Antigen-binding molecules that specifically bind to EGFR and MUC1, drug conjugates thereof, and their pharmaceutical use

Abstract

This disclosure relates to antigen-binding molecules that specifically bind to EGFR and MUC1, drug conjugates thereof, and their pharmaceutically useful applications. Specifically, this disclosure relates to anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments, antigen-binding molecules that specifically bind to EGFR and MUC1, and conjugates thereof with drugs, and their pharmaceutically useful applications.

Description

[Technical Field]

[0001] This disclosure claims priority over CN202310394160.8, filed on 13 April 2023, and CN202311772280.3, filed on 21 December 2023.

[0002] This disclosure belongs to the field of biotechnology and relates to anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments, antigen-binding molecules that specifically bind to EGFR and MUC1, and conjugates thereof with drugs and their pharmaceutically acceptable uses. [Background technology]

[0003] The information provided herein does not necessarily constitute prior art, but merely provides background information related to this disclosure.

[0004] MUC1 is a transmembrane glycoprotein with abundant glycosylation. Its extracellular domain is a dimer formed by the interaction of two chains via hydrogen bonds, consisting of MUC1-N and MUC1-C. MUC1-N has abundant O-glycosylation and a small amount of N-glycosylation, and its amino acid backbone consists of multiple VNTR repeats. MUC1-C contains an extracellular domain, a transmembrane domain, and an intracellular domain.

[0005] In normal tissue, MUC1 is located at the apex of epithelial cells in its full-length form, while EGFR is located at the basal end of epithelial cells. Tumor cells lose their apical-basal polarity, so EGFR and MUC1 are uniformly distributed on the surface of the tumor cell, and this phenomenon causes EGFR and MUC1 to be spatially closer together. In addition, the O-glycosylation of MUC1-N in tumor cells is clearly less pronounced. As the tumor progresses, MUC1-N is shed through the catalytic action of inflammatory factor-related enzymes in the tumor microenvironment, exposing MUC1-C.

[0006] MUC1-N is released into the bloodstream, causing off-target effects for antibodies targeting MUC1-N. This has been the main reason why MUC1-N-targeted antibodies have not been clinically effective to date, and clinical data on MUC1-C-targeted antibodies are extremely limited. This disclosure describes an antibody that targets MUC1-C, has no off-target effects, and can serve as a beneficial complement to MUC1-C antibodies.

[0007] Because EGFR is expressed at a certain level even in normal tissues, clinically available EGFR monoclonal antibodies have clear safety issues and limit their efficacy. The EGFR-MUC1 biantibody disclosed herein preferentially targets biexpressing tumor cells, reduces antibody binding to normal tissues, and couples to low-molecular-weight toxins, thereby reducing side effects and improving efficacy. Furthermore, compared to EGFR monoclonal antibodies or MUC1 monoclonal antibodies, the EGFR-MUC1 biantibody has a broader range of indications and patient populations. [Overview of the project] [Problems that the invention aims to solve]

[0008] This disclosure provides an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to MUC1-C of human MUC1 but does not bind to MUC1-N.

[0009] This disclosure provides an anti-MUC1 antibody or its antigen-binding fragment comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3, and a light chain variable region including LCDR1, LCDR2, and LCDR3, among which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 4, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5, or b. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of either SEQ ID NO: 6 or 47, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 7, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 8, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 9, or d. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 of any one of sequence numbers 63, 10, or 64, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 of sequence number 11.

[0010] In some embodiments, the above-mentioned anti-MUC1 antibody or its antigen-binding fragment, among them, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 4, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5, or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 6, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 7, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 8, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 9, or d. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 63, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11.

[0011] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 47, respectively, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 7, respectively.

[0012] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 10, respectively, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11, respectively.

[0013] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 64, respectively, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11, respectively.

[0014] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the same numbering rules selected from Kabat, IMGT, Chothia, AbM, and Contact.

[0015] In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Chothia numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the AbM numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Contact numbering rules.

[0016] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, a. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17, or b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 18, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains either the amino acid sequence of SEQ ID NO: 20 or 113, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 21, LCDR2 contains the amino acid sequence of SEQ ID NO: 22, and LCDR3 contains the amino acid sequence of SEQ ID NO: 23, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 24, HCDR2 contains the amino acid sequence of SEQ ID NO: 25, and HCDR3 contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 27, LCDR2 contains the amino acid sequence of SEQ ID NO: 28, and LCDR3 contains the amino acid sequence of SEQ ID NO: 29, or d. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 30, HCDR2 contains the amino acid sequence of SEQ ID NO: 31, and HCDR3 contains any one of the amino acid sequences of SEQ ID NO: 114, 32, or 115; and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 34, and LCDR3 contains the amino acid sequence of SEQ ID NO: 35.

[0017] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, a. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17. b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 18, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains the amino acid sequence of SEQ ID NO: 20, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 21, LCDR2 contains the amino acid sequence of SEQ ID NO: 22, and LCDR3 contains the amino acid sequence of SEQ ID NO: 23, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 24, HCDR2 contains the amino acid sequence of SEQ ID NO: 25, and HCDR3 contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 27, LCDR2 contains the amino acid sequence of SEQ ID NO: 28, and LCDR3 contains the amino acid sequence of SEQ ID NO: 29, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 30 for HCDR1, the amino acid sequence of SEQ ID NO: 31 for HCDR2, and the amino acid sequence of SEQ ID NO: 114 for HCDR3, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 33 for LCDR1, the amino acid sequence of SEQ ID NO: 34 for LCDR2, and the amino acid sequence of SEQ ID NO: 35 for LCDR3.

[0018] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, and HCDR3 containing the amino acid sequence of SEQ ID NO: 113, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 23.

[0019] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 30, HCDR2 containing the amino acid sequence of SEQ ID NO: 31, and HCDR3 containing the amino acid sequence of SEQ ID NO: 115, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 33, LCDR2 containing the amino acid sequence of SEQ ID NO: 34, and LCDR3 containing the amino acid sequence of SEQ ID NO: 35.

[0020] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 30, HCDR2 containing the amino acid sequence of SEQ ID NO: 31, and HCDR3 containing the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 33, LCDR2 containing the amino acid sequence of SEQ ID NO: 34, and LCDR3 containing the amino acid sequence of SEQ ID NO: 35.

[0021] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17.

[0022] In some embodiments, the anti-MUC1 antibody described in any one of the above descriptions or its antigen-binding fragment is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, it is a chimeric antibody or a humanized antibody. In some embodiments, it is a humanized antibody.

[0023] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, comprising a framework region (FR) of a human antibody.

[0024] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, a. The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 36, 37, or 38, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 39, 40, 41, or 42, or The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 4, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 5, or b. The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 43, 44, 45, 46, 47, 48, or 49, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 50, 51, or 52, or The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 6, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 7, or c. The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 53, 54, or 55, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 56, 57, 58, or 59, or The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 8, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 9, or d. The heavy chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 63, 60, 61, 62, or 64, and the light chain variable region contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 67, 68, 65, or 66, or The heavy chain variable region includes an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 10, and the light chain variable region includes an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NO: 11.

[0025] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 38K, 40R, 48I, 71A, 73K, 76D, and 82aR, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV1-39*01, 1GKV6-21*02, or IGKV3-11*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 3V, 43S, 47W, 49Y, and 60G. In some embodiments, the above-mentioned anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 38K, 40R, 48I, 71A, 73K, 76D, and 82aR, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 3V, 43S, 47W, 49Y, and 60G.In some embodiments, the above-mentioned anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 38K, 40R, 48I, 71A, 73K, 76D, and 82aR, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 3V, 43S, and 47W. In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the FR of the heavy chain variable region comprises the amino acid substitutions 1E, 71A, 73K, and 76D, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17, and the FR of the light chain variable region comprises the amino acid substitutions 43S and 47W. In some embodiments, the variable regions and CDRs are defined according to Kabat numbering rules.

[0026] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 28R, 30I, 39E, 40R, 43H, 69F, 71A, 76N, 82bQ, 83T, and 84N, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV4-1*01 or IGKV3-11*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 4M, 45K, 68R, and 83V. In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region is such that HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 20 or 113, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1E, 28R, 30I, 39E, 40R, 43H, 69F, 71A, 76N, 82bQ, 83T, and 84N, and the light chain variable region is such that LCDR1 comprises the amino acid sequence of SEQ ID NO: 21, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 23, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1D, 4M, 45K, 68R, and 83V. In some embodiments, the variable regions and CDRs are defined according to Kabat numbering rules.

[0027] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-3*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 2I, 12V, 40R, 44G, 47Y, 48I, 69L, 71V, and 76R, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV1-39*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 36F, 42T, 43S, 47W, 60P, 70S, and 75V. In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 25, and HCDR3 containing the amino acid sequence of SEQ ID NO: 26, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1E, 2I, 12V, 40R, 44G, 47Y, 48I, 69L, 71V, and 76R, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 27, LCDR2 containing the amino acid sequence of SEQ ID NO: 28, and LCDR3 containing the amino acid sequence of SEQ ID NO: 29, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 4L, 36F, 42T, 43S, 47W, 60P, 70S, and 75V. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0028] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-3*01 and FR4 derived from IGHJ1*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 12V, 20M, 24T, 40R, 44G, 48I, 69L, and 71S, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV1-39*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 36L, 39E, 42G, 44I, 46R, 60K, 66R, 69S, and 71Y. In some embodiments, the above-mentioned anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 30, HCDR2 containing the amino acid sequence of SEQ ID NO: 31, and HCDR3 containing the amino acid sequence of SEQ ID NO: 114, 32, or 115, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 12V, 20M, 24T, 40R, 44G, 48I, 69L, and 71S, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 33, LCDR2 containing the amino acid sequence of SEQ ID NO: 34, and LCDR3 containing the amino acid sequence of SEQ ID NO: 35, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 4L, 36L, 39E, 42G, 44I, 46R, 60K, 66R, 69S, and 71Y.In some embodiments, the above-mentioned anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 30, HCDR2 containing the amino acid sequence of SEQ ID NO: 31, and HCDR3 containing the amino acid sequence of SEQ ID NO: 114, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 12V, 20M, 24T, 40R, 44G, 48I, 69L, and 71S, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 33, LCDR2 containing the amino acid sequence of SEQ ID NO: 34, and LCDR3 containing the amino acid sequence of SEQ ID NO: 35, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 4L, 36L, 39E, 42G, 44I, 46R, 60K, 66R, 69S, and 71Y. In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 30, HCDR2 containing the amino acid sequence of SEQ ID NO: 31, and HCDR3 containing the amino acid sequence of SEQ ID NO: 115, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 12V, 20M, 24T, 40R, 44G, 48I, 69L, and 71S, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 33, LCDR2 containing the amino acid sequence of SEQ ID NO: 34, and LCDR3 containing the amino acid sequence of SEQ ID NO: 35, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 4L, 36L, 39E, 42G, 44I, 46R, 60K, 66R, 69S, and 71Y. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0029] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39 or 40, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 37, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39 or 40, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 4, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 5, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 43, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 50, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 44, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 51, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 45, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 50 or 51, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 46, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 51, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 6, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 7, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 53, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 56, 57, or 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 54, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 57, or Alternatively, the heavy chain variable region may include the amino acid sequence of SEQ ID NO: 8, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 9, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 60, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 65 or 68, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 61, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 65, 66, 67, or 68, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 63, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 67 or 68, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 10, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 11.

[0030] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 40, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 37, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 37, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 40, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 4, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 5.

[0031] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 36, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39.

[0032] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, a. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39 or 40, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39 or 40, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5, or b. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 50, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 51, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 50 or 51, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 51, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7, or c. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 56, 57, or 59, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 57, or Alternatively, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9, or d. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 60, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 65 or 68, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain variable region is shown in SEQ ID NOs: 65, 66, 67, or 68, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 63, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 67 or 68, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11.

[0033] In some embodiments, an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5.

[0034] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39.

[0035] In some embodiments, the anti-MUC1 antibody described in any one of the above descriptions or an antigen-binding fragment thereof, which is an antibody fragment, and in some embodiments, the antibody fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, and dAb.

[0036] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the light chain constant region is a human κ or λ light chain constant region.

[0037] In some embodiments, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 69 or 186, and the light chain constant region includes the amino acid sequence of SEQ ID NO: 70.

[0038] In some embodiments, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 69, and the light chain constant region includes the amino acid sequence of SEQ ID NO: 70.

[0039] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain and a light chain, of which, a. The heavy chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 71, 73, 75, or 77, and the light chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 72, 74, 76, or 78, or b. The heavy chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 79, 81, 83, 85, or 87, and the light chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 80, 82, 84, 86, or 88, or c. The heavy chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 89, 91, 93, or 95, and the light chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 90, 92, 94, or 96, or d. The heavy chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 97, 99, 101, 103, 105, 107, 109, or 111, and the light chain contains an amino acid sequence having at least 70% (for example, at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity with SEQ ID NOs. 98, 100, 102, 104, 106, 108, 110, or 112.

[0040] In some embodiments, an anti-MUC1 antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain and a light chain, of which, a. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 73, and the amino acid sequence of the light chain is shown in SEQ ID NO: 74, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 75, and the amino acid sequence of the light chain is shown in SEQ ID NO: 76, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain is shown in SEQ ID NO: 78. b. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 79, and the amino acid sequence of the light chain is shown in SEQ ID NO: 80, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 81, and the amino acid sequence of the light chain is shown in SEQ ID NO: 82, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 83, and the amino acid sequence of the light chain is shown in SEQ ID NO: 84, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 85, and the amino acid sequence of the light chain is shown in SEQ ID NO: 86, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 87, and the amino acid sequence of the light chain is shown in SEQ ID NO: 88. c. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 90, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 92, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 93, and the amino acid sequence of the light chain is shown in SEQ ID NO: 94, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 95, and the amino acid sequence of the light chain is shown in SEQ ID NO: 96. d. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 97, and the amino acid sequence of the light chain is shown in SEQ ID NO: 98, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 99, and the amino acid sequence of the light chain is shown by SEQ ID NO: 100, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 101, and the amino acid sequence of the light chain is shown by SEQ ID NO: 102, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 103, and the amino acid sequence of the light chain is shown by SEQ ID NO: 104, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 105, and the amino acid sequence of the light chain is shown by SEQ ID NO: 106, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 107, and the amino acid sequence of the light chain is shown by SEQ ID NO: 108, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 109, and the amino acid sequence of the light chain is shown by SEQ ID NO: 110, or The amino acid sequence of the heavy chain is shown by SEQ ID NO: 111, and the amino acid sequence of the light chain is shown by SEQ ID NO: 112.

[0041] In some embodiments, it is the anti-MUC1 antibody or antigen-binding fragment thereof described in any one of the above, which comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown by SEQ ID NO: 71, and the amino acid sequence of the light chain is shown by SEQ ID NO: 72.

[0042] In some embodiments, the present disclosure further provides an isolated anti-MUC1 antibody or antigen-binding fragment thereof that competitively binds to human MUC1 with the anti-MUC1 antibody described in any one of the above.

[0043] In some embodiments, the isolated anti-MUC1 antibody or antigen-binding fragment thereof according to the present disclosure is 5×10 -9 ,

[0042] , -8 , , -8 , -8 , , , -9 , -8 , -8 , , -8 , -8 ,

[0043] , M or less (e.g., 4×10 -8 M or less, 3×10 [[ID=​​​​​​​​​​​​​-9 Less than M, 6 x 10 -9 Less than M, 5 x 10 -9 Less than M, 4 x 10 -9 Less than M, 3 x 10 -9 Less than M, 2 x 10 -9 It binds to human MUC1 with a KD value (less than M), and the above KD value is measured by Biacore.

[0044] In another embodiment, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, among which, a. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, 128, 130, or 131, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126 or 133, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 127, 132, or 134, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or d. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126, HCDR2 contains the amino acid sequence of SEQ ID NO: 127, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121. e. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 128, 129, 130, or 131, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121.

[0045] In some embodiments, the above-mentioned anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain variable region including HCDR1, HCDR2, and HCDR3, and a light chain variable region including LCDR1, LCDR2, and LCDR3, of which, a. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 128 or 130, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 133 for HCDR1, the amino acid sequence of SEQ ID NO: 117 for HCDR2, and the amino acid sequence of SEQ ID NO: 118 for HCDR3, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 119 for LCDR1, the amino acid sequence of SEQ ID NO: 120 for LCDR2, and the amino acid sequence of SEQ ID NO: 121 for LCDR3.

[0046] In some embodiments, an anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 116, HCDR2 comprising the amino acid sequence of SEQ ID NO: 117, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 129, and the light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 119, LCDR2 comprising the amino acid sequence of SEQ ID NO: 120, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 121.

[0047] In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof described in any one of the above, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the same numbering rules selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, it is defined according to the Kabat numbering rules. In some embodiments, it is defined according to the IMGT numbering rules. In some embodiments, it is defined according to the Chothia numbering rules. In some embodiments, it is defined according to the AbM numbering rules. In some embodiments, it is defined according to the Contact numbering rules.

[0048] In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, it is a chimeric antibody or a humanized antibody. In some embodiments, it is a humanized antibody.

[0049] In some embodiments, an anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, comprising a framework region (FR) of a human antibody.

[0050] In some embodiments, an anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of SEQ ID NOs: 138, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 149. Preferably, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 138, 142, 144, or 147, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 149. More preferably, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 138, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 149.

[0051] In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, which is an antibody fragment, and in some embodiments, the antibody fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv and dAb.

[0052] In some embodiments, an anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the light chain constant region is a human κ or λ light chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 69 or 186, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 69, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 70.

[0053] In some embodiments, an anti-EGFR antibody or antigen-binding fragment thereof as described in any one of the above, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NOs: 153, 150, 151, 152, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163, and the light chain comprises the amino acid sequence of SEQ ID NOs: 164. Preferably, the heavy chain contains the amino acid sequence of SEQ ID NO: 153, 157, 159, or 162, and the light chain contains the amino acid sequence of SEQ ID NO: 164. More preferably, the heavy chain comprises the amino acid sequence of SEQ ID NO: 153, and the light chain comprises the amino acid sequence of SEQ ID NO: 164.

[0054] In some embodiments, the disclosure further provides isolated anti-EGFR antibodies or antigen-binding fragments thereof that competitively bind to human EGFR with any one of the anti-EGFR antibodies described above.

[0055] In another embodiment, the Disclosure provides an antigen-binding molecule that specifically binds to EGFR and MUC1, comprising at least one antigen-binding module that specifically binds to EGFR and at least one antigen-binding module that specifically binds to MUC1, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL, of which, a. The above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, 128, 130, or 131, and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or b. The above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 126 or 133, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or c. The above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 127, 132, or 134, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or d. The above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 127, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or e. The above EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 128, 129, 130, or 131, and the above EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121.

[0056] In some embodiments, antigen-binding molecules that specifically bind to the above-mentioned EGFR and MUC1, among which, a. The above EGFR-VH is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, and the above EGFR-VL is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 128 or 130, and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or c. The above EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 133, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the above EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121.

[0057] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121.

[0058] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein MUC1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and MUC1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17.

[0059] In some embodiments, an antigen-binding molecule that specifically binds to any one of the EGFR and MUC1 described above, among which, The above EGFR-VH contains the amino acid sequences of SEQ ID NOs. 138, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148, and the above EGFR-VL contains the amino acid sequence of SEQ ID NO. 149. Preferably, the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, 142, 144, or 147, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149. More preferably, EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149.

[0060] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein MUC1-VH comprises the amino acid sequence of SEQ ID NO: 36, and MUC1-VL comprises the amino acid sequence of SEQ ID NO: 39.

[0061] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, wherein the antigen-binding module that specifically binds to EGFR or the antigen-binding module that specifically binds to MUC1 independently comprises a titin chain and an obscurin chain capable of forming a dimer (from WO2022237882A1).

[0062] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 165, and the obscurin chain comprises the amino acid sequence of SEQ ID NO: 166.

[0063] In some embodiments, an antigen-binding molecule that specifically binds to any one of the EGFR and MUC1 described above, further comprising an Fc region, the Fc region preferably being an IgG Fc region, more preferably being an IgG1 Fc region, and more preferably comprising one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor.

[0064] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, further comprising an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, and Fc1 and Fc2 each independently comprising one or more amino acid substitutions that reduce homodimerization of the Fc region. In the context of this application, when Fc1 and Fc2 are referred to, it should be understood that they function in the formation of a dimer and therefore Fc1 and Fc2 are interchangeable.

[0065] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 has a knob structure by knob-in-hole technology and Fc2 has a hole structure by knob-in-hole technology, or vice versa, where Fc2 has a knob structure by knob-in-hole technology and Fc1 has a hole structure by knob-in-hole technology.

[0066] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 has a knob structure by knob-in-hole technology, and Fc2 has a hole structure by knob-in-hole technology.

[0067] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 has an amino acid C at position 358 and an amino acid W at position 370, and Fc2 has an amino acid C at position 357, an amino acid S at position 374, an amino acid A at position 376, and an amino acid V at position 415, and is numbered according to the EU index. Alternatively, the reverse is also true, where Fc2 has an amino acid C at position 358 and an amino acid W at position 370, and Fc1 has an amino acid C at position 357, an amino acid S at position 374, an amino acid A at position 376, and an amino acid V at position 415, and is numbered according to the EU index.

[0068] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 has an amino acid C at position 358 and an amino acid W at position 370, and Fc2 has an amino acid C at position 357, an amino acid S at position 374, an amino acid A at position 376, and an amino acid V at position 415, and is numbered according to the EU index.

[0069] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 contains the amino acid sequence of SEQ ID NO: 169 and Fc2 contains the amino acid sequence of SEQ ID NO: 170. Alternatively, the reverse is also true, where Fc2 contains the amino acid sequence of SEQ ID NO: 169 and Fc1 contains the amino acid sequence of SEQ ID NO: 170.

[0070] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 169 and Fc2 comprises the amino acid sequence of SEQ ID NO: 170.

[0071] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 182 and Fc2 comprises the amino acid sequence of SEQ ID NO: 183. Alternatively, the reverse is also true, where Fc2 comprises the amino acid sequence of SEQ ID NO: 182 and Fc1 comprises the amino acid sequence of SEQ ID NO: 183.

[0072] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 182 and Fc2 comprises the amino acid sequence of SEQ ID NO: 183.

[0073] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to EGFR and one antigen-binding module that specifically binds to MUC1.

[0074] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to EGFR and one antigen-binding module that specifically binds to MUC1, the antigen-binding module that specifically binds to MUC1 is a Fab, and the antigen-binding module that specifically binds to EGFR is a replaced Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer, or the antigen-binding module that specifically binds to EGFR is a Fab, and the antigen-binding module that specifically binds to MUC1 is a replaced Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer.

[0075] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to EGFR and one antigen-binding module that specifically binds to MUC1, the antigen-binding module that specifically binds to MUC1 is a Fab, and the antigen-binding module that specifically binds to EGFR is a replaced Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer.

[0076] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, wherein the antigen-binding molecule comprises one first chain having a structure represented by formula (a), one second chain having a structure represented by formula (b), one third chain having a structure represented by formula (c), and one fourth chain having a structure represented by formula (d). Formula (a) [MUC1-VH]-[CH1]-[Fc1], Formula (b) [MUC1-VL]-[CL], Formula (c) [EGFR-VH]-[Linker1]-[Titin]-[Fc2], Formula (d) [EGFR-VL]-[Linker2]-[Obscurin], Among them, the above Linker1 and Linker2 are the same or different and are peptide linkers, or Linker1 or Linker2 does not exist, the structures represented by Formulas (a), (b), (c) and (d) are arranged from the N-terminus to the C-terminus.

