number 64, and the selected VL sequence is sequence number 74; (5) The selected VH sequence is sequence number 65, and the selected VL sequence is sequence number 75; (6) The selected VH sequence is sequence number 66, and the selected VL sequence is sequence number 75; (7) The selected VH sequence is sequence number 66, and the selected VL sequence is sequence number 72; (8) The selected VH sequence is sequence number 67, and the selected VL sequence is sequence number 75; (9) The selected VH sequence is sequence number 68, and the selected VL sequence is sequence number 76; (10) The selected VH sequence is sequence number 69, and the selected VL sequence is sequence number 77; (11) The selected VH sequence is sequence number 70, and the selected VL sequence is sequence number 78; and (12) The selected VH sequence is sequence number 116, and the selected VL sequence is sequence number 117.

43. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 61 and VL comprises the sequence of SEQ ID NO:

71.

44. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 62 and VL comprises the sequence of SEQ ID NO:

72.

45. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 63 and VL comprises the sequence of SEQ ID NO:

73.

46. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 64 and VL comprises the sequence of SEQ ID NO:

74.

47. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 65 and VL comprises the sequence of SEQ ID NO:

75.

48. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 66 and VL comprises the sequence of SEQ ID NO:

75.

49. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 66 and VL comprises the sequence of SEQ ID NO:

72.

50. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 67 and VL comprises the sequence of SEQ ID NO:

75.

51. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 68 and VL comprises the sequence of SEQ ID NO:

76.

52. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 69 and VL comprises the sequence of SEQ ID NO:

77.

53. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 70 and VL comprises the sequence of SEQ ID NO:

78.

54. The antibody or antigen-binding fragment thereof according to claim 42, wherein VH comprises the sequence of SEQ ID NO: 116 and VL comprises the sequence of SEQ ID NO:

117.

55. An antibody or antigen-binding fragment thereof that binds to a complex containing a KRAS peptide and an MHC molecule, A heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, An antibody or antigen-binding fragment comprising a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 61, and the selected VL sequence is sequence number 71; (2) The selected VH sequence is sequence number 62, and the selected VL sequence is sequence number 72; (3) The selected VH sequence is sequence number 63, and the selected VL sequence is sequence number 73; (4) The selected VH sequence is sequence number 64, and the selected VL sequence is sequence number 74; (5) The selected VH sequence is sequence number 65, and the selected VL sequence is sequence number 75; (6) The selected VH sequence is sequence number 66, and the selected VL sequence is sequence number 75; (7) The selected VH sequence is sequence number 66, and the selected VL sequence is sequence number 72; (8) The selected VH sequence is sequence number 67, and the selected VL sequence is sequence number 75; (9) The selected VH sequence is sequence number 68, and the selected VL sequence is sequence number 76; (10) The selected VH sequence is sequence number 69, and the selected VL sequence is sequence number 77; (11) The selected VH sequence is sequence number 70, and the selected VL sequence is sequence number 78; and (12) The selected VH sequence is sequence number 116, and the selected VL sequence is sequence number 117.

56. The antibody or antigen-binding fragment according to any one of claims 42 to 55, wherein the antibody or antigen-binding fragment specifically binds to a complex containing a KRAS peptide and an MHC molecule.

57. The antibody or antigen-binding fragment thereof according to any one of claims 42 to 56, wherein the KRAS peptide contains valine at the position corresponding to Gly12 of human KRAS (SEQ ID NO: 108).

58. The antibody or antigen-binding fragment thereof according to any one of claims 42 to 57, wherein the KRAS peptide comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

80.

59. The antibody or antigen-binding fragment thereof according to any one of claims 42 to 58, wherein the MHC is HLA (for example, HLA-A3).

60. The antibody or antigen-binding fragment according to any one of claims 42 to 59, wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

61. The antibody or antigen-binding fragment according to any one of claims 42 to 60, wherein the antibody or antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, a human IgG2 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

62. An antibody or an antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment described in any one of claims 1 to 27 and 42 to 60.

63. The antibody or antigen-binding fragment according to any one of claims 1 to 27 and 42 to 62, wherein the antibody or antigen-binding fragment thereof includes a fragment crystallizable region (Fc region).

64. A protein construct that binds to a complex containing KRAS peptide and MHC molecules, （１） A first functional portion comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, and (2) Second functional part containing T cell binding molecules A protein construct that includes [this component].

65. The protein construct according to claim 64, wherein the T cell binding molecule (e.g., VHH or scFv) targets human CD3.

66. The protein construct according to claim 64 or 65, wherein the KRAS peptide comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

80.

