Anti-EGFR / MUC1 antibodies and uses thereof

Abstract

The present disclosure relates to anti-EGFR / MUC1 antibodies, and antibody drug conjugates derived therefrom, and uses thereof.

Description

[Technical Field]

[0001] Priority claims This application claims priority to PCT / CN2023 / 071272, filed January 9, 2023. The entire contents of the above-mentioned application are incorporated herein by reference.

[0002] The present disclosure relates to multispecific anti-EGFR (epidermal growth factor receptor) / MUC1 (mucin 1) antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof), and antibody-drug conjugates derived therefrom. [Background technology]

[0003] Bispecific antibodies are engineered proteins that can simultaneously bind to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting different disease mediators, and delivering payloads to target sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) marks major milestones in the development of bispecific antibodies.

[0004] Because bispecific antibodies have a variety of uses, there is a continuing need to develop a variety of therapeutic agents based on bispecific antibodies. Summary of the Invention

[0005] The present disclosure relates to anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof, which specifically bind to EGFR and MUC1. In some embodiments, the antibodies or antigen-binding fragments thereof have identical light chain variable regions. In some embodiments, the antibodies or antigen-binding fragments thereof share a common light chain. The present invention also relates to antibody-drug conjugates derived from these anti-EGFR / MUC1 antibodies.

[0006] In one aspect, the invention relates to an anti-EGFR / MUC1 antibody, or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MUC1.

[0007] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0008] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR3; and the first light chain variable region (VL1) comprises CDR1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL1 CDR3; The selected VH1 CDR1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following:

[0009] (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (4) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (5) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (6) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (7) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (8) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (9) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 34 to 36, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; and (10) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

[0010] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDR1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL2 CDR3; The selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are one of the following:

[0011] (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; and (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

[0012] In some embodiments, one of the following is true: (1) the amino acid sequences of the selected VH1 CDR1, 2, and 3 are set forth in SEQ ID NOs: 4 to 6, respectively, the amino acid sequences of the selected VL1 CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively, the amino acid sequences of the selected VH2 CDR1, 2, and 3 are set forth in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (4) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (5) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (6) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (7) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (8) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (9) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (10) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (11) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (12) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (13) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are represented by SEQ ID NOs: 31 to 33, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are represented by SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are represented by SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are represented by SEQ ID NOs: 1 to 3, respectively; (14) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 34 to 36, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (15) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (16) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (17) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (18) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (19) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are represented by SEQ ID NOs: 34 to 36, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are represented by SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are represented by SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are represented by SEQ ID NOs: 1 to 3, respectively; or (20) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

[0013] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:47, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:52, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0014] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:48, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:52, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0015] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:49, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:52, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0016] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:50, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:52, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0017] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:51, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:52, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0018] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:47, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0019] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:48, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0020] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:49, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0021] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:50, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0022] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:51, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46.

[0023] In some embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0024] (1) the selected VH sequence is SEQ ID NO: 47 and the selected VL sequence is SEQ ID NO: 46; (2) the selected VH sequence is SEQ ID NO: 48 and the selected VL sequence is SEQ ID NO: 46; (3) the selected VH sequence is SEQ ID NO: 49 and the selected VL sequence is SEQ ID NO: 46; (4) the selected VH sequence is SEQ ID NO: 50 and the selected VL sequence is SEQ ID NO: 46; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 46.

[0025] In some embodiments, the VH1 comprises a VH1 CDR1, a VH1 CDR2, and a VH1 CDR3 that are identical to the VH CDR1, the VH CDR2, and the VH CDR3 of a selected VH sequence; the VL1 comprises a VL1 CDR1, a VL1 CDR2, and a VL1 CDR3 that are identical to the VL CDR1, the VL CDR2, and the VL CDR3 of a selected VL sequence; and the selected VH sequence and the selected VL sequence are one of the following:

[0026] (1) the selected VH sequence is SEQ ID NO: 47 and the selected VL sequence is SEQ ID NO: 46; (2) the selected VH sequence is SEQ ID NO: 48 and the selected VL sequence is SEQ ID NO: 46; (3) the selected VH sequence is SEQ ID NO: 49 and the selected VL sequence is SEQ ID NO: 46; (4) the selected VH sequence is SEQ ID NO: 50 and the selected VL sequence is SEQ ID NO: 46; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 46.

[0027] In some embodiments, VH2 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence; the selected VH sequence and the selected VL sequence are one of the following:

[0028] (1) the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 46; and (2) The selected VH sequence is SEQ ID NO: 53, and the selected VL sequence is SEQ ID NO: 46.