[0077] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above, wherein the MUC1-VH includes an amino acid sequence of SEQ ID NO: 12 in HCDR1, an amino acid sequence of SEQ ID NO: 13 in HCDR2, and an amino acid sequence of SEQ ID NO: 14 in HCDR3, and the MUC1-VL includes an amino acid sequence of SEQ ID NO: 15 in LCDR1, an amino acid sequence of SEQ ID NO: 16 in LCDR2, and an amino acid sequence of SEQ ID NO: 17 in LCDR3, and a. the EGFR-VH includes an amino acid sequence of SEQ ID NO: 116 in HCDR1, an amino acid sequence of SEQ ID NO: 117 in HCDR2, and an amino acid sequence of SEQ ID NO: 129 in HCDR3, and the EGFR-VL includes an amino acid sequence of SEQ ID NO: 119 in LCDR1, an amino acid sequence of SEQ ID NO: 120 in LCDR2, and an amino acid sequence of SEQ ID NO: 121 in LCDR3, or b. the EGFR-VH includes an amino acid sequence of SEQ ID NO: 126 in HCDR1, an amino acid sequence of SEQ ID NO: 117 in HCDR2, and an amino acid sequence of SEQ ID NO: 128 or SEQ ID NO: 130 in HCDR3, and the EGFR-VL includes an amino acid sequence of SEQ ID NO: 119 in LCDR1, an amino acid sequence of SEQ ID NO: 120 in LCDR2, and an amino acid sequence of SEQ ID NO: 121 in LCDR3, or c. The above EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 133, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the above EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121.

[0078] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein MUC1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and MUC1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17, and EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121.

[0079] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein MUC1-VH includes the amino acid sequence of SEQ ID NO: 36, MUC1-VL includes the amino acid sequence of SEQ ID NO: 39, EGFR-VH includes the amino acid sequence of SEQ ID NO: 138, 142, 144, or 147, and EGFR-VL includes the amino acid sequence of SEQ ID NO: 149.

[0080] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein MUC1-VH comprises the amino acid sequence of SEQ ID NO: 36, MUC1-VL comprises the amino acid sequence of SEQ ID NO: 39, EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149.

[0081] In some embodiments, the antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein linker 1 is a peptide linker known in the art, insofar as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide having 1 to 50 or 3 to 20 amino acid residues. In some embodiments, each peptide linker independently has the structure L1-(GGGGS)t-L2, where L1 is binding, A, G, GS, GGG, GGS or GGGGS (SEQ ID NO: 168), t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L2 is binding, G, GG, GGG or GGGG (SEQ ID NO: 181), and the peptide linker is not binding. In some embodiments, linker 1 is identical to linker 2, and its amino acid sequence is shown in SEQ ID NO: 168.

[0082] In some embodiments, the antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 includes the amino acid sequence of SEQ ID NO: 167.

[0083] In some embodiments, the antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein CL is the constant region of the light chain of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the above, wherein CL is the constant region of the light chain of kappa or lamada. In some embodiments, wherein CL includes the amino acid sequence of SEQ ID NO: 70.

[0084] In some embodiments, the antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, and its format is an asymmetric structure molecule containing four chains, as shown in Figure 5.

[0085] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein the format is an asymmetric structure molecule containing four chains, as shown in Figure 5, and of which, MUC1-VH contains the amino acid sequence of SEQ ID NO: 12 for HCDR1, the amino acid sequence of SEQ ID NO: 13 for HCDR2, and the amino acid sequence of SEQ ID NO: 14 for HCDR3, and MUC1-VL contains the amino acid sequence of SEQ ID NO: 15 for LCDR1, the amino acid sequence of SEQ ID NO: 16 for LCDR2, and the amino acid sequence of SEQ ID NO: 17 for LCDR3, and EGFR-VH contains the amino acid sequence of SEQ ID NO: 116 for HCDR1, the amino acid sequence of SEQ ID NO: 117 for HCDR2, and the amino acid sequence of SEQ ID NO: 129 for HCDR3, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 119 for LCDR1, the amino acid sequence of SEQ ID NO: 120 for LCDR2, and the amino acid sequence of SEQ ID NO: 121 for LCDR3.

[0086] In some embodiments, an antigen-binding molecule specifically binds to EGFR and MUC1 as described in any one of the above, wherein the format is an asymmetric structure molecule containing four chains, as shown in Figure 5, of which MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, EGFR-VH contains the amino acid sequence of SEQ ID NO: 138, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 149.

[0087] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 174, 172, 175, 176, or 177, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173; or the antigen-binding molecule comprising a first chain containing the amino acid sequence of SEQ ID NO: 178, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 179, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0088] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 174, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0089] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 172, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0090] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, which comprises a first chain comprising the amino acid sequence of SEQ ID NO: 171, a second chain comprising the amino acid sequence of SEQ ID NO: 74, a third chain comprising the amino acid sequence of SEQ ID NO: 175, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 173.

[0091] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, which comprises a first chain comprising the amino acid sequence of SEQ ID NO: 171, a second chain comprising the amino acid sequence of SEQ ID NO: 74, a third chain comprising the amino acid sequence of SEQ ID NO: 176, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 173.

[0092] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, which comprises a first chain comprising the amino acid sequence of SEQ ID NO: 171, a second chain comprising the amino acid sequence of SEQ ID NO: 74, a third chain comprising the amino acid sequence of SEQ ID NO: 177, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 173.

[0093] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, which comprises a first chain comprising the amino acid sequence of SEQ ID NO: 178, a second chain comprising the amino acid sequence of SEQ ID NO: 74, a third chain comprising the amino acid sequence of SEQ ID NO: 179, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 173.

[0094] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, which comprises a first chain represented by SEQ ID NO: 171, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 174, and a fourth chain represented by SEQ ID NO: 173.

[0095] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain represented by SEQ ID NO: 171, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 172, and a fourth chain represented by SEQ ID NO: 173.

[0096] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain represented by SEQ ID NO: 171, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 175, and a fourth chain represented by SEQ ID NO: 173.

[0097] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain represented by SEQ ID NO: 171, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 176, and a fourth chain represented by SEQ ID NO: 173.

[0098] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain represented by SEQ ID NO: 171, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 177, and a fourth chain represented by SEQ ID NO: 173.

[0099] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above, comprising a first chain represented by SEQ ID NO: 178, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 179, and a fourth chain represented by SEQ ID NO: 173.

[0100] In another embodiment, the Disclosure provides an immune complex comprising an anti-MUC1 antibody or its antigen-binding fragment and effector molecule as described above, or an anti-EGFR antibody or its antigen-binding fragment and effector molecule as described above, or an antigen-binding molecule and effector molecule that specifically bind to EGFR and MUC1 as described above, wherein the effector molecule is coupled to the anti-MUC1 antibody or its antigen-binding fragment, the anti-EGFR antibody or its antigen-binding fragment, or an antigen-binding molecule that specifically binds to EGFR and MUC1, and preferably the effector molecule is selected from antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0101] In some embodiments, the effector molecule is a cytotoxic drug, preferably an exatecan-based drug or an MMAE / MMAF-based drug.

[0102] In another embodiment, the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof. [ka] Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR) a R b ) m -CR 1 R 2 Selected from -C(O)-, R a and R b They are the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, hydroxyl groups, amino groups, cyano groups, nitro groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. Alternatively, R a and R b These, together with the carbon atoms linked to them, form a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group. R 1 This is selected from halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, hydroxyl groups, alkoxy groups, cyano groups, amino groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. R 2 This is selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, hydroxyl groups, alkoxy groups, cyano groups, amino groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group. Alternatively, R a and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group. m is 0, 1, 2, 3, or 4. n is between 1 and 10. L is the linker unit, Pc is an anti-MUC1 antibody or its antigen-binding fragment described in any one of the above, or an anti-EGFR antibody or its antigen-binding fragment described in any one of the above, or an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above, and preferably, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above.

[0103] In some embodiments, an antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, among which, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, Eventually, R a and R b They are identical or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, and alkyl groups. R 1 is a cycloalkyl-alkyl group or cycloalkyl group, R 2 This is selected from hydrogen atoms, haloalkyl groups, and cycloalkyl groups. Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group. m is 0, 1, 2, 3, or 4. n is between 1 and 10. L is the linker unit, Pc is an anti-MUC1 antibody or its antigen-binding fragment described in any one of the above, or an anti-EGFR antibody or its antigen-binding fragment described in any one of the above, or an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above, and preferably, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above.

[0104] In some embodiments, an antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, among which, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, Eventually, R a and R b They are the same or different, and each is independently a hydrogen atom, a deuterium atom, a halogen, and C 1-6 Selected from alkyl groups, R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. m is 0, 1, 2, 3, or 4. n is between 1 and 10. L is the linker unit, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1, comprising one antigen-binding module that specifically binds to EGFR and one antigen-binding module that specifically binds to MUC1, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL, of which, The above MUC1-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14; the above MUC1-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; the above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129; and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121. Preferably, among them, MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, EGFR-VH contains the amino acid sequence of SEQ ID NO: 138, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 149. More preferably, the antigen-binding molecule comprises a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 174, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173; or a first chain containing the amino acid sequence of SEQ ID NO: 178, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 179, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0105] In some embodiments, an antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, among which, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, Eventually, R a and R bThey are the same or different, and each is independently a hydrogen atom, a deuterium atom, a halogen, and C 1-6 Selected from alkyl groups, R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. m is 0, 1, 2, 3, or 4. n is between 1 and 10. L is the linker unit, Pc is an anti-MUC1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing HCDR1, HCDR2, and HCDR3, and a light chain variable region containing LCDR1, LCDR2, and LCDR3, among which, The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17. Preferably, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39. More preferably, Pc is an anti-MUC1 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 71, and the light chain comprises the amino acid sequence of SEQ ID NO: 72.

[0106] In some embodiments, an antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, among which, Y is -O-(CR a R b ) m -CR1 R 2 is -C(O)-, wherein, R a and R b are the same or different and each independently is a hydrogen atom, a deuterium atom, a halogen, and a C 1-6 alkyl group, R 1 is a 3 - to 6 - membered cycloalkyl - C 1-6 alkyl group or a 3 - to 6 - membered cycloalkyl group, R 2 is selected from a hydrogen atom, a C 1-6 haloalkyl group, and a 3 - to 6 - membered cycloalkyl group, alternatively, R 1 and R 2 together with the carbon atom to which they are attached form a 3 - to 6 - membered cycloalkyl group, m is 0, 1, 2, 3 or 4, n is 1 to 10, L is a linker unit, Pc is an anti - EGFR antibody or an antigen - binding fragment thereof comprising a heavy - chain variable region containing HCDR1, HCDR2 and HCDR3, and a light - chain variable region containing LCDR1, LCDR2 and LCDR3, wherein the above - mentioned heavy - chain variable region has HCDR1 comprising the amino acid sequence of SEQ ID NO: 116, HCDR2 comprising the amino acid sequence of SEQ ID NO: 117, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 129, and the above - mentioned light - chain variable region has LCDR1 comprising the amino acid sequence of SEQ ID NO: 119, LCDR2 comprising the amino acid sequence of SEQ ID NO: 120, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 121, preferably, the above - mentioned heavy - chain variable region comprises the amino acid sequence of SEQ ID NO: 138, and the above - mentioned light - chain variable region comprises the amino acid sequence of SEQ ID NO: 149, more preferably, Pc is an anti - EGFR antibody comprising a heavy chain and a light chain, wherein the above - mentioned heavy chain comprises the amino acid sequence of SEQ ID NO: 153, and the above - mentioned light chain comprises the amino acid sequence of SEQ ID NO: 164. In some embodiments, the antibody - drug conjugate represented by the above general formula (Pc - L - Y - D) or a pharmaceutically acceptable salt thereof, wherein the linker unit - L - is - L1 -L 2 -L 3 -L 4 -and, Eventually, L 1 is -(succinimido-3-yl-N)-WC(O)-,-CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)- are selected, and W is selected from alkylene groups and alkylene-cycloalkyl groups, of which the alkylene group or alkylene-cycloalkyl group is independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups and cycloalkyl groups. L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, among them, p 1 is an integer between 1 and 20, L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 4 -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - and selected from chemical bonds, where t is 1, 2, 3, 4, 5 or 6, R 3 , R 4and R 5 They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R 6 and R 7 They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. Preferably, among them, the linker unit-L 1 -L 2 -L 3 -L 4 - is as follows: L 1 teeth [ka] And among them, s 1 is 2, 3, 4, 5, 6, 7 or 8, L 2 It is a chemical bond, L 3 is a tetrapeptide residue, preferably L 3 This is a tetrapeptide residue represented by GGFG (SEQ ID NO: 180), L 4 -NR 5 (CR 6 R 7 )t-, and of these, R 5 , R 6 or R 7 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group, and t is 1 or 2. Of these, the above -L- is L 1 The end is connected to Pc, L 4 The end is connected to a Y.

[0107] In some embodiments, the antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is the general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, [ka] Eventually, Pc is an anti-MUC1 antibody or its antigen-binding fragment described in any one of the above, or an anti-EGFR antibody or its antigen-binding fragment described in any one of the above, or an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above, preferably Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above. m is 0, 1, 2, 3, or 4. n is between 1 and 10. R 1 is a cycloalkyl-alkyl group or cycloalkyl group, R 2 This is selected from hydrogen atoms, haloalkyl groups, and cycloalkyl groups. Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group. W is selected from alkylene groups and alkylene-cycloalkyl groups, and of these, the alkylene group and alkylene-cycloalkyl group are each independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, among them, p 1 is an integer between 1 and 20, L 3This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R 4 and R 5 This is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups. R 6 and R 7 These elements may be identical or different, and each may be independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0108] In some embodiments, the antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is the general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, [ka] Eventually, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above. m is 0, 1, 2, 3, or 4. n is between 1 and 10. R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. W is C 1-6 Alkylene group and C 1-6 Selected from alkylene-3 to 6-membered cycloalkyl groups, of which the above C 1-6 Alkylene group and C 1-6 Alkylene-3 to 6-membered cycloalkyl groups can each be independently and optionally selected as a halogen, hydroxyl group, cyano group, amino group, or C. 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl group, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, among them, p 1 is an integer between 1 and 20, L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, R 4 and R 5 C is a hydrogen atom. 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups and C 1-6 Selected from hydroxyalkyl groups, R 6 and R 7They are identical or different, and each is independently a hydrogen atom, halogen, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups and C 1-6 Selected from hydroxyalkyl groups.

[0109] In some embodiments, the antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is the general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, [ka] Eventually, Pc is an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above. m is 0, 1, 2, 3, or 4. n is between 1 and 10. R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. W is C 1-6 Alkylene group and C 1-6 Selected from alkylene-3 to 6-membered cycloalkyl groups, of which the above C 1-6 Alkylene group and C 1-6 Alkylene-3 to 6-membered cycloalkyl groups can each be independently and optionally selected as a halogen, hydroxyl group, cyano group, amino group, or C. 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, among them, p 1 is an integer between 1 and 20, L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, R 4 and R 5 C is a hydrogen atom. 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups and C 1-6 Selected from hydroxyalkyl groups, R 6 and R 7 They are identical or different, and each is independently a hydrogen atom, halogen, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups and C 1-6 Selected from hydroxyalkyl groups.

[0110] In some embodiments, the antibody-drug conjugate represented by the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is the general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, [ka] Eventually, Pc is an anti-EGFR antibody or its antigen-binding fragment as described in any one of the above. m is 0, 1, 2, 3, or 4. n is between 1 and 10. R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. W is C 1-6 Alkylene group and C 1-6 Selected from alkylene-3 to 6-membered cycloalkyl groups, of which the above C 1-6 Alkylene group and C 1-6 Alkylene-3 to 6-membered cycloalkyl groups can each be independently and optionally selected as a halogen, hydroxyl group, cyano group, amino group, or C. 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, among them, p 1 is an integer between 1 and 20, L 3This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, R 4 and R 5 C is a hydrogen atom. 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups and C 1-6 Selected from hydroxyalkyl groups, R 6 and R 7 They are identical or different, and each is independently a hydrogen atom, halogen, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups and C 1-6 Selected from hydroxyalkyl groups.

[0111] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, wherein m is 0.

[0112] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which R 1 is a 3-6 membered cycloalkyl group, preferably R 1 It is a cyclopropyl group.

[0113] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which R 2 It is a hydrogen atom.

[0114] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which R 1 and R 2 These, together with the carbon atoms to which they are linked, form a 3- to 6-membered cycloalkyl group.

[0115] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, wherein W is C 1-6 Selected from alkylene groups.

[0116] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which L 2 It is a chemical bond.

[0117] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6Deuterated alkyl groups, C 1-6 It is substituted with one or more substituents selected from an alkoxy group and a 3-6 membered cycloalkyl group, preferably L 3 is a tetrapeptide residue, more preferably L 3 This is a tetrapeptide residue represented by GGFG (Sequence ID 180).

[0118] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which R 5 It is a hydrogen atom.

[0119] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, of which R 6 and R 7 They are all hydrogen atoms.

[0120] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, which is an antibody-drug conjugate represented by the general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof, [ka] Eventually, n is 1 to 8, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above.

[0121] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, which is an antibody-drug conjugate represented by the general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof, [ka] Eventually, n is 1 to 8, Pc is an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above.

[0122] In some embodiments, the above general formula (Pc-LYD) or general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, which is an antibody-drug conjugate represented by the general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof, [ka] Eventually, n is 1 to 8, Pc is an anti-EGFR antibody or its antigen-binding fragment as described in any one of the above.

[0123] In another aspect, this disclosure relates to the general formula (Pc-L a The present invention provides a method for preparing an antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, which, [ka] After reducing Pc, the general formula (L a The compound represented by -YD) or a salt thereof is subjected to a coupling reaction, and the general formula (Pc-L a The step of obtaining an antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, Eventually, Pc is an anti-MUC1 antibody or its antigen-binding fragment described in any one of the above, or an anti-EGFR antibody or its antigen-binding fragment described in any one of the above, or an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above, preferably Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 described in any one of the above, W, L 2 , L 3 , R1 , R 2 , R 5 ~R 7 m and n are defined as any one of the above.

[0124] In some embodiments, n is between 0 and 10 (including decimals and integers, and so on).

[0125] In some embodiments, n is between 1 and 10, and for example, when calculated using the average value, it is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0126] In some embodiments, n is 1 to 8, preferably 2 to 8, more preferably 4 to 8, and most preferably 4 to 6.

[0127] In another embodiment, the present disclosure provides a compound represented by the general formula (Pc-M') or a pharmaceutically acceptable salt thereof, [ka] Eventually, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1, comprising one antigen-binding module that specifically binds to EGFR and one antigen-binding module that specifically binds to MUC1, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL, of which, The above MUC1-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14; the above MUC1-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; the above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129; and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121. Preferably, among them, MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, EGFR-VH contains the amino acid sequence of SEQ ID NO: 138, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 149. More preferably, the antigen-binding molecule comprises a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 174, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173, or a first chain containing the amino acid sequence of SEQ ID NO: 178, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 179, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173. W' is selected from alkylene groups and alkylene-cycloalkyl groups, and each of the above alkylene groups and alkylene-cycloalkyl groups is independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 2a -NR 4a (CH2CH2O)p 2 CH2CH2C(O)-, -NR 4a(CH2CH2O)p 2 CH2C(O)-, -S(CH2)p 2 Selected from C(O)- and chemical bonds, among them, p 2 is an integer between 1 and 20, L 3a This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R 4a This is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups. R c , R d , R e and R f They are identical or different, and at least one of them is selected from halogens, alkenyl groups, alkyl groups, and cycloalkyl groups, with the remainder being hydrogen atoms. Or R c , R d , R e and R f Two of these atoms form a cycloalkyl group with the carbon atoms they are linked to, and the remaining two are optionally selected from hydrogen atoms, alkyl groups, and cycloalkyl groups. R 8 , R 10 , R 12 and R 14 ~R 17 They are identical or different, and each is independently selected from a hydrogen atom, alkyl group, haloalkyl group, hydroxyalkyl group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group. R 9 , R 11 and R 13They are identical or different, and each is independently selected from a hydrogen atom, halogen, hydroxyl group, cyano group, amino group, alkyl group, haloalkyl group, hydroxyalkyl group, alkoxy group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group. R 18 is an aryl group or a heteroaryl group, and the above aryl group or heteroaryl group may be optionally further substituted with substituents selected from hydrogen atoms, halogens, hydroxyl groups, alkyl groups, haloalkyl groups, hydroxyalkyl groups, alkoxy groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups. s is between 1 and 10.

[0128] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, where W' is C 1-6 Selected from alkylene groups.

[0129] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which L 2a It is a chemical bond.

[0130] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which L 3a This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 It is substituted with one or more substituents selected from an alkoxy group and a 3-6 membered cycloalkyl group, preferably L 3 is a tetrapeptide residue, more preferably L 3This is a tetrapeptide residue represented by GGFG (Sequence ID 180).

[0131] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R c and R d One of them, R e and R f One of them forms a 3-6 membered cycloalkyl group with the carbon atoms linked to it, and the remaining two are hydrogen atoms, preferably R c and R d One of them, R e and R f One of them forms a cyclopropyl group with the carbon atom it is linked to, and the other two are hydrogen atoms.

[0132] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R 8 , R 10 , R 12 and R 14 ~R 17 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group.

[0133] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R 8 , R 12 , R 14 and R 15 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group.

[0134] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R 10 and R 16 They are all hydrogen atoms.

[0135] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R 17 It is a hydrogen atom.

[0136] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R 9 , R 11 and R 13 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group, preferably R 9 , R 11 and R 13 They are identical or different, and each is independently C 1-6 It is an alkyl group.

[0137] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which R 18 This is a 6-10 membered aryl group, and the above 6-10 membered aryl group may optionally be further composed of a hydrogen atom, a halogen, a hydroxyl group, or C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Hydroxyalkyl groups and C 1-6 Substituting with a substituent selected from alkoxy groups, preferably R 18 This is a phenyl group, and the above phenyl group can optionally be further modified with a hydrogen atom, a halogen, or C 1-6 Alkyl and C 1-6 Substituting with a substituent selected from haloalkyl groups, more preferably R 18 This is a phenyl group, and the above phenyl group can be optionally further substituted with a halogen.

[0138] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above. W' is C 1-6Selected from alkylene groups, L 2a It is a chemical bond, L 3a This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, R c and R d One of them, R e and R f One of them forms a 3-6 membered cycloalkyl group with the carbon atoms linked to it, and the remaining two optionally form a hydrogen atom, C 1-6 Selected from alkyl groups and 3-6 membered cycloalkyl groups, R 8 , R 10 , R 12 and R 14 ~R 17 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group, R 9 , R 11 and R 13 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group, R 18 This is a 6-10 membered aryl group, and the above 6-10 membered aryl group may optionally be further composed of a hydrogen atom, a halogen, a hydroxyl group, or C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Hydroxyalkyl groups and C 1-6 Substituting with substituents selected from alkoxy groups, s is between 1 and 10.

[0139] In some embodiments, the compound represented by the above general formula (PC-M') or a pharmaceutically acceptable salt thereof, among which, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above. W' is C 1-6 Selected from alkylene groups, L 2a It is a chemical bond, L 3a This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, R c and R d One of them, R e and R f One of them forms a cyclopropyl group with the carbon atom it is linked to, and the other two are hydrogen atoms. R 8 , R 10 , R 12 and R 14 ~R 17 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group, R 9 , R 11 and R 13 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group, R 18 This is a 6-10 membered aryl group, and the above 6-10 membered aryl group may optionally be further composed of a hydrogen atom, a halogen, a hydroxyl group, or C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C1-6 Hydroxyalkyl groups and C 1-6 Substituting with substituents selected from alkoxy groups, s is between 4 and 8.