67. The protein construct according to any one of claims 64 to 66, wherein the MHC is HLA (for example, HLA-A3).

68. The protein construct according to any one of claims 64 to 67, wherein the first functional portion and the second functional portion are connected via a linker.

69. Protein constructs, （１） A first functional portion comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, (2) A second functional portion containing a T cell binding molecule, and (3) A third functional part including a single-stranded human crystallizable fragment A protein construct that includes [this component].

70. The T cell binding molecule is an scFv or VHH that targets human CD3. The protein construct according to claim 69.

71. The protein construct according to claim 69 or 70, wherein the first functional portion, the second functional portion, and the third functional portion are connected via one or more linkers.

72. The protein construct according to any one of claims 69 to 71, wherein the KRAS peptide comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

80.

73. The protein construct according to any one of claims 69 to 72, wherein the MHC is HLA (for example, HLA-A3).

74. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, or a protein construct according to any one of claims 64 to 73, covalently bound to a therapeutic agent.

75. The antibody-drug conjugate according to claim 74, wherein the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

76. The antibody-drug conjugate according to any one of claims 74 or 75, wherein the drug-antibody ratio (DAR) is approximately 4.

77. A recombinant receptor comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62.

78. The recombinant receptor further comprises a transmembrane domain and an intracellular signaling domain. The recombinant receptor according to claim 77.

79. The recombinant receptor according to claim 77 or 78, wherein the recombinant receptor is a chimeric antigen receptor ("CAR").

80. The recombinant receptor according to claim 79, wherein the recombinant receptor is a chimeric T cell receptor (chimeric TCR or "cTCR").

81. A polynucleotide encoding a recombinant receptor according to any one of claims 77 to 80.

82. A vector comprising the polynucleotide described in claim 81.

83. The vector according to claim 82, wherein the vector is a viral vector.

84. Recombinant cells expressing the recombinant receptor according to any one of claims 77 to 80.

85. The recombinant cell according to claim 84, wherein the recombinant cell is an immune cell.

86. The recombinant cell according to claim 85, wherein the immune cell is an NK cell or a T cell.

87. The recombinant cell according to claim 86, wherein the recombinant cell is a T cell.

88. The recombinant cells according to claim 87, wherein the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T (NK-T) cells, and γδ T cells.

89. A method for producing recombinant cells, comprising introducing the vector described in claim 82 or 83 into cells in vitro or outside of a living organism.

90. The method according to claim 89, wherein the vector is a viral vector, and the introduction is carried out by transduction.

91. A method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, a protein construct according to any one of claims 64 to 73, an antibody-drug conjugate according to any one of claims 74 to 76, or recombinant cells according to any one of claims 84 to 88.

92. The method according to claim 91, wherein the cancer comprises one or more cancer cells expressing KRAS G12V.

93. The method according to claim 91 or 92, wherein the subject has a solid tumor.

94. The method according to any one of claims 91 to 93, wherein the cancer is pancreatic cancer, colorectal cancer, non-small cell lung cancer (NSCLC), ovarian cancer, esophageal cancer, or bile duct cancer.

95. The method according to any one of claims 91 to 94, further comprising administering to the subject a therapeutically effective amount of anti-OX40 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

96. A method for reducing the rate of tumor growth, wherein the method is A method comprising contacting tumor cells with a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, a protein construct according to any one of claims 64 to 73, an antibody-drug conjugate according to any one of claims 74 to 76, or recombinant cells according to any one of claims 84 to 88.

97. A method for killing tumor cells, wherein the method is A method comprising contacting tumor cells with a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, a protein construct according to any one of claims 64 to 73, an antibody-drug conjugate according to any one of claims 74 to 76, or recombinant cells according to any one of claims 84 to 88.

98. A method for increasing the immune response in a target, wherein the method is A method comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, a protein construct according to any one of claims 64 to 73, an antibody-drug conjugate according to any one of claims 74 to 76, or recombinant cells according to any one of claims 84 to 88.

99. A pharmaceutical composition, A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and 42 to 62, a protein construct according to any one of claims 64 to 73, an antibody-drug conjugate according to any one of claims 74 to 76, or a recombinant cell according to any one of claims 84 to 88, and a pharmaceutically acceptable carrier.

100. An antibody or antigen-binding fragment thereof that binds to a complex containing a KRAS peptide and an MHC molecule, wherein the antibody or antigen-binding fragment specifically binds to an epitope in the KRAS peptide, and the epitope is an amino acid residue corresponding to Val6 of SEQ ID NO: 80.

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