[0029] In some embodiments, VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; VL2 comprises VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence; and the selected VH sequence and the selected VL sequence are one of the following:

[0030] (1) the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 46; and (2) The selected VH sequence is SEQ ID NO: 53, and the selected VL sequence is SEQ ID NO: 46.

[0031] In some embodiments, VH1 comprises the sequence of SEQ ID NO:47 and VL1 comprises the sequence of SEQ ID NO:46.

[0032] In some embodiments, VH1 comprises the sequence of SEQ ID NO:48 and VL1 comprises the sequence of SEQ ID NO:46.

[0033] In some embodiments, VH1 comprises the sequence of SEQ ID NO:49 and VL1 comprises the sequence of SEQ ID NO:46.

[0034] In some embodiments, VH1 comprises the sequence of SEQ ID NO:50 and VL1 comprises the sequence of SEQ ID NO:46.

[0035] In some embodiments, VH1 comprises the sequence of SEQ ID NO:51 and VL1 comprises the sequence of SEQ ID NO:46.

[0036] In some embodiments, VH2 comprises the sequence of SEQ ID NO:52 and VL2 comprises the sequence of SEQ ID NO:46.

[0037] In some embodiments, VH2 comprises the sequence of SEQ ID NO:53 and VL2 comprises the sequence of SEQ ID NO:46.

[0038] In some embodiments, the first antigen-binding domain specifically binds to human EGFR or monkey EGFR, and / or the second antigen-binding domain specifically binds to human MUC1 or monkey MUC1.

[0039] In some embodiments, the first antigen-binding domain is human or humanized and / or the second antigen-binding domain is human or humanized.

[0040] In some embodiments, the antibody is a bispecific antibody.

[0041] In some embodiments, the first antigen-binding domain is a single-chain variable fragment (scFv) and / or the second antigen-binding domain is an scFv.

[0042] In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0043] In some embodiments, the present disclosure relates to anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof that cross-compete with the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein.

[0044] In one aspect, the disclosure relates to nucleic acids comprising polynucleotides encoding the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein.

[0045] In one aspect, the present disclosure relates to a vector comprising a nucleic acid described herein.

[0046] In one aspect, the present disclosure relates to a cell comprising the vector described herein.

[0047] In some embodiments, the cells are CHO cells.

[0048] In one aspect, the present disclosure relates to a cell comprising a nucleic acid described herein.

[0049] In one aspect, the disclosure relates to a method for producing an EGFR / MUC1 antibody or antigen-binding fragment thereof, the method comprising: (a) culturing a cell described herein under conditions sufficient to produce an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof; (b) recovering the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof produced by the cells.

[0050] In one aspect, the disclosure relates to an anti-EGFR / MUC1 antibody drug conjugate (ADC) comprising a therapeutic agent covalently attached to an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof described herein.

[0051] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0052] In some embodiments, the therapeutic agent is MMAE or MMAF.

[0053] In some embodiments, the therapeutic agent is selected from the following:

[0054] [ka]

[0055] In some embodiments, the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker has the following structure: [ka]

[0056] In some embodiments, the antibody drug conjugate has the following structure: [ka] [ka] In some embodiments, n=1 to 8, and in some embodiments, "Ab" refers to an antibody or antigen-binding fragment thereof.

[0057] In some embodiments, the drug-antibody ratio (DAR) is about 4 or 8.

[0058] In one aspect, the disclosure relates to a method of treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof described herein, or a composition comprising an anti-EGFR / MUC1 antibody-drug conjugate described herein.

[0059] In some embodiments, the subject has a cancer that expresses EGFR and / or MUC1 (eg, both EGFR and MUC1).

[0060] In some embodiments, the cancer is a solid tumor, lung cancer, head and neck cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, glioma, esophageal cancer, gastric cancer, stomach cancer, prostate cancer, cervical cancer, multiple myeloma, or non-Hodgkin's lymphoma.

[0061] In some embodiments, the subject is a human.

[0062] In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject.

[0063] In some embodiments, the method further comprises administering chemotherapy to the subject.

[0064] In one aspect, the disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof described herein, or a composition comprising an anti-EGFR / MUC1 antibody-drug conjugate described herein.

[0065] In one aspect, the disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof described herein, or a composition comprising an anti-EGFR / MUC1 antibody-drug conjugate described herein.

[0066] In one aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof described herein, and / or (b) an anti-EGFR / MUC1 antibody-drug conjugate described herein.

[0067] 1) As used herein, the term "antigen-binding domain" refers to one or more protein domains (e.g., formed from amino acids from a single polypeptide, or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that can specifically bind to one or more different antigens (e.g., effector antigens or control antigens). In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. In some embodiments, the antigen-binding domain can comprise an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are well known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other examples, an antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen).