[0140] In some embodiments, the compound represented by the general formula (Pc-M') or a pharmaceutically acceptable salt thereof is, which is a compound represented by the general formula (Pc-M) or a pharmaceutically acceptable salt thereof, among which, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above. [ka] s is between 4 and 6.

[0141] In another embodiment, the present disclosure provides a method for preparing an antibody-drug conjugate represented by the general formula (Pc-M') or a pharmaceutically acceptable salt thereof, which, [ka] The process includes reducing Pc, then coupling it with compound M' or a salt thereof to obtain an antibody-drug conjugate represented by the general formula (Pc-M') or a pharmaceutically acceptable salt thereof. Eventually, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of the above. W', L 2a , L 3a , R 8 ~R 18 , R c , R d , R e , R f And s are defined as in any one of the above definitions.

[0142] In some embodiments, s is between 0 and 10 (including decimals and integers, and so on).

[0143] In some embodiments, s is between 1 and 10, and for example, when calculated using the average value, it is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0144] In some embodiments, s is 1 to 8, preferably 2 to 8, more preferably 4 to 8, and most preferably 4 to 6.

[0145] In another embodiment, the Disclosure provides a pharmaceutical composition comprising any one of the anti-MUC1 antibody or antigen-binding fragment thereof, any one of the anti-EGFR antibody or antigen-binding fragment thereof, any one of the antigen-binding molecules that specifically bind to EGFR and MUC1, any one of the antibody-drug conjugates or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0146] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-MUC1 antibody or an antigen-binding fragment thereof as described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0147] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-EGFR antibody or antigen-binding fragment thereof as described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0148] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule that specifically binds to any one of the EGFR and MUC1 described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0149] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-MUC1 antibody or a drug conjugate of its antigen-binding fragment described above, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0150] In some embodiments, the Disclosure provides a pharmaceutical composition comprising an anti-EGFR antibody or a drug conjugate of the antigen-binding fragment thereof as described above, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0151] In some embodiments, the Disclosure provides a pharmaceutical composition comprising a drug conjugate of an antigen-binding molecule that specifically binds to any one of the EGFR and MUC1 described above (i.e., an EGFR-MUC1 biantibody ADC) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0152] In another embodiment, the Disclosure provides isolated nucleic acids encoding an anti-MUC1 antibody or its antigen-binding fragment as described in any one of the above, or an anti-EGFR antibody or its antigen-binding fragment as described in any one of the above.

[0153] In some embodiments, the Disclosure provides isolated nucleic acids encoding an anti-MUC1 antibody or an antigen-binding fragment thereof as described in any one of the above.

[0154] In some embodiments, the Disclosure provides isolated nucleic acids encoding an anti-EGFR antibody or an antigen-binding fragment thereof as described in any one of the above.

[0155] In another embodiment, the present disclosure provides a host cell comprising an isolated nucleic acid as described in any one of the above.

[0156] In another embodiment, the Disclosure provides a method for preventing or treating a disease, comprising administering to a subject an effective amount for prevention or treatment of any one of the anti-MUC1 antibody or antigen-binding fragment thereof, any one of the anti-EGFR antibody or antigen-binding fragment thereof, any one of the antigen-binding molecules that specifically bind to EGFR and MUC1, any one of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0157] In some embodiments, a method for preventing or treating the above-mentioned disease comprises administering to a subject an effective amount for prevention or treatment of any one of the above-mentioned anti-MUC1 antibody or antigen-binding fragment thereof or pharmaceutical composition thereof.

[0158] In some embodiments, a method for preventing or treating the above-mentioned disease comprises administering to a subject a preventive or therapeutically effective amount of any one of the above-mentioned anti-EGFR antibodies, antigen-binding fragments thereof, or pharmaceutical compositions thereof.

[0159] In some embodiments, a method for preventing or treating the above-mentioned diseases comprises administering to a subject an effective amount for prevention or treatment of an antigen-binding molecule or pharmaceutical composition thereof that specifically binds to any one of the above-mentioned EGFR and MUC1.

[0160] In some embodiments, a method for preventing or treating the above-mentioned diseases, the method comprising administering to a subject a prophylactic or therapeutically effective amount of any one of the above-mentioned anti-MUC1 antibodies or drug conjugates of the antigen-binding fragment thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0161] In some embodiments, a method for preventing or treating the above-mentioned diseases, the method comprising administering to a subject a prophylactic or therapeutically effective amount of any one of the above-mentioned anti-EGFR antibodies or drug conjugates of the antigen-binding fragment thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0162] In some embodiments, a method for preventing or treating the above-mentioned diseases, the method comprising administering to a subject a preventive or therapeutically effective amount of a drug conjugate of an antigen-binding molecule that specifically binds to any one of the above-mentioned EGFR and MUC1 (i.e., an EGFR-MUC1 biantibody ADC) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0163] In another embodiment, the Disclosure provides a use in the preparation of a drug for the prevention or treatment of a disease, comprising administering to a subject a prophylactic or therapeutically effective amount of any one of the anti-MUC1 antibody or its antigen-binding fragment, any one of the anti-EGFR antibody or its antigen-binding fragment, any one of the antigen-binding molecules that specifically bind to EGFR and MUC1, any one of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0164] In some embodiments, the use in the preparation of a drug for preventing or treating the above-mentioned diseases includes administering to a subject a preventive or therapeutically effective amount of any one of the above-mentioned anti-MUC1 antibody or its antigen-binding fragment or pharmaceutical composition.

[0165] In some embodiments, the use in the preparation of a drug for preventing or treating the above-mentioned diseases includes administering to a subject a preventive or therapeutically effective amount of any one of the above-mentioned anti-EGFR antibodies, antigen-binding fragments thereof, or pharmaceutical compositions thereof.

[0166] In some embodiments, the use in the preparation of a drug for preventing or treating the above-mentioned diseases includes administering to a subject an effective amount for prevention or treatment of an antigen-binding molecule or pharmaceutical composition thereof that specifically binds to any one of the above-mentioned EGFR and MUC1.

[0167] In some embodiments, the use in the preparation of a drug for preventing or treating the above-mentioned diseases includes administering to a subject a prophylactic or therapeutically effective amount of any one of the above-mentioned anti-MUC1 antibodies or drug conjugates of its antigen-binding fragment, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0168] In some embodiments, the use in the preparation of a drug for preventing or treating the above-mentioned diseases includes administering to a subject a prophylactic or therapeutically effective amount of any one of the above-mentioned anti-EGFR antibodies or drug conjugates of its antigen-binding fragment, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0169] In some embodiments, the use in the preparation of a drug for preventing or treating the above-mentioned diseases includes administering to a subject a prophylactic or therapeutically effective amount of a drug conjugate of an antigen-binding molecule that specifically binds to any one of the above-mentioned EGFR and MUC1 (i.e., an EGFR-MUC1 biantibody ADC) or a pharmaceutically acceptable salt thereof.

[0170] In another embodiment, the Disclosure provides any of the above-described anti-MUC1 antibodies or antigen-binding fragments thereof, any of the above-described anti-EGFR antibodies or antigen-binding fragments thereof, any of the above-described antigen-binding molecules that specifically bind to EGFR and MUC1, any of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, to be used as a drug. In some embodiments, the drugs are used to prevent or treat diseases.

[0171] In some embodiments, the anti-MUC1 antibody described in any one of the above descriptions, or its antigen-binding fragment, or a pharmaceutical composition thereof, is used as a drug. In some embodiments, the drug is used to prevent or treat a disease.

[0172] In some embodiments, the anti-EGFR antibody described above, or its antigen-binding fragment, or a pharmaceutical composition thereof, is used as a drug. In some embodiments, the drug is used to prevent or treat a disease.

[0173] In some embodiments, the antigen-binding molecule or pharmaceutical composition thereof that specifically binds to any one of the EGFR and MUC1 described above and is used as a drug. In some embodiments, the drug is used to prevent or treat a disease.

[0174] In some embodiments, the anti-MUC1 antibody described in any one of the above descriptions, or a drug conjugate of its antigen-binding fragment, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is used as a drug. In some embodiments, the drug is used to prevent or treat a disease.

[0175] In some embodiments, the anti-EGFR antibody described in any one of the above descriptions, or a drug conjugate of the antigen-binding fragment thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is used as a drug. In some embodiments, the drug is used to prevent or treat a disease.

[0176] In some embodiments, the drug conjugate (i.e., EGFR-MUC1 biantibody ADC) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is used as a drug. In some embodiments, the drug is used to prevent or treat a disease.

[0177] In some embodiments, the disease described in any one of the above is a tumor, and in some embodiments, the disease is an astrocytoma (e.g., anaplastic astrocytoma), glioblastoma, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., breast cancer characterized by BRCA1 and / or BRCA2 mutations), cervical cancer, colorectal cancer (e.g., colon cancer and rectal cancer), fallopian tube cancer, gallbladder cancer, gastric cancer, head and neck cancer, idiopathic myelofibrosis, renal cancer (e.g., renal cell carcinoma, renal rhabdoid tumor and Wilms' tumor), leukemia, liver cancer (e.g., hepatocellular carcinoma), esophageal cancer (also called "esophageal cancer," e.g., esophageal squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), medulloblastoma, melanoma, Merkel The cancer is selected from cell carcinoma, mesothelioma, multiple myeloma, neuroblastoma, oligodendroglioma, ovarian cancer, peritoneal tumor, pancreatic cancer, polycythemia vera, primary neuroectodermal tumor, prostate cancer, retinoblastoma, sarcoma (e.g., chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma and soft tissue sarcoma), squamous cell carcinoma (e.g., cutaneous squamous cell carcinoma), thyroid cancer, endometrial cancer, vestibular schwannoma, blastoma, vulvar cancer, thymoma, testicular cancer, cholangiocarcinoma, pheochromocytoma, paraganglioma and adenoid cystic carcinoma, and in some embodiments, the cancer is selected from lung cancer, head and neck cancer, esophageal cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer and bladder cancer. In some embodiments, the cancer is lung cancer, preferably non-small cell lung cancer.

[0178] In this disclosure, ordinal numbers such as “first,” “second,” “third,” “1,” “2,” and “3” (e.g., “first subunit,” “Fc2,” “third chain,” “linker2”) are used simply to distinguish different features, elements, components, or steps and are not intended to limit quantities, orders, or levels. [Brief explanation of the drawing]

[0179] [Figure 1-2] The results of in vitro internalization experiments of chimeric antibodies against T47D cells are shown. [Figure 3A-E] The results of affinity measurement experiments for chimeric antibodies and humanized antibodies against HCC827-human-MUC1-C cells, based on FACS, are shown. [Figure 3F-H] The results of experiments measuring the affinity of chimeric antibodies and humanized antibodies against CHOK1-human-MUC1-C cells based on FACS are shown. [Figure 3I-P] The results of affinity measurements of chimeric and humanized antibodies against T47D cells (high MUC1 expression, low EGFR expression) based on FACS are shown. [Figure 4A-F] The results of in vitro internalization experiments of MUC1-C humanized antibodies against T47D cells are shown. [Figure 5] A schematic diagram of the molecular morphology of the EGFR-MUC1 biantibody is shown. [Figure 6A-B] This paper presents experimental results of the killing activity of different mutant EGFR-MUC1 biantibody ADCs (coupled to M toxin) against tumor cell lines HCC827 (high EGFR expression, low MUC1 expression) and HCC70 (moderate EGFR expression, moderate MUC1 expression). [Figure 7A] This shows the curves of change in tumor volume over study days in different groups of the HCC827 CDX mouse model. [Figure 7B] This shows the curves of change in mouse body weight over study days for different groups in the HCC827 CDX mouse model. [Figure 8A] This shows the curves of change in tumor volume over study days in different groups of HPAC CDX mouse models. [Figure 8B] This shows the curves of change in mouse body weight over study days for different groups in the HPAC CDX mouse model. [Modes for carrying out the invention]

[0180] term To make this disclosure more easily understood, some technical and scientific terms are defined below. Unless otherwise specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0181] The singular forms “one,” “one type,” and “the above” used in the specification and claims refer to multiple subjects unless otherwise specified in the context.

[0182] Unless otherwise specified in the context, words such as "contain," "possess," and "include" in patent specifications and claims should be understood to mean "include, but not limited to," rather than being exclusive or exhaustive.

[0183] The term "cytokine" is a general term for proteins released from one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include mRNA-2, IFNγ, TNFα, CCL-2, and IL-6.

[0184] The term "and / or" refers to a combination of two meanings: "and" and "or." For example, the phrase "A, B and / or C" attempts to cover A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0185] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0186] The term "MUC1" refers to the full-length MUC1 protein, and human MUC1 contains the amino acid sequence of Sequence ID No. 2. The amino acid sequences of MUC1 molecules from non-human species (e.g., mice, monkeys, rabbits, dogs, pigs, etc.) are available from public resources.

[0187] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and α-carbon bonded to an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide skeletons, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0188] The term "amino acid mutation" includes amino acid substitutions (also called amino acid replacements), deletions, insertions, and modifications. A final construct can be realized by any combination of substitutions, deletions, insertions, and modifications, provided that the final construct possesses desired properties, such as reduced binding to the Fc receptor. Deletions and insertions of amino acid sequences include deletions and insertions at the amino and / or carboxyl terminals of a polypeptide chain. Specific amino acid mutations may also be amino acid substitutions. In one embodiment, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include substitutions with amino acids that do not exist naturally or derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for modifying amino acid side chain groups other than genetic engineering, such as chemical modifications, are also expected to be available. In this specification, the same amino acid mutation can be represented by various names. In this specification, an amino acid residue at a specific site can be indicated using the format position + amino acid residue. For example, 82aR indicates that the amino acid residue at the 82a position is R. S82aR indicates that the amino acid residue at position 82a (also called 82A) has mutated from the original S to R.

[0189] The term "antigen-binding molecule" is used in its broadest sense to cover a variety of molecules that specifically bind to an antigen, as long as they exhibit the desired antigen-binding activity. This includes, but is not limited to, antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins formed by fusing both. The antigen-binding molecules described herein include variable regions (VH) and (VL) that together constitute the antigen-binding domain. Exemplarily, the antigen-binding molecules described herein are bispecific antigen-binding molecules (e.g., bispecific antibodies).

[0190] The term "antibody" is used in its broadest sense to encompass a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding moieties). For example, natural IgG antibodies are heterotetrameric glycoproteins with approximately 150,000 daltons, consisting of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL), also called a variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL).

[0191] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, for example, a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. The prior art discloses bispecific antibodies of various structures, which may be classified into IgG-like bispecific antibodies and antibody-fragment type bispecific antibodies based on the integrity of the IgG molecule, or into bivalent, trivalent, quaternary, or higher bispecific antibodies based on the number of antigen-binding regions, or into symmetrical bispecific antibodies and asymmetrical bispecific antibodies based on whether the structure is symmetrical or not. Among these, bispecific antibodies of the antibody fragment type, such as Fab fragments lacking an Fc fragment, form bispecific antibodies with relatively low immunogenicity, a small molecular weight, and relatively high tumor tissue permeability by linking two or more Fab fragments within a single molecule. Typical antibody structures of this type include, for example, F(ab')2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-like bispecific antibodies (e.g., those containing an Fc fragment) have a relatively large molecular weight. The Fc fragment contributes to antibody purification and improves its solubility and stability. The Fc portion also binds to the receptor FcRn, potentially increasing the serum half-life of the antibody.

[0192] A "natural antibody" is a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH), also called a variable heavy chain domain or heavy chain variable region, followed by a heavy chain constant region. The natural IgG heavy chain constant region generally contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL), also called a variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL). The terms "full-length antibody," "complete antibody," and "whole antibody" may be used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody, or containing heavy chains in the Fc region as limited herein. The light chain of a natural complete antibody comprises a variable region VL and a constant region CL, with VL located at the amino terminus of the light chain, and the constant region comprising a κ chain and a λ chain; the heavy chain comprises a variable region VH and a constant region (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain, and the constant region located at the carboxy terminus, of which CH3 is closest to the carboxy terminus of the polypeptide; and the heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0193] The term "variable region" or "variable domain" of an antibody refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. In this specification, the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody each contain four conservative framework regions (FRs) and three complementarity-determining regions (CDRs), respectively. Of these, the term "complementarity-determining region" or "CDR" refers to the region in the variable domain that primarily promotes binding to the antigen, while "framework" or "FR" refers to variable domain residues other than CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3, and the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus).

[0194] The amino acid sequence boundaries of CDRs can be determined by various known methods, such as the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system, the "ABM" numbering system, the "contact" numbering system (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), and the correspondences between various numbering systems are well known to those skilled in the art, and are exemplified as shown in Table 1 below.

[0195] [Table 1]

[0196] Unless otherwise specified, the variable regions and CDRs in the embodiments of this disclosure are all subject to the "Kabat" numbering rules. In specific embodiments, one numbering system (e.g., Kabat) is used to limit amino acid residues, but proposals corresponding to other numbering systems are considered equivalent proposals.

[0197] The term "antibody fragment" refers to a molecule distinct from the complete antibody, and includes a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fd, Fv, Fab, dsFv, dAb, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), bivalent antibodies, linear antibodies, single-chain antibodies (e.g., scFv), and multispecific antibodies consisting of antibody fragments.

[0198] The terms “Fc region” or “fragment crystallizable region” are used to define the C-terminal region of an antibody heavy chain and include native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Preferred Fc regions used in the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may be altered, for example, by deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system), or by deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise specified, the numbering rules for Fc regions are the EU numbering system, also known as the EU index.

[0199] The term "chimera" refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, but the rest of the heavy chain and / or light chain originates from a different source or species.

[0200] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting relatively low immunogenicity in humans. Humanization may be achieved, for example, by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the framework region portion of the constant and variable regions).

[0201] The terms “human antibody,” “humanized antibody,” “whole human antibody,” and “fully human antibody” may be used interchangeably and refer to antibodies whose variable and constant regions are human sequences. These terms cover antibodies derived from human genes but with altered sequences, such as those with potentially reduced immunogenicity, increased affinity, or removal of cysteine ​​or glycosylation sites that may cause undesirable folding. These terms also cover antibodies produced by recombination in non-human cells (which may confer glycosylation not characteristic of human cells). Furthermore, these terms also cover antibodies already fed in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of “human antibody” explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0202] The term "affinity" refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X to its ligand Y can generally be expressed by its dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0203] As used herein, the terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, obtained from the ratio of kd to ka (i.e., kd / ka) and expressed in molar concentration (M). The KD value of an antibody can be measured by methods known in the art. For example, by systematically measuring surface plasmon resonance (e.g., Biacore) using a biosensor system, or by measuring affinity in solution by solution equilibrium titration (SET).

[0204] The term "surface plasmon resonance" refers to an optical phenomenon that analyzes real-time interactions by detecting changes in protein concentration in a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0205] The term "effector function" can refer to biological activity that is altered by the antibody isotype, as well as by the antibody's Fc region (either the natural sequence Fc region or a mutated amino acid sequence Fc region). Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0206] The term "monoclonal antibody" essentially refers to a homogeneous population of antibodies, meaning that the amino acid sequences of the antibody molecules in that population are identical, with the exception of a few possible native variations. In contrast, polyclonal antibody preparations generally contain multiple different antibodies, each with different amino acid sequences that are typically specific to different epitopes in their variable domains. "Monoclonal" describes the characteristics of antibodies obtained from a homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0207] The term "antigen" refers to a molecule or molecular portion of an antibody that can be selectively recognized or bound by an antigen-binding molecule (e.g., an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding molecules (e.g., antibodies).

[0208] The term "epitope" refers to an area or region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes are formed by a continuous sequence of amino acids (linear epitopes) or may include discontinuous amino acids that are spatially close together, for example, due to antigen folding (i.e., tertiary folding of the antigen due to the properties of the protein) (contaxial epitopes). Contaxial epitopes and linear epitopes differ in that binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by methods commonly used in this field, including, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, antigen chemical modification (see Prot.Sci.9(2000)487-496), and cross-inhibition.

[0209] The terms "specifically bindable," "specifically bind," or "bind" refer to the ability of an antibody to bind to a particular antigen or its epitope with higher affinity than other antigens or epitopes. Generally, antibodies have an affinity of approximately 1 × 10⁻⁶. -7 M or less (for example, approximately 1 × 10) -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 The antibody binds to the antigen or its epitope with an equilibrium dissociation constant (KD) of M or less. In some embodiments, the KD of the antibody that binds to the antigen is 10% or less (e.g., 1%) of the KD of the antibody that binds to a nonspecific antigen (e.g., BSA, casein). The KD can be measured by known methods, for example, by BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or its epitope may cross-react to other related antigens, for example, to corresponding antigens from other species (homologous) (e.g., humans, or monkeys such as cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp), or marmosets (Callithrix jacchus) (commonmarmoset, marmoset)).

[0210] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to a target antigen or its epitope. A specific antigen-binding module includes, for example, the antigen-binding domain of an antibody, which includes a heavy chain variable region and a light chain variable region. The term "antigen-binding module that specifically binds to MUC1" refers to a module capable of binding to MUC1 or its epitope with sufficient affinity so that the molecule containing the module can be used as a diagnostic and / or therapeutic agent targeting MUC1. An antigen-binding module includes an antibody fragment as defined herein, e.g., Fab, a substituted Fab, or scFv.

[0211] The terms "anti-MUC1 antibody" and "antibody that binds to MUC1" refer to antibodies that can bind to MUC1 or its epitope with sufficient affinity.

[0212] The terms "antibody-dependent cell cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity (e.g., natural killer cells (NK), monocytes, macrophages, and neutrophils) mediated by Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies provided herein can be evaluated in vitro using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected by markers released from the lysed cells (e.g., radioactive substrates, fluorescent dyes, or innate intracellular proteins).

[0213] The term "antibody-dependent cell phagocytosis (ADCP)" refers to a mechanism by which antibody-coated target cells are removed through the internalization of phagocytic cells (e.g., macrophages or dendritic cells).

[0214] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death, in which the Fc effector domain of a target-binding antibody binds to and activates complement component C1q, which in turn activates the complement cascade, leading to the death of target cells. Complement activation can also cause the deposition of complement components on the surface of target cells, and these complement components promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0215] The term “nucleic acid” may be used interchangeably with the term “polynucleotide” herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof, exhibiting single-stranded or double-stranded forms. The above terms cover nucleic acids containing known nucleotide analogs or modified skeletal residues or linkers, which may be synthetic, naturally occurring, or not naturally occurring, possess similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated nucleic acid” refers to a nucleic acid molecule isolated from components of its natural environment. Isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, and includes one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly stated. Specifically, as detailed below, degenerate codon substitutions can be obtained by producing sequences in which the 3rd position of one or more selected (or all) codons is replaced with a mixed base and / or a deoxyinosine residue.

[0216] The terms “polypeptide” and “protein” may be used interchangeably herein and refer to polymers of amino acid residues. These terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, and also apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence implicitly covers its conservatively modified variants.

[0217] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions, provided that gaps are introduced as necessary to achieve the highest possible percentage of sequence identity when performing optimal alignment on two sequences, and no conservative substitutions are considered part of sequence identity. To measure the percentage of sequence identity, alignment can be performed using known techniques in this art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters applied to the measurement and alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0218] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it is ligated. One type of vector is a "plasmid," which refers to a circular double-stranded DNA ring that can be ligated to an attached DNA segment. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which another DNA segment can be ligated to a viral genome. Some vectors can self-replicate in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can replicate with the host genome after being introduced into the host cell by being aligned with the host cell's genome. The term "expression vector" or "expression construct" refers to a vector containing nucleic acid sequences that can transform a host cell and guide and / or control the expression of one or more heterogeneous coding regions that are manipulably ligated to it (together with the host cell). An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and the RNA splice of coding regions that are manipulably ligated to introns, if present.

[0219] The terms “host cell,” “host cell line,” and “host cell culture” may be used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” and without regard to passage number, include primary transformed cells and their derived offspring. Offspring may contain mutations, but may not be exactly the same as the parent cells in nucleic acid contents. In this specification, this includes offspring of mutants having the same function or biological activity as those screened or selected in the initial transformed cells. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include, but are not limited to, human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, and Pichia stiptis. Pichia methanolica, Pichia genus, Saccharomyces cerevisiae, Saccharomyces genus, Hansenula polymorpha, Kluyveromyces genus, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum Gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.This includes the genera Pichia, any Saccharomyces, Hansenula polymorpha, any Cliveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0220] The term "alkyl group" refers to a saturated, linear or branched aliphatic hydrocarbon group that has 1 to 20 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (i.e., C 1-20 Alkyl alkyl group). The above alkyl group is an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 A alkyl group is preferred, and an alkyl group having 1 to 6 carbon atoms (i.e., C 1-6Alkyl groups are more preferred. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl This includes the n-octyl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof. The alkyl group may or may not be substituted, and if substituted, it may be substituted at any available linking point, and preferably the substituent is one or more selected from a D atom, halogen, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0221] The term "alkenyl group" refers to an alkyl group in which the molecule contains at least one carbon-carbon double bond, the definition of which is as described above, and which has 2 to 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) (i.e., C 2-12 Alkenyl group). The above alkenyl group is an alkenyl group having 2 to 6 carbon atoms (i.e., C 2-6 An alkenyl group is preferred. Non-limiting examples include vinyl groups, propenyl groups, isopropenyl groups, butenyl groups, etc. The alkenyl group may be substituted or unsubstituted, and if substituted, it may be substituted at any available linkage point, and the substituent is preferably one or more selected from D atoms, alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups.

[0222] The term "alkoxy group" refers to an -O-(alkyl group), of which the definition of an alkyl group is as described above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy groups. The alkoxy group may be substituted or unsubstituted, and if substituted, it may be substituted at any available linkage point, and the substituent is preferably one or more selected from a D atom, halogen, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0223] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic whole-carbocyclic group (i.e., a monocyclic cycloalkyl group) or a polycyclic group (i.e., a polycyclic cycloalkyl group) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3 to 20-membered cycloalkyl group). The above cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3 to 12-membered cycloalkyl group), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3 to 8-membered cycloalkyl group), and most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3 to 6-membered cycloalkyl group).

[0224] The above monocyclic cycloalkyl groups include, as non-limiting examples, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups.

[0225] The above polycyclic cycloalkyl groups include spirocycloalkyl groups, condensed cycloalkyl groups, and crosslinked cycloalkyl groups.

[0226] The term "spirocycloalkyl group" refers to a polycyclic system in which rings share one carbon atom (called a spiro atom), and which may contain one or more double bonds within the ring, or may contain one or more heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group to form a sulfoxide or sulfone, but without -OO-, -OS-, or -SS-), provided that at least one whole carbon ring is included and the linking point is located on that whole carbon ring, which has 5 to 20 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (i.e., a 5-20 membered spirocycloalkyl group). The above spirocycloalkyl group is preferably a spirocycloalkyl group having 6 to 14 ring atoms (i.e., a 6 to 14-membered spirocycloalkyl group), and more preferably a spirocycloalkyl group having 7 to 10 ring atoms (i.e., a 7 to 10-membered spirocycloalkyl group). The above spirocycloalkyl group includes monospirocycloalkyl groups and polyspirocycloalkyl groups (e.g., bisspirocycloalkyl groups), and is preferably a monospirocycloalkyl group or a bisspirocycloalkyl group, and more preferably a 3-member / 4-member, 3-member / 5-member, 3-member / 6-member, 4-member / 4-member, 4-member / 5-member, 4-member / 6-member, 5-member / 3-member, 5-member / 4-member, 5-member / 5-member, 5-member / 6-member, 5-member / 7-member, 6-member / 3-member, 6-member / 4-member, 6-member / 5-member, 6-member / 6-member, 6-member / 7-member, 7-member / 5-member, or 7-member / 6-member monospirocycloalkyl group. Non-limiting examples are: The connection point can be in any position. [ka] This includes, among others.

[0227] The term "condensed cycloalkyl group" refers to a polycyclic system in which two adjacent carbon atoms are shared between rings, and is obtained by condensing one or more monocyclic cycloalkyl groups, or by condensing a monocyclic cycloalkyl group with one or more heterocyclyl groups, aryl groups, or heteroaryl groups, where the linking point is located on the monocyclic cycloalkyl group, which may contain one or more double bonds within the ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5 to 20-membered condensed cycloalkyl group). The above condensed cycloalkyl group is preferably a condensed cycloalkyl group having 6 to 14 ring atoms (i.e., a 6 to 14-membered condensed cycloalkyl group), and more preferably a condensed cycloalkyl group having 7 to 10 ring atoms (i.e., a 7 to 10-membered condensed cycloalkyl group). The above-mentioned condensed cycloalkyl groups include bicyclic condensed cycloalkyl groups and polycyclic condensed cycloalkyl groups (e.g., tricyclic condensed cycloalkyl groups, tetracyclic condensed cycloalkyl groups, etc.), preferably bicyclic condensed cycloalkyl groups or tricyclic condensed cycloalkyl groups, and more preferably 3-member / 4-member, 3-member / 5-member, 3-member / 6-member, 4-member / 4-member, 4-member / 5-member, 4-member / 6-member, 5-member / 3-member, 5-member / 4-member, 5-member / 5-member, 5-member / 6-member, 5-member / 7-member, 6-member / 3-member, 6-member / 4-member, 6-member / 5-member, 6-member / 6-member, 6-member / 7-member, 7-member / 5-member, or 7-member / 6-member bicyclic condensed cycloalkyl groups. Non-limiting examples are: The connection point can be in any position. [ka] , [ka] This includes, among others.

[0228] The term "crosslinked cycloalkyl group" refers to a total carbon polycyclic system in which rings share two carbon atoms that are not directly linked, and which may contain one or more double bonds within the ring, and which have 5 to 20 carbon atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (i.e., a 5-20 membered crosslinked cycloalkyl group). The above crosslinked cycloalkyl group is preferably a crosslinked cycloalkyl group having 6 to 14 carbon atoms (i.e., a 6-14 membered crosslinked cycloalkyl group), and more preferably a crosslinked cycloalkyl group having 7 to 10 carbon atoms (i.e., a 7-10 membered crosslinked cycloalkyl group). The above crosslinked cycloalkyl group includes bicyclic crosslinked cycloalkyl groups and polycyclic crosslinked cycloalkyl groups (e.g., tricyclic crosslinked cycloalkyl groups, tetracyclic crosslinked cycloalkyl groups, etc.), and is preferably a bicyclic crosslinked cycloalkyl group or a tricyclic crosslinked cycloalkyl group. Non-limiting examples are: The connection point can be in any position. [ka] Includes.

[0229] The cycloalkyl group may or may not be substituted, and if substituted, it may be substituted at any available linking point, and preferably the substituent is one or more selected from a D atom, halogen, alkyl group, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, oxo group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0230] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl group) or a polycyclic heterocycle (i.e., a polycyclic heterocyclyl group) that contains at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur within its ring (the nitrogen may be optionally oxidized to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group to form a sulfoxide or sulfone, but without -OO-, -OS-, or -SS-), and has 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3 to 20-membered heterocyclyl group). The above heterocyclyl group is preferably a heterocyclyl group having 3 to 12 ring atoms (i.e., a 3 to 12-membered heterocyclyl group), for example, a 4 to 12-membered heterocyclyl group containing at least one nitrogen atom, more preferably a heterocyclyl group having 3 to 8 ring atoms (i.e., a 3 to 8-membered heterocyclyl group), more preferably a heterocyclyl group having 3 to 6 ring atoms (i.e., a 3 to 6-membered heterocyclyl group), and most preferably a heterocyclyl group having 5 or 6 ring atoms (i.e., a 5 or 6-membered heterocyclyl group).

[0231] The above monocyclic heterocyclyl groups include, as non-limiting examples, pyrrolidinyl groups, tetrahydropyranyl groups, 1,2,3,6-tetrahydropyridyl groups, piperidinyl groups, piperazinyl groups, morpholinyl groups, thiomorpholinyl groups, and homopiperazinyl groups.

[0232] The above polycyclic heterocyclyl groups include spiroheterocyclyl groups, condensed heterocyclyl groups, and cross-linked heterocyclyl groups.

[0233] The term "spiroheterocyclyl group" refers to a polycyclic heterocyclic system in which rings share one atom (called a spiro atom), which may contain one or more double bonds within the rings, and which contain at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group to form a sulfoxide or sulfone, but which does not contain -OO-, -OS-, or -SS-), provided that at least one monocyclic heterocyclyl group is included and the linking point is located on the monocyclic heterocyclyl group, which has 5 to 20 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (i.e., a 5 to 20-membered spiroheterocyclyl group). The above-mentioned spiroheterocyclyl group is preferably a spiroheterocyclyl group having 6 to 14 ring atoms (i.e., a 6 to 14-membered spiroheterocyclyl group), and more preferably a spiroheterocyclyl group having 7 to 10 ring atoms (i.e., a 7 to 10-membered spiroheterocyclyl group). The above spiroheterocyclyl group includes a monospiroheterocyclyl group and a polyspiroheterocyclyl group (e.g., a bisspiroheterocyclyl group), preferably a monospiroheterocyclyl group or a bisspiroheterocyclyl group, and more preferably a 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered, or 7-membered / 6-membered monospiroheterocyclyl group. Non-limiting examples are: [ka] This includes, among others.

[0234] The term "condensed heterocyclyl group" refers to a polycyclic heterocyclic system in which two adjacent atoms are shared between rings, and which may contain one or more double bonds within the ring, and which may contain at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group to form a sulfoxide or sulfone, but -OO-, -OS-, or -SS-). (excluding ), it is a monocyclic heterocyclyl group condensed with one or more monocyclic heterocyclyl groups, or a monocyclic heterocyclyl group condensed with one or more cycloalkyl groups, aryl groups, or heteroaryl groups, wherein the linking point is on the monocyclic heterocyclyl group and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5 to 20-membered condensed heterocyclyl group). The above condensed heterocyclyl group is preferably a condensed heterocyclyl group having 6 to 14 ring atoms (i.e., a 6 to 14-membered condensed heterocyclyl group), and more preferably a condensed heterocyclyl group having 7 to 10 ring atoms (i.e., a 7 to 10-membered condensed heterocyclyl group). The above-mentioned condensed heterocyclyl groups include bicyclic and polycyclic condensed heterocyclyl groups (e.g., tricyclic condensed heterocyclyl groups, tetracyclic condensed heterocyclyl groups, etc.), preferably bicyclic or tricyclic condensed heterocyclyl groups, and more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered, or 7-membered / 6-membered bicyclic condensed heterocyclyl groups. Non-limiting examples are: [ka] This includes, among others.

[0235] The term "bridged heterocyclyl group" refers to a polycyclic heterocyclic system in which rings share two atoms that are not directly linked, and which may contain one or more double bonds within the ring, and which contain at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group to form a sulfoxide or sulfone, but not containing -OO-, -OS-, or -SS-), and which has 5 to 20 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (i.e., a 5 to 20-membered bridged heterocyclyl group). The above-mentioned crosslinked heterocyclyl groups are preferably crosslinked heterocyclyl groups having 6 to 14 ring atoms (i.e., 6 to 14 membered crosslinked heterocyclyl groups), and more preferably crosslinked heterocyclyl groups having 7 to 10 ring atoms (i.e., 7 to 10 membered crosslinked heterocyclyl groups). Depending on the number of rings that make up the group, it can be divided into bicyclic crosslinked heterocyclyl groups and polycyclic crosslinked heterocyclyl groups (e.g., tricyclic crosslinked heterocyclyl groups, tetracyclic crosslinked heterocyclyl groups, etc.), and preferably bicyclic crosslinked heterocyclyl groups or tricyclic crosslinked heterocyclyl groups. Non-limiting examples are: [ka] This includes, among others.

[0236] The heterocyclyl group may or may not be substituted, and if substituted, it may be substituted at any available linking point, and preferably the substituent is one or more selected from a D atom, halogen, alkyl group, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, oxo group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0237] The term "aryl group" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aryl group) or a polycyclic aromatic ring system (i.e., a polycyclic aryl group) having a conjugated π-electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 6 to 14-membered aryl group). The aryl group is preferably an aryl group having 6 to 10 ring atoms (i.e., a 6 to 10-membered aryl group). The monocyclic aryl group is, for example, a phenyl group. The polycyclic aryl group includes, as non-limiting examples, a naphthyl group, anthryl group, a phenanthryl group, and the like. The above polycyclic aryl group further includes a phenyl group fused with one or more heterocyclyl groups or cycloalkyl groups, or a naphthyl group fused with one or more heterocyclyl groups or cycloalkyl groups, where the linking point is on the phenyl group or naphthyl group, and in this case, the number of ring atoms still indicates the number of ring atoms in the polycyclic aromatic ring system, as an unrestricted example, [ka] This includes, among others.

[0238] The aryl group may or may not be substituted, and if substituted, it may be substituted at any available linking point, and preferably the substituent is one or more selected from a D atom, halogen, alkyl group, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, oxo group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0239] The term "heteroaryl group" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl group) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl group) having a conjugated π-electron system, which contains at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur within its ring (the nitrogen may be optionally oxidized to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group to form a sulfoxide or sulfone, but without -OO-, -OS-, or -SS-), and which has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 5-14 membered heteroaryl group). The above heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5 to 10-membered heteroaryl group), more preferably a monocyclic heteroaryl group having 5 or 6 ring atoms (i.e., a 5 or 6-membered monocyclic heteroaryl group) or a bicyclic heteroaryl group having 8 to 10 ring atoms (i.e., an 8 to 10-membered bicyclic heteroaryl group), and most preferably a 5 or 6-membered monocyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen, and sulfur in the ring, or an 8 to 10-membered bicyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen, and sulfur in the ring.

[0240] The above monocyclic heteroaryl groups are, as non-limiting examples, furanyl group, thienyl group, thiazolyl group, isothiazolyl group, oxazolyl group, isoxazolyl group, oxadiazolyl group, thiadiazolyl group, imidazolyl group, pyrazolyl group, triazolyl group, tetrazolyl group, furazanyl group, pyrrolyl group, N-alkylpyrrolyl group, pyridyl group, pyrimidinyl group, pyridonyl group, N-alkylpyridone (for example, [ka] (etc.), including pyrazinyl groups, pyridazinyl groups, etc.

[0241] The above polycyclic heteroaryl groups include, as non-limiting examples, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalyl, phthalazinyl, benzimidazolyl, benzothienyl, quinazolinyl, benzothiazolyl, and carbazolyl groups. The above polycyclic heteroaryl groups further include those in which a monocyclic heteroaryl group is fused with one or more aryl groups, where the linkage point is on an aromatic ring, and in this case, the number of ring atoms still represents the number of ring atoms in a polycyclic heteroaromatic ring system. The above polycyclic heteroaryl groups further include those in which a monocyclic heteroaryl group is fused with one or more cycloalkyl or heterocyclyl groups, where the linkage point is on a monocyclic heteroaromatic ring, and in this case, the number of ring atoms still represents the number of ring atoms in a polycyclic heteroaromatic ring system. Non-limiting examples are, [ka] This includes, among others.

[0242] The heteroaryl group may or may not be substituted, and if substituted, it may be substituted at any available linkage point, and preferably the substituent is one or more selected from a D atom, halogen, alkyl group, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0243] The above-mentioned cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups include residues derived by removing one hydrogen atom from a parent ring atom, or residues derived by removing two hydrogen atoms from the same ring atom or two different ring atoms of the parent, i.e., "divalent cycloalkyl groups," "divalent heterocyclyl groups," "arylene groups," and "heteroarylene groups."

[0244] In the chemical structure of the compound described in this disclosure, [ka] The bond " indicates that the arrangement is not specified, that is, if a chiral isomer exists in the chemical structure, [ka] The combination " is " [ka] " or " [ka] It could be " or " [ka] " and " [ka] It is also possible to include both of these arrangements simultaneously.

[0245] The compounds relating to this disclosure include all suitable isotopic derivatives of the compound. The term "isotopic derivative" refers to a compound in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass. Examples of isotopes that can be introduced into the compounds relating to this disclosure include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, for example, 2 H (deuterium, D), 3 H (tritium, T), 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 32 p, 33 p, 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I,129 I and 131 I is one example, and deuterium is preferred.

[0246] Compared to non-deuterated drugs, deuterated drugs offer advantages such as reduced toxicity and side effects, improved drug stability, enhanced therapeutic effects, and extended biological half-life. All isotopic transformations of the compounds relating to this disclosure, whether radioactive or non-radioactive, are included within the scope of this disclosure. Each available hydrogen atom bonded to a carbon atom may be independently substituted with a deuterium atom, the deuterium substitution may be partial or complete, with partial deuterium substitution meaning that at least one hydrogen atom is substituted with at least one deuterium atom.

[0247] "Optional" or "optionally" means that the event or situation described thereafter may occur, but does not necessarily have to occur, and the description includes both cases in which the event or situation occurs and cases in which it does not.

[0248] The term "pharmaceutical composition" refers to a mixture comprising one or more anti-MUC1 antibodies or their antigen-binding fragments as described herein, anti-EGFR antibodies or their antigen-binding fragments, antigen-binding molecules that specifically bind to EGFR and MUC1, and antibody-drug conjugates thereof or pharmaceutically acceptable salts thereof, and other chemical components, the other components being, for example, physiologically / pharmaceutically acceptable carriers and excipients.

[0249] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation that is distinct from the active ingredient and is non-toxic to the target substance. pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0250] The terms “subject” or “individual” include humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates, sheep, dogs, cattle, chickens, amphibians and reptiles. Unless otherwise noted, the above terms “patient” or “subject” may be used interchangeably herein. In some embodiments, the individual or subject is human.

[0251] "Administer" or "give" means contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biofluid, when applied to an animal, human, subject, cell, tissue, organ, or biofluid.

[0252] The term "sample" refers to fluids, cells, or tissue-like samples isolated from a subject, and fluids, cells, or tissues present within the body of the subject. Exemplary samples are biological fluids, such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excrement, sputum, mucosal secretions from secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids from the abdominal cavity and other body cavities, fluids collected from bronchial lavage fluid, synovial fluid, liquid solutions that have come into contact with the subject or biological source, such as cell and organ culture media (including cell or organ-conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration tissue, surgically excised tissue, organ cultures, or cell cultures.

[0253] "Treatment" and "processing" (and their grammatical variations) refer to clinical interventions aimed at altering the natural processes of the individual being treated, and which may be carried out for preventive purposes or in the course of a clinicopathological process. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction / decreasation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and regression or improvement of the prognosis. In some embodiments, antibodies or their antigen-binding fragments, antigen-binding molecules or antibody-drug conjugates of the present disclosure are used to delay disease development or mitigate disease progression.

[0254] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or their potential causes, prevent the appearance of symptoms and / or their potential causes, and / or improve or improve damage caused by or associated with the disease state (e.g., lung disease). In some examples, the effective dose is a therapeutic effective dose or a prophylactic effective dose. A "therapeutic effective dose" is an amount sufficient to treat a disease state or symptoms, in particular a state or symptoms associated with the disease state, or to prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptoms otherwise associated with the disease. A "prophylactic effective dose" is an amount, when administered to a subject, that provides a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete therapeutic or prophylactic effects do not necessarily occur with a single dose, but may occur after a series of doses have been administered. Therefore, a therapeutic or prophylactic effective dose can be administered in one or multiple doses. The "therapeutic dose" and "preventive dose" can vary depending on various factors, such as the individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, the patient's improved health status.

[0255] The anti-MUC1 antibody or its antigen-binding fragment, the anti-EGFR antibody or its antigen-binding fragment, and antigen-binding molecules that specifically bind to EGFR and MUC1 as disclosed herein. This disclosure provides an anti-MUC1 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, and an antigen-binding molecule that specifically binds to EGFR and MUC1, having many advantageous properties such as good in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties, and drug discovery potential (e.g., yield, purity, and stability).

[0256] Exemplary anti-MUC1 antibodies or their antigen-binding fragments Examples of the present disclosure include antibody series M4, M6, F4-1, and F4-18. Hereinafter, antibody M4 will be used as an example to describe the antibodies or their antigen-binding fragments in the present disclosure.

[0257] Exemplary examples include an anti-MUC1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2, and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 4, and LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5, respectively.

[0258] Exemplary examples include an anti-MUC1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 15, LCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0259] Exemplary examples include an anti-MUC1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is represented by the amino acid sequence of HCDR1 shown in SEQ ID NO: 12, the amino acid sequence of HCDR2 shown in SEQ ID NO: 13, and the amino acid sequence of HCDR3 shown in SEQ ID NO: 14, and the light chain variable region is represented by the amino acid sequence of LCDR1 shown in SEQ ID NO: 15, the amino acid sequence of LCDR2 shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 shown in SEQ ID NO: 17.

[0260] Exemplary examples include the anti-MUC1 antibody or its antigen-binding fragment, which is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, it is a chimeric antibody or a fully human antibody. In some embodiments, it is a humanized antibody.

[0261] Exemplary examples include an anti-MUC1 antibody or an antigen-binding fragment thereof comprising a framework region (FR) of a human antibody.

[0262] Exemplary examples include an anti-MUC1 antibody or antigen-binding fragment thereof of the present disclosure, wherein the heavy chain variable region comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 36, 37, or 38, and the light chain variable region comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 39, 40, 41, or 42; or the heavy chain variable region comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 5.

[0263] Exemplary examples include an anti-MUC1 antibody or antigen-binding fragment thereof of the present disclosure, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 38K, 40R, 48I, 71A, 73K, 76D, and 82aR, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV1-39*01, 1GKV6-21*02, or IGKV3-11*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 3V, 43S, 47W, 49Y, and 60G. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0264] In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region is such that HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 38K, 40R, 48I, 71A, 73K, 76D, and 82aR, and the light chain variable region is such that LCDR1 comprises the amino acid sequence of SEQ ID NO: 15, LCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 3V, 43S, 47W, 49Y, and 60G. In some embodiments, the variable regions and CDRs are defined according to Kabat numbering rules.

[0265] In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the FR of the heavy chain variable region comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 38K, 40R, 48I, 71A, 73K, 76D, and 82aR, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17, and the FR of the light chain variable region comprises one or more amino acid substitutions selected from the group consisting of 3V, 43S, and 47W. In some embodiments, the variable regions and CDRs are defined according to Kabat numbering rules.

[0266] In some embodiments, the above-described anti-MUC1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the FR of the heavy chain variable region comprises the amino acid substitutions 1E, 71A, 73K, and 76D, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17, and the FR of the light chain variable region comprises the amino acid substitutions 43S and 47W. In some embodiments, the variable regions and CDRs are defined according to Kabat numbering rules.

[0267] Exemplary examples include an anti-MUC1 antibody or an antigen-binding fragment thereof of the present disclosure, which is an antibody fragment, and in some embodiments, it is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, and dAb.

[0268] Exemplary examples include an anti-MUC1 antibody or an antigen-binding fragment thereof comprising a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the light chain constant region is a human κ or λ light chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 69 or 186, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 69, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 70.

[0269] Exemplary examples include an anti-MUC1 antibody or an antigen-binding fragment thereof of the present disclosure, wherein the anti-MUC1 antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NOs. 71, 73, 75, or 77, and the light chain comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NOs. 72, 74, 76, or 78.

[0270] Exemplary, an anti-MUC1 antibody or its antigen-binding fragment thereof, wherein the anti-MUC1 antibody comprises a heavy chain and a light chain, of which, The amino acid sequence of the heavy chain is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 73, and the amino acid sequence of the light chain is shown in SEQ ID NO: 74, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 75, and the amino acid sequence of the light chain is shown in SEQ ID NO: 76, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain is shown in SEQ ID NO: 78.

[0271] Exemplary examples include an anti-MUC1 antibody or an antigen-binding fragment thereof according to the present disclosure, wherein the anti-MUC1 antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO: 71, and the amino acid sequence of the light chain being shown in SEQ ID NO: 72.

[0272] Exemplary, the present disclosure further provides isolated anti-MUC1 antibodies or antigen-binding fragments thereof that competitively bind to human MUC1 with any one of the anti-MUC1 antibodies described above.

[0273] Exemplary, an isolated anti-MUC1 antibody or its antigen-binding fragment relating to the present disclosure, wherein 5 × 10 -8 Less than M (for example, 4 × 10) -8 Less than M, 3 x 10 -8 Less than M, 2.5 × 10 -8 Less than M, 2 x 10 -8 Less than M, 1.5 × 10 -8 Less than M, 1 x 10 -8 Less than M, 9 x 10 -9 Less than M, 8 x 10 -9 Less than M, 7 x 10 -9 Less than M, 6 x 10 -9 Less than M, 5 x 10 -9 Less than M, 4 x 10 -9 Less than M, 3 x 10 -9 Less than M, 2 x 10 -9It binds to human MUC1 with a KD value (less than M), and the above KD value is measured by Biacore.

[0274] Exemplary anti-EGFR antibodies or their antigen-binding fragments Examples of the present disclosure disclose an anti-EGFR antibody or an antigen-binding fragment thereof.

[0275] Exemplary examples include an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 116, HCDR2 comprising the amino acid sequence of SEQ ID NO: 117, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 129, and the light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 119, LCDR2 comprising the amino acid sequence of SEQ ID NO: 120, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 121.

[0276] Exemplary examples include the anti-EGFR antibody or its antigen-binding fragment, which is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, it is a chimeric antibody or a fully human antibody. In some embodiments, it is a humanized antibody.

[0277] Exemplary examples include an anti-EGFR antibody or antigen-binding fragment thereof of the present disclosure, which comprises a framework region (FR) of a human antibody.

[0278] Exemplary, an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 138 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 149.

[0279] Exemplary examples include an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 153 and the light chain comprises the amino acid sequence of SEQ ID NO: 164.

[0280] Exemplary antigen-binding molecules that specifically bind to EGFR and MUC1 The embodiments of this disclosure disclose antigen-binding molecules that specifically bind to EGFR and MUC1.

[0281] Exemplary, an antigen-binding molecule that specifically binds to EGFR and MUC1 of the present disclosure comprises at least one antigen-binding module that specifically binds to EGFR and at least one antigen-binding module that specifically binds to MUC1, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL, of which, The above MUC1-VH includes the amino acid sequence of SEQ ID NO: 12 in HCDR1, the amino acid sequence of SEQ ID NO: 13 in HCDR2, and the amino acid sequence of SEQ ID NO: 14 in HCDR3; the above MUC1-VL includes the amino acid sequence of SEQ ID NO: 15 in LCDR1, the amino acid sequence of SEQ ID NO: 16 in LCDR2, and the amino acid sequence of SEQ ID NO: 17 in LCDR3; the above EGFR-VH includes the amino acid sequence of SEQ ID NO: 116 in HCDR1, the amino acid sequence of SEQ ID NO: 117 in HCDR2, and the amino acid sequence of SEQ ID NO: 129 in HCDR3; and the above EGFR-VL includes the amino acid sequence of SEQ ID NO: 119 in LCDR1, the amino acid sequence of SEQ ID NO: 120 in LCDR2, and the amino acid sequence of SEQ ID NO: 121 in LCDR3.

[0282] Exemplary, an antigen-binding molecule that specifically binds to EGFR and MUC1 of the present disclosure comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d). (a)[MUC1-VH]-[CH1]-[Fc1], (b)[MUC1-VL]-[CL], (c)[EGFR-VH]-[GGGGS]-[Titin]-[Fc2], (d) [EGFR-VL]-[GGGGS]-[Obscurin], Of these, the structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus.

[0283] Exemplary examples include antigen-binding molecules that specifically bind to EGFR and MUC1 of the present disclosure, the format of which is an asymmetric structure molecule comprising four chains, as shown in Figure 5, wherein MUC1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14; MUC1-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129; and EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121.

[0284] Exemplary, an antigen-binding molecule that specifically binds to EGFR and MUC1 of the present disclosure, having a format asymmetric structure molecule comprising four chains, as shown in Figure 5, wherein MUC1-VH comprises the amino acid sequence of SEQ ID NO: 36, MUC1-VL comprises the amino acid sequence of SEQ ID NO: 39, EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149.

[0285] Exemplary examples include antigen-binding molecules that specifically bind to EGFR and MUC1 of the present disclosure, comprising a first chain represented by SEQ ID NO: 171, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 174, and a fourth chain represented by SEQ ID NO: 173.

[0286] Exemplary examples include antigen-binding molecules that specifically bind to EGFR and MUC1 of the present disclosure, comprising a first chain represented by SEQ ID NO: 178, a second chain represented by SEQ ID NO: 74, a third chain represented by SEQ ID NO: 179, and a fourth chain represented by SEQ ID NO: 173.

[0287] Exemplary antibody-drug conjugates or pharmaceutically acceptable salts thereof The examples of this disclosure disclose antibody-drug conjugates represented by the general formula (Pc-LYD) or pharmaceutically acceptable salts thereof. [ka] Eventually, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b They are the same or different, and each is independently a hydrogen atom, a deuterium atom, a halogen, and C 1-6 Selected from alkyl groups, R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. m is 0, 1, 2, 3, or 4. n is 2 to 8, preferably 4 to 8, and more preferably 4 to 6. L is the linker unit, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1, comprising one antigen-binding module that specifically binds to EGFR and one antigen-binding module that specifically binds to MUC1, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL, of which, The above MUC1-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14; the above MUC1-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; the above EGFR-VH includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129; and the above EGFR-VL includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121. Preferably, among them, MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, EGFR-VH contains the amino acid sequence of SEQ ID NO: 138, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 149. More preferably, the antigen-binding molecule comprises a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 174, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173; or a first chain containing the amino acid sequence of SEQ ID NO: 178, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 179, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0288] Exemplary examples include an antibody-drug conjugate represented by the general formula (Pc-LYD) of this disclosure or a pharmaceutically acceptable salt thereof, which is of the general formula (Pc-L a An antibody-drug conjugate represented by -YD) or a pharmaceutically acceptable salt thereof, [ka] Eventually, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described above. m is 0, 1, 2, 3, or 4. n is 2 to 8, preferably 4 to 8, and more preferably 4 to 6. R 1 This is a 3-6 member cycloalkyl-C 1-6 It is an alkyl group or a 3-6 membered cycloalkyl group. R 2 C is a hydrogen atom. 1-6 Selected from haloalkyl groups and 3-6 membered cycloalkyl groups, Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkyl group. W is C 1-6 Alkylene group and C 1-6 Selected from alkylene-3 to 6-membered cycloalkyl groups, L 2 It is a chemical bond, L 3This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally include halogen, hydroxyl group, cyano group, amino group, and C 1-6 Alkyl alkyl group, C 1-6 Haloalkyl group, C 1-6 Deuterated alkyl groups, C 1-6 Substituted with one or more substituents selected from alkoxy groups and 3- to 6-membered cycloalkyl groups, R 5 is a hydrogen atom or C 1-6 It is an alkyl group, R 6 and R 7 They are identical or different, and each is independently a hydrogen atom or C 1-6 It is an alkyl group.

[0289] Exemplary examples include an antibody-drug conjugate represented by the general formula (Pc-LYD) of this disclosure or a pharmaceutically acceptable salt thereof, which is an antibody-drug conjugate represented by the general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof. [ka] Eventually, n is 2 to 8, preferably 4 to 8, and more preferably 4 to 6. Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1, as described above.

[0290] Antibody structure In one embodiment, the antibody provided herein is a full-length antibody.

[0291] In one embodiment, the antibody provided herein is an antibody fragment.

[0292] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, and is particularly a Fab fragment. "Fab" is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. "Fab fragment" may be produced by lysing the antibody with papain. "Fab'" contains VL, CL, VH, and CH1, and further includes a region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains of two Fab' fragments, making it possible to form an F(ab')2 molecule. "Fab'-SH" is a Fab' fragment in which a cysteine ​​residue in the constant region contains a free mercapto group. "F(ab')2" is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region.

[0293] In another embodiment, the antibody fragment is a biantibody, triplobody, or quadruplobody. A biantibody is an antibody fragment containing two antigen-binding sites, the fragment containing linked VH and VL on the same polypeptide chain (VH-VL). By using a linker that is too short, pairing of two domains on the same chain becomes impossible, forcing those domains to pair with complementary domains on another chain, thereby producing two antigen-binding sites, the two antigens may be the same or different.

[0294] In another embodiment, the antibody fragment is a single-stranded Fab fragment. The "single-stranded Fab fragment" or "scFab" is a polypeptide comprising VH, CH1, VL, CL and a linker, wherein the antibody domain and the linker have one of the following sequences from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In one embodiment, the linker is a polypeptide containing at least 30 amino acids. In another embodiment, the linker is a polypeptide containing 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between CL and CH1. Furthermore, by inserting cysteine ​​residues (for example, at position 44 in the heavy chain variable region and position 100 in the light chain variable region, numbered by Kabat), interchain disulfide bonds are produced, which can further stabilize these single-chain Fab molecules.

[0295] In another embodiment, the antibody fragment is an Fv fragment consisting of the VH and VL domains of a single arm of the antibody.

[0296] In another embodiment, the antibody fragment is a single-chain variable fragment (scFv). "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are sequentially linked by a short flexible peptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, in this specification, scFv may comprise the VL and VH variable regions in any order; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0297] In another embodiment, the antibody fragment is a dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is substituted with a cysteine ​​residue via disulfide bonds between the cysteine ​​residues. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody by a known method (Protein Engineering. 7:697 (1994)).

[0298] In another embodiment, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment that includes all or part of the heavy chain variable domain of an antibody, or all or part of the light chain variable domain.

[0299] In one embodiment, the antibody provided herein is a chimeric antibody. In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, the chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from the class or subclass of the parent antibody.

[0300] In one embodiment, the antibody is a humanized antibody. Typically, humanization of a non-human antibody reduces its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, of which the CDR or a portion thereof is derived from a non-human antibody, while the FR or a portion thereof is derived from a human antibody. Optionally, the humanized antibody may further contain a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody may be substituted with corresponding residues from a non-human antibody (e.g., an antibody providing the CDR sequence).

[0301] Humanized antibodies and methods for their production are outlined in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further discussed in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409, and Kashmiri et al., Methods. This topic is described in publications such as 36:25-34 (2005) (regarding grafting of specific determination regions (SDRs)), Padlan, Mol.Immunol.28:489-498 (1991) (regarding "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (regarding "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer 83:252-260 (2000) (regarding the "induction selection" method for FR shuffling).

[0302] Human framework regions usable for humanization include framework regions selected by the "best-fit" method (see, e.g., Sims et al., J.Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subclasses of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al., Proc.Natl.Acad.Sci.USA, 89:4285 (1992) and Presta et al., J.Immunol., 151:2623 (1993)), framework regions of human maturation (somatic mutation) or human germline framework regions (see, e.g., Almagro and Fransson, Front.Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening the FR library (e.g., Baca et al. This includes, but is not limited to, al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0303] Mutants of anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments In one embodiment, the following are covered: amino acid sequence variants of anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments, provided herein. For example, these can be expected to improve the binding affinity and / or other biological properties of the antibodies. Amino acid sequence variants of antibodies can be prepared by introducing suitable modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or substitution of residues in the amino acid sequence of anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments. The final construct can be obtained by any combination of deletions, insertions, and substitutions, provided that the final construct has desired properties, such as antigen-binding properties.

[0304] Substitution, insertion, and deletion of mutants In one embodiment, antigen variants are provided that include one or more amino acid substitutions. The sites of interest for substitutional mutagenesis include CDR and FR. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into antibodies of interest, and the products can be screened for desired activities such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0305] [Table 2]

[0306] [Table 3]

[0307] Amino acids can be grouped as follows based on their general side-chain characteristics: (1) Hydrophobic substances: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral, hydrophilic substances: Cys, Ser, Thr, Asn, Gln, (3) Acidic substances: Asp, Glu, (4) Basic substances: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0308] Non-conservative substitution requires replacing a member in one of these classes with a member in another class.

[0309] A single substitution variant involves the substitution of one or more CDR residues in a parent antibody (e.g., a humanized or human antibody). Generally, variants selected for further study have altered (e.g., improved) biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody, and / or fundamentally retain certain biological properties of the parent antibody. One exemplary substitution variant is an affinity-matured antibody, which can be readily produced, for example, by affinity-maturation techniques based on phage display (e.g., techniques as described herein). In short, one or more CDR residues are mutated, the mutant antibody is displayed on a phage, and its specific biological activity (e.g., binding affinity) is screened. For example, alterations (e.g., substitutions) can be made to CDRs to improve antibody affinity. Such alterations can be made to CDR "hotspots," i.e., residues encoded by codons that are frequently mutated during somatic cell maturation, and / or residues that come into contact with the antigen, and the resulting mutant VH or VL can be tested for binding affinity. In several embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then created. Furthermore, the library is screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR orientation method that randomizes several CDR residues (e.g., 4-6 residues at a time). For example, CDR residues involved in antigen binding can be specifically identified by alanine scanning mutagenesis or modeling. HCDR3 and LCDR3 are particularly often targeted.

[0310] In some embodiments, substitutions, insertions, or deletions may be made in one or more CDRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, a conservative change (e.g., a conservative substitution, as provided herein) that does not substantially reduce binding affinity can be made to a CDR. Such changes may be, for example, outside the antigen contact residue in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is either unchanged or contains one, two, or three or fewer amino acid substitutions.

[0311] A method that can be used to identify residues or regions in antibodies that could be target sites for mutagenesis is called "alanine scanning mutagenesis." In this method, one residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that are functionally sensitive to the initial substitution. Contact points between the antibody and the antigen can also be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues may be targeted or removed as substitution candidates. Mutants may be screened to determine whether they contain desired properties.

[0312] Amino acid insertions include amino-terminal and / or carboxyl-terminal fusions of polypeptides, ranging in length from one residue to 100 or more residues, and intra-sequence insertions of one or more amino acid residues. An example of terminal insertion is an antibody containing an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N or C terminus of an antibody with a polypeptide that extends the serum half-life of an enzyme or antibody.

[0313] Recombination Anti-MUC1 antibodies or their antigen-binding fragments, and anti-EGFR antibodies or their antigen-binding fragments can be produced by recombinant methods. For these methods, one or more isolated nucleic acids encoding anti-MUC1 antibodies or their antigen-binding fragments, and anti-EGFR antibodies or their antigen-binding fragments are provided.

[0314] In one embodiment, the Disclosure provides isolated nucleic acids encoding the above-described anti-MUC1 antibody or its antigen-binding fragment, or the anti-EGFR antibody or its antigen-binding fragment. Each of these nucleic acids can independently encode any one of the above-described polypeptide chains. In another embodiment, the Disclosure provides one or more vectors (e.g., expression vectors) containing such nucleic acids. In another embodiment, the Disclosure provides host cells containing such nucleic acids. In one embodiment, the Disclosure provides a method for preparing a polypeptide or fusion protein, comprising culturing host cells containing the nucleic acid encoding the polypeptide or fusion protein under conditions suitable for expression, as provided above, and optionally recovering the above-described anti-MUC1 antibody or its antigen-binding fragment from the host cells (or host cell medium).

[0315] To recombinantly produce anti-MUC1 antibodies or their antigen-binding fragments, or anti-EGFR antibodies or their antigen-binding fragments, protein-encoding nucleic acids are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods, or obtained by chemical synthesis.

[0316] Suitable host cells for cloning or expressing an anti-MUC1 antibody or its antigen-binding fragment, or an anti-EGFR antibody or its antigen-binding fragment, include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, they can be isolated from a paste-like substance of bacterial cells in a soluble fraction and further purified.

[0317] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable clones or expression hosts for vectors encoding fusion proteins, and include fungal and yeast strains. Suitable host cells for the expression of fusion proteins may be derived from multicellular organisms (invertebrates and vertebrates), and examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have already been identified and may be used in combination with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts, such as US5959177, US6040498, US6420548, US7125978, and US6417429, or vertebrate cells, such as mammalian cell lines suitable for growth in suspension, may be used as hosts. Other suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7), human fetal kidney cell line (293 or 293T cells), baby hamster kidney cell line (BHK), mouse Sertoli cell line (TM4 cell line), monkey kidney cell line (CV1), African green monkey kidney cell line (VERO-76), human cervical cancer cell line (HELA), canine kidney cell line (MDCK), buffalo rat hepatocyte cell line (BRL3A), human lung cell line (W138), human hepatocyte cell line (Hep G2), mouse mammary tumor cell line (MMT 060562), TRI cell line, MRC 5 cell line, and FS4 cell line. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cell line, including DHFR-CHO cell line, and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of several mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0318] measurement The anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts provided herein can be identified, screened, or characterized for their physical / chemical features and / or biological activity by various assay methods known in the art. In one embodiment, the activity of the anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments of this disclosure is tested by known methods such as ELISA and Western blotting.

[0319] Treatment method and route of administration Any anti-MUC1 antibody or its antigen-binding fragment, anti-EGFR antibody or its antigen-binding fragment, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts thereof provided herein can be used in therapeutic methods. In another embodiment, this disclosure provides the use of anti-MUC1 antibody or its antigen-binding fragment, anti-EGFR antibody or its antigen-binding fragment, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts thereof in the manufacture or preparation of pharmaceuticals. In some embodiments, the disease is a disease or condition related to MUC1 or EGFR. In some embodiments, the disease is a tumor. In some embodiments, the above-mentioned diseases include astrocytoma (e.g., undifferentiated astrocytoma), glioblastoma, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., breast cancer characterized by BRCA1 and / or BRCA2 mutations), cervical cancer, colorectal cancer (e.g., colon cancer and rectal cancer), fallopian tube cancer, gallbladder cancer, gastric cancer, head and neck cancer, idiopathic myelofibrosis, renal cancer (e.g., renal cell carcinoma, renal rhabdoid tumor and Wilms' tumor), leukemia, liver cancer (e.g., hepatocellular carcinoma), esophageal cancer (also called "esophageal cancer," e.g., esophageal squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, and neurological cancers. The cancer is selected from blastoma, oligodendroglioma, ovarian cancer, peritoneal tumor, pancreatic cancer, polycythemia vera, primary neuroectodermal tumor, prostate cancer, retinoblastoma, sarcoma (e.g., chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma and soft tissue sarcoma), squamous cell carcinoma (e.g., cutaneous squamous cell carcinoma), thyroid cancer, endometrial cancer, vestibular schwannoma, blastoma, vulvar cancer, thymoma, testicular cancer, cholangiocarcinoma, pheochromocytoma, paraganglioma and adenoid cystic carcinoma, and in some embodiments, the cancer is selected from lung cancer, head and neck cancer, esophageal cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer and bladder cancer. In some embodiments, the cancer is lung cancer, preferably non-small cell lung cancer.

[0320] In another embodiment, the present invention provides a pharmaceutical composition comprising, for example, an anti-MUC1 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, an antigen-binding molecule that specifically binds to EGFR and MUC1, and an antibody-drug conjugate thereof or a pharmaceutically acceptable salt thereof, for use in any of the above-described pharmaceutical uses or therapeutic methods. In one embodiment, the pharmaceutical composition comprises any anti-MUC1 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, an antigen-binding molecule that specifically binds to EGFR and MUC1, and an antibody-drug conjugate thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0321] The anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts can be used alone or in combination with other reagents for therapeutic purposes. For example, the antibodies of the Disclosure may be administered together with at least one other therapeutic agent.

[0322] The anti-MUC1 antibodies or their antigen-binding fragments, anti-EGFR antibodies or their antigen-binding fragments, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts (and any other therapeutic agents) of this disclosure may be administered by any preferred means, including parenteral, intrapulmonary, and intranasal, and, if local treatment is required, into the lesion. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be carried out by any suitable route, for example, by injection such as intravenous or subcutaneous injection, which is determined in part by whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but are not limited to, single doses or multiple doses at multiple time points, bolus doses, and pulse infusions.

[0323] The anti-MUC1 antibody or its antigen-binding fragment, anti-EGFR antibody or its antigen-binding fragment, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts thereof are prepared, administered, and given in accordance with good medical practice. Possible factors in this context include the specific disease being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of reagent delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals. The anti-MUC1 antibody or its antigen-binding fragment, anti-EGFR antibody or its antigen-binding fragment, antigen-binding molecules that specifically bind to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts may be prepared with or without one or more reagents currently used for the prevention or treatment of the above-mentioned diseases. The effective amount of such other reagents is determined by the amount present in the pharmaceutical composition, the type of disease or treatment, and other factors. They are generally used in the same doses and routes of administration as described herein, or in approximately 1% to 99% of the doses described herein, or in different doses and via any route determined to be empirically / clinically preferable.

[0324] For the prevention or treatment of disease, the appropriate dose of the anti-MUC1 antibody or its antigen-binding fragment, the anti-EGFR antibody or its antigen-binding fragment, the antigen-binding molecule that specifically binds to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts thereof (used alone or in combination with one or more other therapeutic agents) is determined by the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is for prevention or treatment, past treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient as appropriate, either once or in a series of treatments.

[0325] product In another aspect of this disclosure, a product (e.g., a kit) is provided which includes materials that can be used for the treatment, prevention and / or diagnosis of the above-mentioned disease. The product includes a container and a label or package insert on or in combination with the container. Preferred containers include, for example, bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials, such as glass or plastic. The container may contain a composition that effectively treats, prevents and / or diagnoses a disease, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a plug through which a subcutaneous injection needle can be inserted). At least one active agent in the composition is an anti-MUC1 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, an antigen-binding molecule that specifically binds to EGFR and MUC1, and their antibody-drug conjugates or pharmaceutically acceptable salts. The label or package insert indicates that the composition is used to treat a selected disease. The product may also comprise (a) a first container containing a composition comprising the active molecule of the Disclosure, and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent. The product in such embodiments of the Disclosure may further comprise a package insert indicating that the composition can be used to treat a particular medical condition. Selectively, or further, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer. From a business and user perspective, it may further comprise other necessary materials, including other buffers, diluents, filters, needles and syringes.

[0326] The present disclosure is further described below in accordance with examples and test examples, but these examples and test examples are not intended to limit the scope of the present disclosure. In the examples or test examples of the present disclosure, experimental methods for which specific conditions are not specified are generally performed under normal conditions, for example, according to Cold Spring Harbor's Antibody Technology Experiment Manual, Molecular Cloning Manual, or according to conditions recommended by the raw material or product manufacturer, and reagent materials for which specific sources are not specified were obtained by purchasing from the market.

[0327] Examples Example 1: Preparation of MUC1 antigen Using the UniProt MUC1 antigen (human MUC1 protein, Uniprot number: P15941) as a MUC1 template, the amino acid sequences of the antigen and detection proteins used in this disclosure were designed, and different tags such as His tags or Fc tags were selectively fused to the MUC1 protein. Each was cloned into a pTT5 vector (Biovector, CAT#102762), expressed by transient transfection in 293 cells, purified, and obtained the antigen and detection proteins of this disclosure.

[0328] The extracellular domain sequence of the MUC1-C protein with a His tag (abbreviated as MUC1-CL-6xHis) was used as an immunoantigen and detection reagent. [ka] Note: The underlined portion is the 6×His tag, and the bolded portion is the extracellular domain of the MUC1 protein.

[0329] The sequence of a fusion protein (abbreviated as hMUC1-CL-Fc) between the extracellular domain of the MUC1-C protein and Human-IgG1-Fc was used as the immunogen. [ka] Note: The underlined portion is the Human-IgG1-Fc portion, and the bolded portion is the extracellular domain of the MUC1 protein.

[0330] Simultaneously, the sequence of the fusion protein (abbreviated as Cyno MUC1-C-Fc) between the extracellular domain of the Cyno-MUC1-C protein and Human-IgG1-Fc was also used as a detection reagent. [ka] Note: The underlined portion is the Human-IgG1-Fc portion, and the bolded portion is the extracellular domain of the Cyno-MUC1 protein.

[0331] Example 2: Purification of recombinant protein related to MUC1 1. Purification step of His-tagged recombinant protein The cell expression supernatant sample was centrifuged at high speed to remove impurities. The nickel column was equilibrated with a PBS solution containing 20 mM imidazole and washed with 2–5 times the column volume. The replaced cell supernatant sample was chromatographed onto a Ni Sepharose excel column (GE, 17-3712-02). The column was rinsed with PBS solution until the A280 reading was reduced to the baseline. The chromatography column was then washed with PBS + 20 mM imidazole to remove nonspecifically bound heteroproteins, and the effluent was collected. Furthermore, the target protein was eluted with a PBS solution containing 300 mM imidazole, the elution peak was collected, and the target protein was transferred to PBS in a concentration tube and concentrated to the appropriate concentration. The obtained protein was identified accurately by electrophoresis, peptide mapping, and LC-MS, and then aliquoted for use. His-tagged MUC1-CL-6xHis was obtained and used as a detection reagent for the antibodies of this disclosure.

[0332] 2. Purification step of MUC1-CL-Fc fusion protein The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the supernatant was subjected to affinity chromatography using MabSelect Sure (GE, 17-5438-01). The MabSelect Sure chromatography column was first regenerated with 0.2 M NaOH, then equilibrated with PBS, and after binding the supernatant, it was washed with PBS until the A280 reading was reduced to the baseline. The target protein was eluted with 0.1 M acetate buffer at pH 3.5 and neutralized with 1 M Tris-HCl. The target protein was transferred to PBS in a concentration tube and concentrated to an appropriate concentration, and the resulting protein was identified accurately by electrophoresis and LC-MS before being aliquoted for use. This method is used to purify the MUC1-CL-Fc fusion protein, but the method may also be used to purify the antibody protein in this disclosure.

[0333] 3. Purification step for hybridoma screening antibody sample expression: The cell expression supernatant sample was centrifuged at high speed to remove impurities, and Protein A magnetic bead packing material (SM003100) was added to the supernatant. The mixture was incubated with shaking at room temperature for 3 hours. The packing material was washed three times with PBS and then once with ultrapure water. The target protein was eluted with 0.1 M acetate buffer at pH 3.0 and neutralized with 1 M Tris-HCl. The target protein was transferred to PBS in a concentrating tube and concentrated to an appropriate concentration. The resulting protein was identified accurately by electrophoresis and LC-MS, and then aliquoted for use.

[0334] Example 3: Mouse immunization plan for mouse anti-MUC1-C antibody and acquisition of hybridoma antibody 1. Mouse immunity Anti-human MUC1-C antibody was produced by immunizing mice. In the experiment, female Balb / c and SJL white mice, 6-8 weeks old (Shanghai SLAC Laboratory Animal Co., Ltd., Animal Production License Number: SCXK (Hu) 2017-0005), were used. Breeding environment: SPF grade. After purchasing the mice, they were bred in the laboratory environment for one week, with the light / dark cycle adjusted to 12 / 12 hours, the temperature at 20-25 °C, and the humidity at 40%-60%. The mice acclimated to the environment were immunized according to the following plan.

[0335] Immunization Plan 1: Immunization was carried out using the protein antigen (hMUC1 C-L-Fc). The protein antigen (hMUC1 C-L-Fc) was cross-immunized using TiterMax® Gold Adjuvant (Sigma) and Thermo Imject® Alum (Thermo) adjuvant. The ratio of the protein antigen (hMUC1 C-L-Fc) to the adjuvant (TiterMax® Gold Adjuvant) was 1:1, and after emulsification, it was inoculated. The ratio of the protein antigen (hMUC1 C-L-Fc) to the adjuvant (Thermo Imject® Alum) was 3:1, and after shaking and uniformly mixing, it was inoculated. It was 50 μg / mouse / time (primary immunization), 25 μg / mouse / time (regular immunization), and 50 μg / mouse / time (boost immunization). The immunization times were 0, 14, 34, 48, and 78 days. Blood was collected on days 26, 40, 57, and 82 to measure the antibody titer in the mouse serum. After the 4th to 5th immunizations, the antibody titer in the mouse serum was determined by the ELISA method, and mice with a tendency to have a high and stable antibody titer in the serum were selected for spleen cell fusion. Boost immunization was carried out 3 days before spleen cell fusion, and an antigen solution prepared with the protein antigen (hMUC1 C-L-Fc) and physiological saline was injected intraperitoneally (i.p.) at 50 μg / mouse, or a cell antigen solution resuspended in phosphate buffer solution was injected intraperitoneally (i.p.) at 1×10 7 cells / mouse.

[0336] Immunization Plan 2: Immunization was performed using cross-immunization with a protein antigen (hMUC1-CL-His) and a cell antigen (CHO-K1-MUC1-C). Of these, the protein antigen (hMUC1-CL-His) was cross-immunized using TiterMax® Gold Adjuvant (Sigma) and Thermo Imject® Alum (Thermo) adjuvants. The ratio of protein antigen (hMUC1-CL-His) to adjuvant (TiterMax® Gold Adjuvant) was 1:1, and it was inoculated after emulsification. The ratio of antigen hMUC1-CL-Fc to adjuvant (Thermo Imject® Alum) was 3:1, and it was inoculated after being homogenized by shaking. The dosages are 50 μg / animal / administered (initial immunization), 25 μg / animal / administered (regular immunization), and 50 μg / animal / administered (booster immunization). Immunization to the cellular antigen (CHO-K1-MUC1-C) is 1 × 10⁻¹⁶. 7 Cells / animal / inoculation were used. Cell antigens were resuspended in phosphate buffer solution before inoculation. Inoculation times were days 0, 14, 34, 48, 69, and 86. Blood samples were collected on days 26, 40, 57, 82, and 96. After 5-9 immunizations, antibody titers in mouse serum were determined by ELISA. Mice with high and stable antibody titers in their serum were selected, and their spleens were taken for spleen cell fusion. An additional immunization was performed 3 days before spleen cell fusion, and 1 × 10⁶ cells of cell antigen solution resuspended in phosphate buffer solution were administered. 7 Cells / animal were injected intraperitoneally (IP).

[0337] Immunization Plan 3: Immunization was performed using cross-immunization with a protein antigen (hMUC1-CL-Fc) and a cell antigen (CHO-K1-MUC1-C). Of these, the protein antigen (hMUC1-CL-Fc) was cross-immunized using TiterMax® Gold Adjuvant (Sigma) and Thermo Imject® Alum (Thermo) adjuvants. The ratio of protein antigen (hMUC1-CL-Fc) to adjuvant (TiterMax® Gold Adjuvant) was 1:1, and it was inoculated after emulsification. The ratio of antigen hMUC1-CL-Fc to adjuvant (Thermo Imject® Alum) was 3:1, and it was inoculated after being homogenized by shaking. The dosages are 50 μg / animal / administered (initial immunization), 25 μg / animal / administered (regular immunization), and 50 μg / animal / administered (booster immunization). Immunization to the cellular antigen (CHO-K1-MUC1-C) is 1 × 10⁻¹⁶. 7 Cells / animal / inoculation were used. Cell antigens were resuspended in phosphate buffer solution before inoculation. Inoculation times were days 0, 12, 25, 40, 55, 112, 127, and 156. Blood samples were collected on days 21, 35, 49, 63, 108, and 154. After 5-8 immunizations, antibody titers in mouse serum were determined by ELISA. Mice with high and stable antibody titers in their serum were selected, and their spleens were removed for spleen cell fusion. Three days before spleen cell fusion, an additional immunization was performed, and an antigen solution prepared with protein antigen (hMUC1 CL-Fc) and physiological saline was injected intraperitoneally (ip) at a dose of 50 μg / animal.

[0338] Immunization Plan 4: Immunization was performed using cross-immunization with a protein antigen (hMUC1-CL-Fc) and a cell antigen (HEK293-MUC1-C). Of these, the protein antigen (hMUC1-CL-Fc) was cross-immunized using TiterMax® Gold Adjuvant (Sigma) and Thermo Imject® Alum (Thermo) adjuvants. The ratio of protein antigen (hMUC1-CL-Fc) to adjuvant (TiterMax® Gold Adjuvant) was 1:1, and it was inoculated after emulsification. The ratio of antigen hMUC1-CL-Fc to adjuvant (Thermo Imject® Alum) was 3:1, and it was inoculated after homogeneous mixing by shaking. The doses were 50 μg / animal / dose (primary immunization), 25 μg / animal / dose (normal immunization), and 50 μg / animal / dose (booster immunization). Immunity to the cellular antigen (HEK293-MUC1-C) is 1 × 10⁻⁶ 7 Cells / animal / inoculation were used, and the cell antigen was resuspended in phosphate buffer solution before inoculation. Inoculation times were days 0, 14, 28, and 42. Blood was collected on days 10 and 38, and after the 3rd and 4th immunizations, antibody titers in mouse serum were determined by ELISA. Mice with high and stable antibody titers in their serum were selected, and their spleens were removed for spleen cell fusion. Three days before spleen cell fusion, an additional immunization was performed, and an antigen solution prepared with protein antigen (hMUC1 CL-Fc) and physiological saline was injected intraperitoneally (ip) at a dose of 50 μg / animal.

[0339] 2. Fusion of spleen cells Using an optimized electrofusion method, splenic lymphocytes were fused with myeloma cells (Sp2 / 0 cells, ATCC® CRL-8287®) to obtain hybridoma cells.

[0340] Based on the counting results of spleen cells, fused hybridoma cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI) at a density of 3-4 × 10^5 / mL and inoculated into 96-well plates at 150 μL / well. After incubation at 37°C and 5% CO2 for 4-5 days, the supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. After incubation at 37°C and 5% CO2 for 2 days, ELISA detection was performed.

[0341] Example 4: Screening of mouse anti-MUC1-C hybridoma antibodies 1. ELISA experiment to determine the binding of hybridoma supernatant antibodies to the hMUC1-CL-his protein. Human hMUC1-CL-his protein was diluted to a concentration of 1 μg / mL with pH 7.4 PBS buffer and added to a 96-well microplate (Corning, product no. CLS3590-100EA) at a volume of 100 μL / well. The plate was incubated at 4°C for 16-18 hours. After discarding the liquid, 200 μL / well of 5% skim milk powder (Seiko Seibutsu, product no. A600669-0250) blocking solution diluted with PBS was added, and the plate was incubated at 37°C for 1.5 hours to block the protein. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (PBS with 0.1% tween-20 at pH 7.4). Then, 100 μL / well of hybridoma supernatant was added, and the plate was incubated at 37°C for 1 hour. After incubation, the plates were washed five times with PBST, and 50 μL / well of secondary antibody (Jackson ImmunoResearch, product number 1115-035-003) diluted in 2% MPBS was added and incubated at 37°C for 1 hour. After washing the plates five times with PBST, 50 μL / well of TMB chromogenic substrate (KPL, Cat No. 52-00-03) was added and incubated at room temperature for 5-10 minutes. The reaction was stopped by adding 50 μL / well of 1 M H2SO4, and the absorption value was read at a wavelength of 450 nm using a VERSAmax plate reader (Molecular Devices). The results are shown in the table below.

[0342] [Table 4]

[0343] 2. Mirrorball experiment of conjugation of hybridoma supernatant antibody to human MUC1-C cell line HCC827-human-MUC1-C (internally constructed overexpression stable transformed cell line), CHOK1-cyno-MUC1-C (internally constructed overexpression stable transformed cell line), or CHOK1-WT cells were digested and washed once with PBS buffer. Then, the cells were centrifuged at 1000 rpm for 5 minutes, diluted in PBS buffer, and resuspended with CellTracker® Green CMFDA dye (Thermo Fisher Scientific (China) Co., Ltd., product number C7025) to a final concentration of 50 nM (cell density 1E6 / mL), and incubated in an incubator for 30 minutes. After incubation, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS containing 1% FBS (Gibco, product number 10100147). After further centrifugation, the cells were resuspended with PBS buffer (final density 1-2E5 / mL), and APC-labeled fluorescent secondary antibody (BD biosciences, product number 550826) was added according to a dilution ratio of 1:200. The cell mixture was added to a 384-well plate (Corning, product number 3764), with 20 μL of cells per well, i.e., 2000-4000 cells per well. In the 384-well plate, excluding the positive and negative control wells, 20 μL of hybridoma supernatant was added to each well, and the plates were left at room temperature in the dark for 2 hours. The values ​​were read using a Mirrorball instrument (Sptlabtech).

[0344] [Table 5]

[0345] 3. Screening for MUC1-C antibodies based on hybridoma cells. Four hybridoma strains obtained through qualitative detection using ELISA and mirrorball were sequenced, and the corresponding antibodies were named after sequencing. The antibody for hybridoma 17F3 was named M4 after sequencing, the antibody for hybridoma 28E4 was named M6 after sequencing, the antibody for hybridoma 3H6 was named F4-1 after sequencing, and the antibody for hybridoma 36G9 was named F4-18 after sequencing.

[0346] The four antibody strains obtained were sequenced, and the resulting sequences were subjected to CDR classification. The mouse variable region sequences were selected and ligated to the constant region sequences of human antibodies to express chimeric antibodies. The amino acid sequences of the heavy and light chain variable regions of the antibodies obtained through screening are as follows.

[0347] >M4 heavy chain variable region sequence (M4 mVH): [ka] >M4 light chain variable region sequence (M4 mVL): [ka] >M6 heavy chain variable region sequence (M6 mVH): [ka] >M6 light chain variable region sequence (M6 mVL): [ka] >F4-1 heavy chain variable region sequence (F4-1 mVH): [ka] >F4-1 light chain variable region sequence (F4-1 mVL): [ka] >F4-18 heavy chain variable region sequence (F4-18 mVH): [ka] >F4-18 light chain variable region sequence (F4-18 mVL): [ka] Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the underlined parts are CDR sequences determined according to the Kabat numbering system, and the ununderlined parts are FR sequences.

[0348] The heavy and light chain CDR region sequences of mouse antibodies M4, M6, F4-1, and F4-18 are shown in the table below.

[0349] [Table 6]

[0350] [Table 7]

[0351] Example 5: Humanization of anti-MUC1-C mouse antibody Alignment with the IMGT Human Antibody Heavy / Light Chain Variable Region Germline Gene Database was performed using MOE software. Germline genes with high homology to M4, M6, F4-1, and F4-18 were selected as templates. The CDRs of these four mouse antibodies were transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific examples, the CDR amino acid residues are determined and annotated using the Kabat numbering system.

[0352] 1. Humanization of M4 mouse antibodies Mouse antibody M4 has humanized light chain templates IGKV1-39*01 / IGKV6-21*02 / IGKV3-11*01 and IGKJ4*01, and humanized heavy chain templates IGHV1-46*01 and IGHJ6*01. The CDR of mouse antibody M4 was transplanted into its respective human template, and further reverse mutations were performed on the FR portion amino acids of the humanized antibody. Considering the removal of chemical modification sites such as potential isomerization, the removal of N-terminal pyroglutamic acid formation, and the reduction of potential immunogenicity, the light chain FR portion contained one or more mutations from 3, 43, 47, 49, or 60 (the location of the mutation site was determined according to Kabat numbering rules), and the heavy chain FR portion contained one or more mutations from 1, 28, 38, 40, 48, 71, 73, 76, and 82a (the location of the mutation site was determined according to Kabat numbering rules). The amino acid substitutions in the variable region of the humanized antibody M4 are shown in the table below.

[0353] [Table 8]

[0354] The heavy chain variable region / light chain variable region sequences of the M4 humanized antibody are as follows: >huM4VH1 [ka] >huM4VH2 [ka] >huM4VH3 [ka] >huM4VL1 [ka] >huM4VL2 [ka] >huM4VL3 [ka] >huM4VL4 [ka] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the underlined parts of the sequence are CDR sequences determined according to the Kabat numbering system, and the ununderlined parts are FR sequences.

[0355] 2. Humanization of M6 mouse antibodies Mouse antibody M6 has humanized light chain templates IGKV4-1*01 / IGKV3-11*01 and IGKJ4*01, and humanized heavy chain templates IGHV1-46*01 and IGHJ6*01. The CDR of mouse antibody M6 was transplanted into its respective human template, and further reverse mutations were performed on the FR portion amino acids of the humanized antibody. Considering the removal of chemical modification sites such as potential isomerization, the removal of N-terminal pyroglutamic acid formation, and the reduction of potential immunogenicity, the light chain FR portion contained one or more mutations from 1, 4, 45, 68, or 83 (the location of the mutation site was determined according to Kabat numbering rules), and the heavy chain contained one or more mutations from 1, 28, 30, 39, 40, 43, 69, 71, 76, 82b, 83, 84, and 97 (the location of the mutation site was determined according to Kabat numbering rules). The amino acid substitutions in the variable region of the humanized antibody M6 are shown in the table below.

[0356] [Table 9]

[0357] The heavy chain variable region / light chain variable region sequences of the M6 ​​humanized antibody are as follows: >huM6VH1 [ka] >huM6VH2 [ka] >huM6VH3 [ka] >huM6VH4 [ka] >huM6VH5 [ka] >huM6VH6 [ka] >huM6VH7 [ka] >huM6VL1 [ka] >huM6VL2 [ka] >huM6VL3 [ka]

[0358] The CDR for the M6 ​​humanized antibody is as follows:

[0359] [Table 10]

[0360] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the underlined part is the CDR sequence determined according to the Kabat numbering system, and the ununderlined part is the FR sequence.

[0361] 3. Humanization of F4-1 mouse antibodies Mouse antibody F4-1 uses humanized light chain templates IGKV1-39*01 and IGKJ4*01, and humanized heavy chain templates IGHV1-3*01 and IGHJ6*01. The CDR of mouse antibody F4-1 was transplanted into these human templates, and then reverse mutations were performed on the FR portion amino acids of the humanized antibody. Considering the removal of chemical modification sites such as potential isomerization, the removal of N-terminal pyroglutamic acid formation, and the reduction of potential immunogenicity, the light chain FR portion contained mutations 4, 36, 42, 43, 47, 60, 70, and 75 (the location of the mutation sites was determined according to Kabat numbering rules), and the heavy chain FR portion contained one or more mutations from 1, 2, 12, 40, 44, 47, 48, 69, 71, and 76 (the location of the mutation sites was determined according to Kabat numbering rules). The amino acid substitutions in the humanized antibody variable region of antibody F4-1 are shown in the table below.

[0362] [Table 11]

[0363] The heavy chain variable region / light chain variable region sequences of the F4-1 humanized antibody are as follows:

[0364] >huF4-1VH1 [ka] >huF4-1VH2 [ka] >huF4-1VH3 [ka] >huF4-1VL1 [ka] >huF4-1VL2 [ka] >huF4-1VL3 [ka] >huF4-1VL4 [ka] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the underlined part is the CDR sequence determined according to the Kabat numbering system, and the ununderlined part is the FR sequence.

[0365] 4. Humanization of F4-18 mouse antibodies The mouse antibody F4-18 uses humanized light chain templates IGKV1-39*01 and IGKJ4*01, and humanized heavy chain templates IGHV1-3*01 and IGHJ1*01. The CDR of mouse antibody F4-18 was transplanted into these human templates, and further reverse mutations were performed on the FR portion amino acids of the humanized antibody. Considering the removal of chemical modification sites such as potential isomerization, the removal of N-terminal pyroglutamic acid formation, and the reduction of potential immunogenicity, the light chain FR portion contained mutations 4, 36, 39, 42, 44, 46, 60, 66, 69, and 71 (the location of the mutation sites was determined according to Kabat numbering rules), and the heavy chain contained one or more mutations from 12, 20, 24, 40, 44, 48, 69, 71, 96, and 101 (the location of the mutation sites was determined according to Kabat numbering rules). The amino acid substitutions in the humanized antibody variable region of antibody F4-18 are shown in the table below.

[0366] [Table 12]

[0367] The heavy chain variable region / light chain variable region sequences of the F4-18 humanized antibody are as follows: >huF4-18VH1 [ka] >huF4-18VH2 [ka] >huF4-18VH3 [ka] >huF4-18VH4 [ka] >huF4-18VH5 [ka] >huF4-18VL1 [ka] >huF4-18VL2 [ka] >huF4-18VL3 [ka] >huF4-18VL4 [ka]

[0368] The CDR for the F4-18 humanized antibody is as follows:

[0369] [Table 13]

[0370] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the underlined part is the CDR sequence determined according to the Kabat numbering system, and the ununderlined part is the FR sequence.

[0371] 5. Construction and expression of the IgG1 form of humanized anti-MUC1-C antibody. Primer PCR was designed to assemble each humanized antibody VH / VK gene fragment, and homologous recombination was performed with the expression vector pTT5 (containing the signal peptide and constant region gene (CH1-FC / CL) fragment, constructed in the laboratory) to construct the full-length antibody expression vector VH-CH1-FC-pTT5 / VK-CL-pTT5. The antibody heavy chain constant region may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4 or their variants, and the light chain constant region may be selected from the light chain constant regions of human κ, λ chains or their variants. Exemplarily, in the following example, the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region shown in SEQ ID NO: 69 or 186, and the light chain constant region is selected from the human light chain constant region shown in SEQ ID NO: 70.

[0372] Human IgG1 heavy chain constant region sequence: [ka] Human IgG1 heavy chain constant region sequence (with LALA mutation): [ka] Human light chain constant region sequence: [ka]

[0373] By ligating the carboxyl terminus of the heavy chain variable region of the above-screened mouse antibodies M4, M6, F4-1, and F4-18 to the amino terminus of the human heavy chain constant region shown in SEQ ID NO: 69, and by ligating the carboxyl terminus of the light chain variable region of the mouse antibodies to the amino terminus of the human light chain constant region shown in SEQ ID NO: 70, the corresponding chimeric antibodies can be obtained. Specifically, the chimeric antibodies of M4, M6, F4-1, and F4-18 are shown as ChiM4, ChiM6, ChiF4-1, and ChiF4-18, respectively.

[0374] By ligating the carboxyl terminus of the variable region of the humanized antibody heavy chains of M4, M6, F4-1, and F4-18 constructed above to the amino terminus of the constant region of the human heavy chain shown in SEQ ID NO: 69 to form the full-length antibody heavy chain, and by ligating the carboxyl terminus of the variable region of the humanized antibody light chains of M4, M6, F4-1, and F4-18 to the amino terminus of the constant region of the human light chain shown in SEQ ID NO: 70 to form the full-length antibody light chain, the humanized antibodies shown in Tables 10 to 13 below can be obtained.

[0375] [Table 14]

[0376] [Table 15]

[0377] [Table 16]

[0378] [Table 17]

[0379] The full-length sequences of exemplary humanized antibody heavy / light chains are as follows:

[0380] >M4-H1L1 heavy chain sequence: [ka] >M4-H1L1 light chain sequence: [ka] >M4-H2L1 heavy chain sequence: [ka] >M4-H2L1 light chain sequence: [ka] >M4-H1L2 double-stranded sequence: [ka] >M4-H1L2 light chain sequence: [ka] >M4-H2L2 double-stranded sequence: [ka] >M4-H2L2 light chain sequence: [ka] >M6-H1L1 heavy chain sequence: [ka] >M6-H1L1 light chain sequence: [ka] >M6-H3L1 heavy chain sequence: [ka] >M6-H3L1 light chain sequence: [ka] >M6-H2L2 double-stranded sequence: [ka] >M6-H2L2 light chain sequence: [ka] >M6-H3L2 double-stranded sequence: [ka] >M6-H3L2 light chain sequence: [ka] >M6-H4L2 double-stranded sequence: [ka] >M6-H4L2 light chain sequence: [ka] >F4-1-H1L1 heavy chain sequence: [ka] >F4-1-H1L1 light chain sequence: [ka] >F4-1-H1L2 double-stranded sequence: [ka] >F4-1-H1L2 light chain sequence: [ka] >F4-1-H2L2 double-stranded sequence: [ka] >F4-1-H2L2 light chain sequence: [ka] >F4-1-H1L4 heavy chain sequence: [ka] >F4-1-H1L4 light chain sequence: [ka] >F4-18-H1L1 heavy chain sequence: [ka] >F4-18-H1L1 light chain sequence: [ka] >F4-18-H2L1 heavy chain sequence: [ka] >F4-18-H2L1 light chain sequence: [ka] >F4-18-H2L2 double-stranded sequence: [ka] >F4-18-H2L2 light chain sequence: [ka] >F4-18-H2L3 heavy chain sequence: [ka] >F4-18-H2L3 light chain sequence: [ka] >F4-18-H4L3 heavy chain sequence: [ka] >F4-18-H4L3 light chain sequence: [ka] >F4-18-H1L4 heavy chain sequence: [ka] >F4-18-H1L4 light chain sequence: [ka] >F4-18-H2L4 heavy chain sequence: [ka] >F4-18-H2L4 light chain sequence: [ka] >F4-18-H4L4 heavy chain sequence: [ka] >F4-18-H4L4 light chain sequence: [ka] Note: In the full-length antibody sequence shown above, the underlined portion is the antibody variable region sequence, and the ununderlined portion is the antibody constant region sequence.

[0381] Example 6: Modification of anti-EGFR antibody The molecule that specifically binds to EGFR can be derived from any suitable antibody, such as zalutumumab or its variants, among which,

[0382] [Table 18] >Zalutumumab heavy chain variable region sequence (abbreviated as "ZalVH"): [ka] >Zalutumumab light chain variable region sequence (abbreviated as "ZalVL"): [ka] >Zalutumumab heavy chain sequence: [ka] >Zalutumumab light chain sequence: [ka] By performing mutational modifications on amino acids at positions 31, 33, 52A, 56, 60, 97 and / or 99 of the heavy chain variable region of Zalutumumab, and / or on the amino acid at position 1 of the light chain variable region, a total of 15 anti-EGFR antibodies were obtained: ZalH', ZalH1, ZalH2, ZalH3, ZalH4, ZalH5, ZalH6, ZalH7, ZalH8, ZalH9, ZalH10, ZalH11, ZalH12, ZalH13, and ZalH14, respectively. Their specific sequences are as follows.

[0383] [Table 19]

[0384] >The heavy chain variable region sequence of ZalH1 (abbreviated as "ZalVH1"): [ka] >The heavy chain variable region sequence of ZalH2 (abbreviated as "ZalVH2"): [ka] >The heavy chain variable region sequence of ZalH3 (abbreviated as "ZalVH3"): [ka] >The heavy chain variable region sequence of ZalH4 (abbreviated as "ZalVH4"): [ka] >The heavy chain variable region sequence of ZalH5 (abbreviated as "ZalVH5"): [ka] >The heavy chain variable region sequence of ZalH6 (abbreviated as "ZalVH6"): [ka] Sequence ID 140 >The heavy chain variable region sequence of ZalH7 (abbreviated as "ZalVH7"): [ka] >The heavy chain variable region sequence of ZalH8 (abbreviated as "ZalVH8"): [ka] >The heavy chain variable region sequence of ZalH9 (abbreviated as "ZalVH9"): [ka] >The heavy chain variable region sequence of ZalH10 (abbreviated as "ZalVH10"): [ka] >The heavy chain variable region sequence of ZalH11 (abbreviated as "ZalVH11"): [ka] >The heavy chain variable region sequence of ZalH12 (abbreviated as "ZalVH12"): [ka] >The heavy chain variable region sequence of ZalH13 (abbreviated as "ZalVH13"): [ka] >The heavy chain variable region sequence of ZalH14 (abbreviated as "ZalVH14"): [ka] >The light chain variable region sequences of ZalH' and ZalH1~ZalH14 (hereinafter abbreviated as "ZalVL1"): [ka] >ZalH' heavy chain sequence: Sequence ID 124 >ZalH1 heavy chain sequence: [ka] >ZalH2 heavy chain sequence: [ka] >ZalH3 heavy chain sequence: [ka] >ZalH4 heavy chain sequence: [ka] >ZalH5 heavy chain sequence: [ka] >ZalH6 heavy chain sequence: [ka] >ZalH7 heavy chain sequence: [ka] >ZalH8 heavy chain sequence: [ka] >ZalH9 heavy chain sequence: [ka] >ZalH10 heavy chain sequence: [ka] >ZalH11 heavy chain sequence: [ka] >ZalH12 heavy chain sequence: [ka] >ZalH13 heavy chain sequence: [ka] >ZalH14 heavy chain sequence: [ka] >Light chain sequences of ZalH' and ZalH1~ZalH14: [ka] Note: In the antibody sequences above, the underlined portion is the antibody variable region sequence, the double underlined portion is the antibody CDR sequence, the ununderlined portion is the antibody constant region sequence, and the bolded letters are mutant amino acids.

[0385] Example 7: Construction of an anti-EGFR-MUC1 biantibody The EGFR-MUC1 biantibody used a 1:1 molecular configuration, and the MUC1 arm was selected as M4-H1L1 (hereinafter referred to as "M4H1L1"), which was assembled with EGFR antibodies ZalH', ZalH4, ZalH8, ZalH10, and ZalH13 to form the biantibody. Of these, the VH EGFR antibody was combined with titin, and the VL EGFR antibody was combined with obscurin. Furthermore, S358C and T370W mutations (knob) were introduced into the MUC1 antibody heavy chain, and Y357C, T374S, L376A, and Y415V mutations (hole) were introduced into the EGFR antibody heavy chain. The format was as follows: Chain 1: [VH(anti-MUC1)]-[IgG1(CH1)]-[Fc(Knob)], Chain 2: [VL(anti-MUC1)]-[CL], Chain 3: [VH (anti-EGFR)]-[Linker 1]-[Titin]-[Fc (Hole)], Chain 4: [VL(anti-EGFR)]-[linker 2]-[Obscurin] is an asymmetric structure molecule containing four chains, and its schematic diagram is shown in Figure 5 (where T represents Titin and O represents Obscurin).

[0386] [Table 20]

[0387] >Titin chain: [ka] >Obscurin chain: [ka] >CH1: [ka] >CL: Sequence ID 70 Linker 1 and Linker 2: GGGGS (Sequence ID 168) >Fc(knob): [ka] >Fc(hole): [ka]

[0388] The overall arrangement is as follows:

[0389] M4H1L1-ZalH' sequence: Chain 1 (huM4VH1-CH1-Fc(Knob)): [ka] >Chain 2 (huM4VL1-CL): [ka] Chain 3: [ka] Chain 4: [ka] M4H1L1-ZalH4 sequence: Chain 1: Sequence ID 171 Chain 2: Sequence ID 74 Chain 3: [ka] Chain 4: Sequence ID 173 M4H1L1-ZalH8 sequence: Chain 1: Sequence ID 171 Chain 2: Sequence ID 74 Chain 3: [ka] >Chain 4: Sequence ID 173 M4H1L1-ZalH10 sequence: Chain 1: Sequence ID 171 Chain 2: Sequence ID 74 Chain 3: [ka] 6 >Chain 4: Sequence ID 173 M4H1L1-ZalH13 sequence: Chain 1: Sequence ID 171 Chain 2: Sequence ID 74 Chain 3: [ka] >Chain 4: Sequence ID 173 Note: In the antibody sequence above, the underlined portion is the antibody variable region sequence, the ununderlined portion is the antibody constant region sequence, and the wavy underlined portion is the linker sequence.

[0390] The negative control antibody IgG1 used in this disclosure has a VH / VL sequence derived from patent US6114143A, and its heavy chain constant region and light chain constant region sequences are SEQ ID NO: 69 and SEQ ID NO: 70, respectively, and its full-length sequence is as follows. IgG single-stranded: [ka] IgG1 light chain: [ka] Note: In the sequence, the underlined portion is the variable region, and the italicized portion is the constant region.

[0391] Example 8: M4H1L1-ZalH4-LALA biantibody The M4H1L1-ZalH4-LALA biantibody is obtained by introducing the L238A and L239A mutations into chain 1 and the L242A and L243A mutations into chain 3, while leaving the sequences of chains 2 and 4 unchanged. This results in M4H1L1-ZalH4-LALA containing four chains, and the specific sequence is as follows. M4H1L1-ZalH4-LALA sequence: [ka] Chain 2: Sequence ID 74 Chain 3: [ka] Chain 4: Sequence ID 173 >Fc(knob)': [ka] >Fc(hole)': [ka] Note: In the antibody sequence above, the underlined portion is the antibody variable region sequence, the ununderlined portion is the antibody constant region sequence, the wavy underlined portion is the linker sequence, and the bolded letters are mutant amino acids.

[0392] Example 9: Coupling of EGFR-MUC1 biantibody to M toxin M4H1L1-ZalH'-M [ka] Under conditions of 37°C, an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 6.3 μL, 63 nmol) was added to a PBS-buffered aqueous solution of the antibody M4H1L1-ZalH' (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 0.38 mL, 25.7 nmol). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed to 30 mM histidine-acetate buffer with pH 5.0 using a Sephadex G25 gel column, and the solution was concentrated to 10 mg / mL. Compound M (0.245 mg, 257 nmol) was dissolved in 24 μL of acetonitrile and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain M4H1L1-ZalH'-M histidine-acetate buffer (0.3 mg / mL, 10.2 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=3.87.

[0393] M4H1L1-ZalH4-M [ka] Under conditions of 37°C, an aqueous solution of M4H1L1-ZalH4 antibody buffered in PBS (0.05 M PBS buffer solution with pH=6.3, 10.0 mg / mL, 0.5 mL, 33.8 nmol) was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 8.4 μL, 84 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed using a Sephadex G25 gel column to 30 mM histidine-acetate buffer with pH 5.0, and concentrated to 10 mg / mL.

[0394] Compound M (0.32 mg, 338 nmol) was dissolved in 32 μL of acetonitrile and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain M4H1L1-ZalH4-M histidine-acetate buffer (0.42 mg / mL, 10.7 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=4.8.

[0395] M4H1L1-ZalH8-M [ka] Under conditions of 37°C, an aqueous solution of M4H1L1-ZalH8 antibody buffered in PBS (0.05 M PBS buffer solution with pH=6.3, 10.0 mg / mL, 0.5 mL, 33.8 nmol) was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 8.25 μL, 82.5 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed using a Sephadex G25 gel column to 30 mM histidine-acetate buffer with pH 5.0, and the solution was concentrated to 10 mg / mL.

[0396] Compound M (0.32 mg, 338 nmol) was dissolved in 32 μL of acetonitrile and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain M4H1L1-ZalH8-M histidine-acetate buffer (0.39 mg / mL, 10.7 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=4.13. M4H1L1-ZalH10-M [ka] Under conditions of 37°C, an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 4.57 μL, 45.7 nmol) was added to a PBS-buffered aqueous solution of antibody M4H1L1-ZalH10 (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 0.276 mL, 18.6 nmol). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed to 30 mM histidine-acetate buffer with pH 5.0 using a Sephadex G25 gel column, and the solution was concentrated to 10 mg / mL.

[0397] Compound M (0.18 mg, 186 nmol) was dissolved in 18 μL of acetonitrile and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain the title product M4H1L1-ZalH10-M in histidine-acetate buffer (0.22 mg / mL, 10.2 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=4.33.

[0398] M4H1L1-ZalH13-M [ka] Under conditions of 37°C, an aqueous solution of M4H1L1-ZalH13 antibody buffered in PBS (0.05 M PBS buffer solution with pH=6.3, 10.0 mg / mL, 0.47 mL, 32 nmol) was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 7.85 μL, 78.5 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed to 30 mM histidine-acetate buffer with pH 5.0 using a Sephadex G25 gel column, and the solution was concentrated to 10 mg / mL.

[0399] Compound M (0.306 mg, 320 nmol) was dissolved in 31 μL of acetonitrile and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain M4H1L1-ZalH13-M histidine-acetate buffer (0.42 mg / mL, 10.2 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=4.3.

[0400] IgG1-M [ka] Under conditions of 37°C, an aqueous solution of PBS-buffered antibody IgG1 (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 0.5 mL, 33.8 nmol) was mixed with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 8.11 μL, 81.1 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was then cooled to 25°C in a water bath.

[0401] Compound M (0.32 mg, 338 nmol) was dissolved in 32 μL of acetonitrile and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain IgG1-M histidine-acetate buffer (0.48 mg / mL, 10.2 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=4.37.

[0402] Example 10: Coupling of antibody to 9-A toxin 1. Coupling of EGFR-MUC1 biantibody to 9-A toxin [ka]

[0403] Under conditions of 37°C, an aqueous solution of M4H1L1-ZalH4 antibody buffered in PBS (0.05 M PBS buffer solution with pH=6.3, 10.0 mg / mL, 30 mL, 2.027 μmol) was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 851 μL, 8.51 μmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed to 30 mM histidine-acetate buffer with pH 5.0 using a Sephadex G25 gel column, and the solution was concentrated to 10 mg / mL.

[0404] Compound 9-A (26.1 mg, 24.32 μmol) was dissolved in 1.5 mL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain histidine-acetate buffer for ADC-1 (7.1 mg / mL, 39 mL), which was stored refrigerated at 4°C. The average DAR was calculated using HIC: n=5.78. ADC-2 Under conditions of 37°C, a PBS-buffered aqueous solution of the antibody M4H1L1-ZalH4 (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 30.8 mL, 2.074 μmol) was mixed with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 509.5 μL, 5.095 μmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed using a Sephadex G25 gel column to 30 mM histidine-acetate buffer with pH 5.0, and concentrated to 10 mg / mL.

[0405] Compound 9-A (22.3 mg, 20.74 μmol) was dissolved in 1.5 mL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain ADC-2 histidine-acetate buffer (5.8 mg / mL, 47.6 mL), which was stored refrigerated at 4°C. The average DAR was calculated using HIC: n=4.5.

[0406] ADC-3 [ka] Under conditions of 37°C, an aqueous solution of the antibody M4H1L1-ZalH4-LALA buffered in PBS (0.05 M PBS buffer solution with pH=6.3, 10.0 mg / mL, 1.11 mL, 75 nmol) was mixed with an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 29.3 μL, 293 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed to 30 mM histidine acetate buffer with pH 5.0 using a Sephadex G25 gel column, and the solution was concentrated to 10 mg / mL.

[0407] Compound 9-A (0.967 mg, 900 nmol) was dissolved in 55 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain ADC-3 histidine-acetate buffer (0.96 mg / mL, 10.5 mL), which was stored refrigerated at 4°C. The mean DAR was calculated by MS: n=5.51.

[0408] 2. Coupling of EGFR antibody to 9-A toxin ZalH4-9-A [ka] Under conditions of 37°C, a PBS-buffered aqueous solution of the antibody ZalH4 (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 1.57 mL, 106 nmol) was mixed with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 35 μL, 350 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed using a Sephadex G25 gel column to 30 mM histidine-acetate buffer with pH 5.0, and the solution was concentrated to 10 mg / mL.

[0409] Compound 9-A (1.37 mg, 1.272 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain ZalH4-9-A in histidine-acetate buffer (1.1 mg / mL, 12.8 mL), which was stored refrigerated at 4°C. The average DAR was calculated by RP-HPLC: n=5.94.

[0410] 3. Coupling of MUC1 antibody to 9-A toxin M4H1L1-9-A [ka] Under conditions of 37°C, an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 31.2 μL, 312 nmol) was added to a PBS-buffered aqueous solution of antibody M4H1L1 (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 1.4 mL, 94.6 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was cooled to 25°C in a water bath, and then the solution was changed to 30 mM histidine-acetate buffer with pH 5.0 using a Sephadex G25 gel column, and the solution was concentrated to 10 mg / mL.

[0411] Compound 9-A (1.22 mg, 1.135 μmol) was dissolved in 70 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 30 mM histidine-acetate buffer at pH 5.0) to obtain M4H1L1-9-A histidine-acetate buffer (0.94 mg / mL, 12.5 mL), which was stored refrigerated at 4°C. The mean value was calculated by RP-HPLC: n=6.6.

[0412] 4. Coupling of negative control antibody to 9-A toxin IgG1-9-A [ka] Under conditions of 37°C, an aqueous solution of PBS-buffered antibody IgG1 (0.05 M PBS-buffered aqueous solution with pH=6.3, 10.0 mg / mL, 3.9 mL, 263 nmol) was mixed with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 81.6 μL, 816 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to allow the reaction to stop. The reaction solution was then cooled to 25°C in a water bath.

[0413] Compound 9-A (3.4 mg, 3.162 μmol) was dissolved in 200 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to allow the reaction to stop. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.3 and containing 0.001 M EDTA) to obtain the title product IgG-9-A in PBS buffer (2.42 mg / mL, 14.7 mL), which was stored refrigerated at 4°C. The mean value was calculated by RP-HPLC: n=6.15.

[0414] Test Example 1: Measurement of Chimeric Antibody Affinity Based on FACS HCC827-human-MUC1-C (internally constructed overexpression stable transformed cell line), CHOK1-cyno-MUC1-C (internally constructed overexpression stable transformed cell line), and T47D cells (human ductal carcinoma cells) were digested and added to a 96-well plate at a rate of 1E5 cells / well. After centrifugation at 300g for 5 minutes, the supernatant was discarded and the cells were washed once with PBS buffer containing 2% FBS. The antibody was diluted in PBS buffer containing 2% FBS at a 5-fold dilution ratio, starting from an initial concentration of 20 μg / mL, to create a total of eight concentration gradients. 100 microliters of antibody sample was added to each well, the cells were resuspended, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes, the supernatant was discarded and the cells were washed twice with PBS buffer containing 2% FBS. Secondary antibody (Alexa Fluor® 488 goat anti-human IgG(H+L) 1:1000, Invitrogen, A11013) was added and incubated at 4°C for 40 minutes. After centrifugation at 300g for 5 minutes, the supernatant was discarded and the cells were washed twice with PBS buffer containing 2% FBS. The cells were resuspended in 100 μL of PBS buffer containing 2% FBS and then placed on an instrument for measurement. The FACS detection results are as follows.

[0415] [Table 21] The results showed that the chimeric antibodies of this disclosure have relatively strong affinity for stable transformed cell lines or tumor cell lines.

[0416] Test Example 2: Internalization Test of Tumor Cells Against Anti-MUC1-C Chimeric Antibody 1. Purpose of the examination The objective of this experiment is to detect the endocytotic activity of MUC1 chimeric monoclonal antibody drugs in tumor cells. Cells were treated in vitro with different concentrations of MUC1 chimeric monoclonal antibody drugs and cultured for 3 days. Then, tumor cell proliferation was detected using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, product number: G7573) reagent, and IC was performed. 50 The endocytosis activity of the antibody was evaluated based on its value.

[0417] 2. Experimental Method To illustrate the method for testing the endocytosis activity of the MUC1 chimeric monoclonal antibody agent of this disclosure against tumor cells, an in vitro growth inhibition test method against T47D cells is given as an example. This method is similarly applicable to, but not limited to, in vitro growth inhibition activity tests against other tumor cells.

[0418] 1. Cell culture: T47D cells were cultured in 1640 medium (GE, product number SH30024.01) with 10% FBS and 2 μg / mL human insulin (Yokusei, product number 40112ES60).

[0419] 2. Cell preparation: Logarithmic T47D cells were taken, washed once with PBS (phosphate buffer, Shanghai Yuanpei Biotechnology Co., Ltd.), then 2-3 mL of trypsin (0.25% Trypsin-EDTA (1×), Gibco, Life Technologies) was added and digested for 2-3 minutes. After the cells were completely digested, 10-15 mL of cell culture medium was added to elute the digested cells, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and then 10-20 mL of cell culture medium containing 20% ​​ultra-low IgG fetal bovine serum (Bosheng Bio, product number BS-0007-500) was added to resuspend the cells and prepare a single-cell suspension.

[0420] 3. Seeding cells onto plates: Mix the T47D single-cell suspension uniformly and increase the viable cell density to 4 × 10⁶ with the cell culture medium.4 Each cell / mL was adjusted accor...

Claims

1. An anti-MUC1 antibody or its antigen-binding fragment comprising a heavy chain variable region containing HCDR1, HCDR2, and HCDR3, and a light chain variable region containing LCDR1, LCDR2, and LCDR3, a. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 4, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5, or b. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 of either SEQ ID NO: 6 or 47, respectively, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 7, respectively, or c. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 8, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 9, or d. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs. 63, 10, or 64, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and HCDR3 of SEQ ID NOs.

11. Preferably, a. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 4, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5, or b. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 6, respectively, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 7, respectively, or c. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 8, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 9, or d. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 63, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11, More preferably, the heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 4, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO:

5. An anti-MUC1 antibody or its antigen-binding fragment.

2. a. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 12, HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and HCDR3 contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 15, LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains the amino acid sequence of SEQ ID NO: 17, or b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 18, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains either one of the amino acid sequences of SEQ ID NO: 20 or 113, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 21, LCDR2 contains the amino acid sequence of SEQ ID NO: 22, and LCDR3 contains the amino acid sequence of SEQ ID NO: 23, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 24, HCDR2 contains the amino acid sequence of SEQ ID NO: 25, and HCDR3 contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 27, LCDR2 contains the amino acid sequence of SEQ ID NO: 28, and LCDR3 contains the amino acid sequence of SEQ ID NO: 29, or d. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 30, HCDR2 contains the amino acid sequence of SEQ ID NO: 31, and HCDR3 contains any one of the amino acid sequences of SEQ ID NO: 114, 32, or 115, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 33, LCDR2 contains the amino acid sequence of SEQ ID NO: 34, and LCDR3 contains the amino acid sequence of SEQ ID NO:

35. Preferably, a. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO:

17. b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 18, HCDR2 contains the amino acid sequence of SEQ ID NO: 19, and HCDR3 contains the amino acid sequence of SEQ ID NO: 20, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 21, LCDR2 contains the amino acid sequence of SEQ ID NO: 22, and LCDR3 contains the amino acid sequence of SEQ ID NO: 23, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 24, HCDR2 contains the amino acid sequence of SEQ ID NO: 25, and HCDR3 contains the amino acid sequence of SEQ ID NO: 26, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 27, LCDR2 contains the amino acid sequence of SEQ ID NO: 28, and LCDR3 contains the amino acid sequence of SEQ ID NO: 29, or d. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 30, HCDR2 containing the amino acid sequence of SEQ ID NO: 31, and HCDR3 containing the amino acid sequence of SEQ ID NO: 114, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 33, LCDR2 containing the amino acid sequence of SEQ ID NO: 34, and LCDR3 containing the amino acid sequence of SEQ ID NO:

35. More preferably, the heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO:

17. The anti-MUC1 antibody or antigen-binding fragment thereof as described in claim 1.

3. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, preferably a chimeric antibody or a humanized antibody, and more preferably a humanized antibody.

4. a. The heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 36, 37, or 38, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 39, 40, 41, or 42, or the heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 5, or b. The heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 43, 44, 45, 46, 47, 48, or 49, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 50, 51, or 52, or the heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 6, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 7, or c. The heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 53, 54, or 55, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 56, 57, 58, or 59, or the heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 8, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9, or d. The heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs. 63, 60, 61, 62, or 64, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs. 67, 68, 65, or 66, or the heavy chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs. 10, and the light chain variable region includes an amino acid sequence having at least 70% sequence identity with SEQ ID NOs.

11. Preferably, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39 or 40, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 37, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39 or 40, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 4, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

5. b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 43, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 50, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 44, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 51, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 45, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 50 or 51, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 46, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 51, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 6, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

7. c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 53, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 56, 57, or 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 54, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 57, or Alternatively, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 8, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

9. d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 60, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 65 or 68, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 61, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 65, 66, 67, or 68, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 63, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 67 or 68, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 10, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

11. More preferably, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

39. An anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. An antibody fragment, preferably selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, and dAb, which is an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 4.

6. The anti-MUC1 antibody comprises a heavy chain constant region and a light chain constant region. Preferably, the heavy chain steady region is the human IgG1, IgG2, IgG3, or IgG4 heavy chain steady region. The light chain steady-state region is the human κ or λ light chain steady-state region. More preferably, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 69 or 186. The light chain constant region includes the amino acid sequence of SEQ ID NO:

70. An anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. The anti-MUC1 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, of which, a. The heavy chain contains an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 71, 73, 75, or 77, and the light chain contains an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 72, 74, 76, or 78, or b. The heavy chain contains an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 79, 81, 83, 85, or 87, and the light chain contains an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 80, 82, 84, 86, or 88, or c. The heavy chain contains an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 89, 91, 93, or 95, and the light chain contains an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 90, 92, 94, or 96, or d. The heavy chain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 97, 99, 101, 103, 105, 107, 109, or 111, and the light chain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, or 112. Preferably, a. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain is shown in SEQ ID NO: 72, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 73, and the amino acid sequence of the light chain is shown in SEQ ID NO: 74, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 75, and the amino acid sequence of the light chain is shown in SEQ ID NO: 76, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain is shown in SEQ ID NO:

78. b. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 79, and the amino acid sequence of the light chain is shown in SEQ ID NO: 80, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 81, and the amino acid sequence of the light chain is shown in SEQ ID NO: 82, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 83, and the amino acid sequence of the light chain is shown in SEQ ID NO: 84, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 85, and the amino acid sequence of the light chain is shown in SEQ ID NO: 86, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 87, and the amino acid sequence of the light chain is shown in SEQ ID NO:

88. c. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 90, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 91, and the amino acid sequence of the light chain is shown in SEQ ID NO: 92, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 93, and the amino acid sequence of the light chain is shown in SEQ ID NO: 94, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 95, and the amino acid sequence of the light chain is shown in SEQ ID NO:

96. d. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 97, and the amino acid sequence of the light chain is shown in SEQ ID NO: 98, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 99, and the amino acid sequence of the light chain is shown in SEQ ID NO: 100, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 101, and the amino acid sequence of the light chain is shown in SEQ ID NO: 102, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 103, and the amino acid sequence of the light chain is shown in SEQ ID NO: 104, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 105, and the amino acid sequence of the light chain is shown in SEQ ID NO: 106, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 107, and the amino acid sequence of the light chain is shown in SEQ ID NO: 108, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 109, and the amino acid sequence of the light chain is shown in SEQ ID NO: 110, or The amino acid sequence of the heavy chain is shown in SEQ ID NO: 111, and the amino acid sequence of the light chain is shown in SEQ ID NO:

112. More preferably, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain is shown in SEQ ID NO:

72. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 6.

8. An anti-EGFR antibody or its antigen-binding fragment comprising a heavy chain variable region containing HCDR1, HCDR2, and HCDR3, and a light chain variable region containing LCDR1, LCDR2, and LCDR3, a. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, 128, 130, or 131, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126 or 133, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 127, 132, or 134, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or d. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 127, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. e. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 128, 129, 130, or 131, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. Preferably, a. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or b. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 128 or 130, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, or c. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 133, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. more, The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. Anti-EGFR antibody or its antigen-binding fragment.

9. The heavy chain variable region includes the amino acid sequences of SEQ ID NOs: 138, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

149. Preferably, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 138, 142, 144, or 147, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

149. More preferably, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 138, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

149. The anti-EGFR antibody or antigen-binding fragment thereof according to claim 8.

10. An antibody fragment, preferably selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, and dAb, which is an anti-EGFR antibody or antigen-binding fragment thereof according to claim 9.

11. The anti-EGFR antibody comprises a heavy chain constant region and a light chain constant region. Preferably, the heavy chain steady region is the human IgG1, IgG2, IgG3, or IgG4 heavy chain steady region. The light chain steady-state region is the human κ or λ light chain steady-state region. More preferably, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 69 or 186. The light chain constant region includes the amino acid sequence of SEQ ID NO:

70. The anti-EGFR antibody or antigen-binding fragment thereof according to claim 9.

12. It includes heavy chains and light chains, of which, The heavy chain comprises the amino acid sequence of SEQ ID NOs: 153, 150, 151, 152, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163, and the light chain comprises the amino acid sequence of SEQ ID NOs:

164. Preferably, the heavy chain contains the amino acid sequence of SEQ ID NO: 153, 157, 159, or 162, and the light chain contains the amino acid sequence of SEQ ID NO:

164. More preferably, the heavy chain comprises the amino acid sequence of SEQ ID NO: 153, and the light chain comprises the amino acid sequence of SEQ ID NO:

164. The anti-EGFR antibody or antigen-binding fragment thereof according to claim 11.

13. An antigen-binding molecule that specifically binds to EGFR and MUC1, At least one antigen-binding module that specifically binds to EGFR, It comprises at least one antigen-binding module that specifically binds to MUC1, The antigen-binding module that specifically binds to the aforementioned EGFR includes a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL. The antigen-binding module that specifically binds to the aforementioned MUC1 includes a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL, of which, a. The EGFR-VH is characterized in that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, 128, 130, or 131, and the EGFR-VL is characterized in that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The EGFR-VH is characterized in that HCDR1 contains the amino acid sequence of SEQ ID NO: 126 or 133, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the EGFR-VL is characterized in that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or c. The EGFR-VH is characterized in that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 127, 132, or 134, and HCDR3 contains the amino acid sequence of SEQ ID NO: 118, and the EGFR-VL is characterized in that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or d. The EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 127, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. e. The EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 126, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 128, 129, 130, or 131, and the EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. Preferably, a. The EGFR-VH is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, and the EGFR-VL is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The EGFR-VH is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 128 or 130, and the EGFR-VL is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or c. The EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 133, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, more, a. The EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and the EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. An antigen-binding molecule that specifically binds to EGFR and MUC1.

14. The MUC1-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14, The aforementioned MUC1-VL includes the amino acid sequence of SEQ ID NO: 15 in LCDR1, the amino acid sequence of SEQ ID NO: 16 in LCDR2, and the amino acid sequence of SEQ ID NO: 17 in LCDR3. An antigen-binding molecule that specifically binds to EGFR and MUC1 as described in claim 13.

15. The EGFR-VH comprises the amino acid sequences of SEQ ID NOs: 138, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO:

149. Preferably, the EGFR-VH contains the amino acid sequence of SEQ ID NO: 138, 142, 144, or 147, and the EGFR-VL contains the amino acid sequence of SEQ ID NO:

149. More preferably, EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and EGFR-VL comprises the amino acid sequence of SEQ ID NO:

149. The antigen-binding molecule that specifically binds to EGFR and MUC1 as described in claim 14.

16. The antigen-binding molecule according to claim 15, wherein MUC1-VH comprises the amino acid sequence of SEQ ID NO: 36, and MUC1-VL comprises the amino acid sequence of SEQ ID NO:

39.

17. The antigen-binding module that specifically binds to the EGFR or the antigen-binding module that specifically binds to the MUC1 each independently comprises a titin chain and an obscurin chain capable of forming a dimer. Preferably, the titin chain contains the amino acid sequence of SEQ ID NO: 165, and the obscurin chain contains the amino acid sequence of SEQ ID NO:

166. An antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 16.

18. Further including the Fc region, Preferably, the Fc region is an IgG Fc region, More preferably, the Fc region is an IgG1 Fc region, More preferably, the Fc region includes one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor. An antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 17.

19. The molecule includes an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, and the Fc1 and Fc2 each independently contain one or more amino acid substitutions that reduce homodimerization of the Fc region. Preferably, Fc1 has a knob structure using knob-in-hole technology, and Fc2 has a hole structure using knob-in-hole technology, or Fc2 has a knob structure using knob-in-hole technology, and Fc1 has a hole structure using knob-in-hole technology. More preferably, Fc1 has an amino acid C at position 358 and an amino acid W at position 370, and Fc2 has an amino acid C at position 357, an amino acid S at position 374, an amino acid A at position 376, and an amino acid V at position 415, and is numbered according to the EU index, or Fc2 has an amino acid C at position 358 and an amino acid W at position 370, and Fc1 has an amino acid C at position 357, an amino acid S at position 374, an amino acid A at position 376, and an amino acid V at position 415, and is numbered according to the EU index, Most preferably, Fc1 comprises the amino acid sequence of SEQ ID NO: 169, Fc2 comprises the amino acid sequence of SEQ ID NO: 170, or Fc1 comprises the amino acid sequence of SEQ ID NO: 182, Fc2 comprises the amino acid sequence of SEQ ID NO: 183, or Fc2 comprises the amino acid sequence of SEQ ID NO: 169, Fc1 comprises the amino acid sequence of SEQ ID NO: 170, or Fc2 comprises the amino acid sequence of SEQ ID NO: 182, Fc1 comprises the amino acid sequence of SEQ ID NO:

183. The antigen-binding molecule that specifically binds to EGFR and MUC1 according to claim 18.

20. One antigen-binding module that specifically binds to EGFR, It comprises one antigen-binding module that specifically binds to MUC1, The antigen-binding module that specifically binds to the aforementioned MUC1 is Fab. The antigen-binding module that specifically binds to the aforementioned EGFR is a replaced Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer. Preferably, The antigen-binding molecule comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), of which, Formula (a) [MUC1-VH]-[CH1]-[Fc1], Formula (b) [MUC1-VL]-[CL], Formula (c) [EGFR-VH]-[Linker 1]-[Titin]-[Fc2], Formula (d) [EGFR-VL]-[Linker 2]-[Obscurin], Eventually, Linker 1 and linker 2 are the same or different, are peptide linkers, or neither linker 1 nor linker 2 exists. The structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus. More preferably, among them, MUC1-VH contains the amino acid sequence of SEQ ID NO: 12 in HCDR1, the amino acid sequence of SEQ ID NO: 13 in HCDR2, and the amino acid sequence of SEQ ID NO: 14 in HCDR3, and MUC1-VL contains the amino acid sequence of SEQ ID NO: 15 in LCDR1, the amino acid sequence of SEQ ID NO: 16 in LCDR2, and the amino acid sequence of SEQ ID NO: 17 in LCDR3, and a. The EGFR-VH is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 116, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 129, and the EGFR-VL is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or b. The EGFR-VH is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 126, HCDR2 contains the amino acid sequence of SEQ ID NO: 117, and HCDR3 contains the amino acid sequence of SEQ ID NO: 128 or 130, and the EGFR-VL is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 119, LCDR2 contains the amino acid sequence of SEQ ID NO: 120, and LCDR3 contains the amino acid sequence of SEQ ID NO: 121, or c. The EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 133, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 118, and the EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121, More preferably, eventually, The aforementioned MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, and the aforementioned MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, and The EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, 142, 144, or 147, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO:

149. Most preferably, the antigen-binding molecule is A first chain containing the amino acid sequence of sequence number 171, A second chain containing the amino acid sequence of SEQ ID NO: 74, A third chain containing the amino acid sequence of SEQ ID NO: 174, 172, 175, 176, or 177, It includes a fourth chain containing the amino acid sequence of SEQ ID NO: 173, Alternatively, the antigen-binding molecule is A first chain containing the amino acid sequence of sequence number 178, A second chain containing the amino acid sequence of SEQ ID NO: 74, A third chain containing the amino acid sequence of sequence number 179, A fourth chain containing the amino acid sequence of SEQ ID NO: 173, An antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 19.

21. The aforementioned antigen-binding molecule is One antigen-binding module that specifically binds to EGFR, It comprises one antigen-binding module that specifically binds to MUC1, The antigen-binding module that specifically binds to the aforementioned MUC1 is Fab. The antigen-binding module that specifically binds to the aforementioned EGFR is a replaced Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer. Preferably, The antigen-binding molecule comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d). Formula (a) [MUC1-VH]-[CH1]-[Fc1], Formula (b) [MUC1-VL]-[CL], Formula (c) [EGFR-VH]-[Linker 1]-[Titin]-[Fc2], Formula (d) [EGFR-VL]-[Linker 2]-[Obscurin], Eventually, Linker 1 and linker 2 are the same or different, are peptide linkers, or neither linker 1 nor linker 2 exists. The structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus. More preferably, among them, MUC1-VH contains the amino acid sequence of SEQ ID NO: 12 in HCDR1, the amino acid sequence of SEQ ID NO: 13 in HCDR2, and the amino acid sequence of SEQ ID NO: 14 in HCDR3, and MUC1-VL contains the amino acid sequence of SEQ ID NO: 15 in LCDR1, the amino acid sequence of SEQ ID NO: 16 in LCDR2, and the amino acid sequence of SEQ ID NO: 17 in LCDR3, and The EGFR-VH comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and the EGFR-VL comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO:

121. More preferably, among them, MUC1-VH includes the amino acid sequence of SEQ ID NO: 36, MUC1-VL includes the amino acid sequence of SEQ ID NO: 39, EGFR-VH includes the amino acid sequence of SEQ ID NO: 138, and EGFR-VL includes the amino acid sequence of SEQ ID NO:

149. Most preferably, the antigen-binding molecule is A first chain containing the amino acid sequence of sequence number 171, A second chain containing the amino acid sequence of SEQ ID NO: 74, A third chain containing the amino acid sequence of sequence number 174, It includes a fourth chain containing the amino acid sequence of SEQ ID NO: 173, Alternatively, the antigen-binding molecule is A first chain containing the amino acid sequence of sequence number 178, A second chain containing the amino acid sequence of SEQ ID NO: 74, A third chain containing the amino acid sequence of sequence number 179, A fourth chain containing the amino acid sequence of SEQ ID NO: 173, An antigen-binding molecule that specifically binds to EGFR and MUC1 as described in claim 20.

22. It is an immune complex, An anti-MUC1 antibody or its antigen-binding fragment and effector molecule according to any one of claims 1 to 7, Or An anti-EGFR antibody or its antigen-binding fragment and effector molecule according to any one of claims 8 to 12, Or Antigen-binding molecule and effector molecule that specifically bind to EGFR and MUC1 according to any one of claims 13 to 21, Includes, Among these, the effector molecule is coupled to the anti-MUC1 antibody or its antigen-binding fragment, the anti-EGFR antibody or its antigen-binding fragment, or an antigen-binding molecule that specifically binds to EGFR and MUC1. Preferably, the effector molecule is selected from antitumor agents, immunomodulators, biological reaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof. immune complex.

23. An antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 Y is selected from -O-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)-, -O-CR １ R ２ -(CR ａ R ｂ ), ｍ -O-CR １ R ２ -, -NH-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)- or -S-(CR ａ R ｂ ), ｍ -CR １ [[ID=4,2]]R ２ -C(O)-; R ａ and R ｂ They are the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, hydroxyl groups, amino groups, cyano groups, nitro groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. Or, R ａ and R ｂ These, together with the carbon atoms linked to them, form a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group. R １ This is selected from halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, hydroxyl groups, alkoxy groups, cyano groups, amino groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. R ２ This is selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, hydroxyl groups, alkoxy groups, cyano groups, amino groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group. Or, R ａ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group. m is 0, 1, 2, 3 or 4, n is between 1 and 10. L is the linker unit, Pc is an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or an anti-EGFR antibody or its antigen-binding fragment according to any one of claims 8 to 12, or an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21. Preferably, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of claims 13 to 21. An antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof.

24. Y is -O-(CR ａ R ｂ ) ｍ -CR １ R ２ -C(O)-, Eventually, R ａ and R ｂ They are the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, and alkyl groups. R １ is a cycloalkyl-alkyl group or a cycloalkyl group. R ２ This is selected from hydrogen atoms, haloalkyl groups, and cycloalkyl groups. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group. m is 0, 1, 2, 3 or 4, n is between 1 and 10. L is the linker unit, Pc is an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or an anti-EGFR antibody or its antigen-binding fragment according to any one of claims 8 to 12, or an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21. Preferably, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of claims 13 to 21. An antibody-drug conjugate represented by the general formula (Pc-L-Y-D) as described in claim 23, or a pharmaceutically acceptable salt thereof.

25. Linker unit -L- is -L １ -L ２ -L ３ -L ４ - and Eventually, L １ is -(succinimido-3-yl-N)-W-C(O)-,-CH ２ -C(O)-NR ３ -W-C(O)- and -C(O)-W-C(O)- are selected, where W is selected from alkylene groups and alkylene-cycloalkyl groups, and of which the alkylene group or alkylene-cycloalkyl group is independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups and cycloalkyl groups. L ２ -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ ) p １ Selected from C(O)- and chemical bonds, among them, p １ is an integer between 1 and 20, L ３ This is a peptide residue consisting of 2 to 7 amino acid residues, of which the amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ４ -NR ５ (CR ６ R ７ ) ｔ -, -C(O)NR ５ -, -C(O)NR ５ (CH ２ ) ｔ - Selected from chemical bonds, where t is 1, 2, 3, 4, 5 or 6, R ３ , R ４ and R ５ They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R ６ and R ７ They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. Preferably, among them, the linker unit-L １ -L ２ -L ３ -L ４ - is as follows: L １ teeth 【Chemistry 2】 And among them, s １ is 2, 3, 4, 5, 6, 7 or 8, L ２ It is a chemical bond, L ３ is a tetrapeptide residue, preferably L ３ This is a tetrapeptide residue shown in Sequence ID No. 180, L ４ is - NR ５ (CR ６ R ７ )t−, where R ５ , R ６ or R ７ are the same or different and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2, Among them, the -L- is L １ The end is connected to Pc, and L ４ The end is connected to Y An antibody-drug conjugate represented by the general formula (Pc-L-Y-D) as described in claim 23 or 24, or a pharmaceutically acceptable salt thereof.

26. General formula (Pc-L ａ An antibody-drug conjugate represented by (-Y-D) or a pharmaceutically acceptable salt thereof, 【Transformation 3】 Eventually, Pc is an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or an anti-EGFR antibody or its antigen-binding fragment according to any one of claims 8 to 12, or an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21. Preferably, Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 as described in any one of claims 13 to 21. m is 0, 1, 2, 3 or 4, n is between 1 and 10. R １ is a cycloalkyl-alkyl group or a cycloalkyl group. R ２ This is selected from hydrogen atoms, haloalkyl groups, and cycloalkyl groups. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group. W is selected from alkylene groups and alkylene-cycloalkyl groups, and of these, the alkylene group and alkylene-cycloalkyl group are each independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ２ -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ ) p １ Selected from C(O)- and chemical bonds, among them, p １ is an integer between 1 and 20, L ３ This is a peptide residue consisting of 2 to 7 amino acid residues, of which the amino acid residues are selected from amino acid residues formed from amino acids among phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R ４ and R ５ This is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups. R ６ and R ７ They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. An antibody-drug conjugate represented by the general formula (Pc-L-Y-D) as described in any one of claims 23 to 25, or a pharmaceutically acceptable salt thereof.

27. General formula (Pc-L ａ A method for preparing an antibody-drug conjugate represented by (-Y-D) or a pharmaceutically acceptable salt thereof, 【Chemistry 4】 After reducing Pc, the general formula (L ａ The compound represented by -Y-D) or a salt thereof is subjected to a coupling reaction, and the general formula (Pc-L ａ The process includes the step of obtaining an antibody-drug conjugate represented by (-Y-D) or a pharmaceutically acceptable salt thereof, Eventually, Pc is an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or an anti-EGFR antibody or its antigen-binding fragment according to any one of claims 8 to 12, or an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21, preferably Pc is an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21. W, L ２ , L ３ , R １ , R ２ , R ５ ~R ７ m and n are as defined in claim 26. method.

28. A pharmaceutical composition, An anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, an anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 8 to 12, an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 23 to 26, A compound comprising one or more pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutical composition.

29. An isolated nucleic acid encoding an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or an anti-EGFR antibody or its antigen-binding fragment according to any one of claims 8 to 12.

30. A host cell comprising the isolated nucleic acid described in claim 29.

31. A method for preventing or treating a disease, wherein the method is The procedure comprises administering to a subject an effective amount for prevention or treatment of an anti-MUC1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, an anti-EGFR antibody or its antigen-binding fragment according to any one of claims 8 to 12, an antigen-binding molecule that specifically binds to EGFR and MUC1 according to any one of claims 13 to 21, an antibody-drug conjugate according to any one of claims 23 to 26 or its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 28. Preferably, the disease is a tumor. More preferably, the disease is selected from astrocytoma, glioblastoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, gallbladder cancer, gastric cancer, head and neck cancer, idiopathic myelofibrosis, kidney cancer, leukemia, liver cancer, esophageal cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, neuroblastoma, oligodendroglioma, ovarian cancer, peritoneal tumor, pancreatic cancer, polycythemia vera, primary neuroectodermal tumor, prostate cancer, retinoblastoma, sarcoma, squamous cell carcinoma, thyroid cancer, endometrial cancer, vestibular schwannoma, blastoma, vulvar cancer, thymoma, testicular cancer, cholangiocarcinoma, pheochromocytoma, paraganglioma, and adenoid cystic carcinoma. Most preferably, the disease is selected from lung cancer, head and neck cancer, esophageal cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, stomach cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, and bladder cancer. method.

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