[0068] 2) The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a protein complex containing two heavy chains and two light chains. Further examples of antibodies are described herein.

[0069] 3) As used herein, the term "multispecific antibody" refers to an antibody comprising two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or epitopes on different antigens (e.g., different proteins present on the surface of the same cell or on the surface of different cells). In some embodiments, a multispecific antibody binds to two different epitopes (e.g., a "bispecific antibody"). In some embodiments, a multispecific antibody binds to three different epitopes (e.g., a "trispecific antibody"). In some embodiments, a multispecific antibody binds to four different epitopes (e.g., a "tetraspecific antibody"). In some embodiments, a multispecific antibody binds to five different epitopes (e.g., a "pentaspecific antibody"). Each binding specificity can be present in any suitable valency. Non-limiting examples of multispecific antibodies are described herein.

[0070] 4) As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes, which can be on the same antigen or on different antigens.

[0071] 5) As used herein, the term "common light chain" refers to a light chain capable of interacting with two or more different heavy chains to form different antigen-binding sites, which can specifically bind to different antigens or epitopes. Similarly, the term "common light chain variable region" refers to a light chain variable region capable of interacting with two or more different heavy chain variable regions to form different antigen-binding sites, which can specifically bind to different antigens or epitopes. In some embodiments, antibodies or antigen-binding fragments thereof may share a common light chain. In some embodiments, anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof may share a common light chain variable region.

[0072] 6) As used herein, the term "anti-EGFR / MUC1 antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that binds to both MUC1 and EGFR.

[0073] 7) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials well-known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0074] 8) Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0075] [Figure 1] 1 shows exemplary structures of anti-EGFR / MUC1 antibodies described herein. [Figure 2]1 shows the mean tumor volume in different groups of B-NDG mice injected with HCC70 cells and treated with phosphate-buffered saline (PBS) or ADC. [Figure 3] 1 shows the mean tumor volume in different groups of B-NDG mice injected with Panc 02.03 cells and treated with PBS or ADC. [Figure 4] 1 shows the mean tumor volume in different groups of B-NDG mice injected with NCI-H1975 cells and treated with PBS or ADC. [Figure 5] 1 shows the mean tumor volume in different groups of B-NDG mice injected with NCI-H1650 cells and treated with PBS or ADC. [Figure 6] The heavy chain variable region CDR sequences of the anti-EGFR antigen-binding domains (E-6C4, E-9A6, E-9D2, E-9F3, and E-9H2) and the anti-MUC1 antigen-binding domains (10M1 and M-11E8) of the anti-EGFR / MUC1 antibodies according to Kabat's definition are listed below. [Figure 7] The heavy chain variable region CDR sequences of the anti-EGFR antigen-binding domains (E-6C4, E-9A6, E-9D2, E-9F3, and E-9H2) and the anti-MUC1 antigen-binding domains (10M1 and M-11E8) of the anti-EGFR / MUC1 antibodies defined by Chothia are listed below. [Figure 8] The light chain variable region CDR sequences of anti-EGFR / MUC1 antibodies according to the Kabat and Chothia definitions are listed below. [Figure 9] The sequences of the heavy chain variable region and light chain variable region of the anti-EGFR / MUC1 antibody are listed below. [Figure 10] The specific amino acid sequences discussed in this disclosure are listed below. [Figure 11] 1 shows the mean tumor volume in different groups of B-NDG mice implanted with patient-derived papillary tumor fragments and treated with PBS or ADC. [Figure 12] Panels AB show the endocytosis rates of anti-EGFR antibody, anti-MUC1 antibody, and anti-EGFR / MUC1 bispecific antibody in NUGC-4 cells (FIG. 12A) or HCC70 cells (FIG. 12B). [Figure 13] AB show the endocytosis rates of anti-EGFR / MUC1 bispecific antibodies and ADCs in NUGC-4 cells (FIG. 13A) or Panc 02.03 cells (FIG. 13B). [Figure 14] shows the mean tumor volume in different groups of B-NDG mice implanted with patient-derived pancreatic tumor fragments and treated with PBS or ADC. DETAILED DESCRIPTION OF THE INVENTION

[0076] A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens). In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms. Each arm can have one heavy chain variable region and one light chain variable region to form an antigen-binding domain (or antigen-binding region). In some embodiments, the bispecific antibody shares a common light chain.

[0077] The present invention relates to anti-EGFR / MUC1 antibodies (eg, bispecific antibodies or antigen-binding fragments thereof) that specifically bind to EGFR and MUC1, and antibody-drug conjugates derived from these anti-EGFR / MUC1 antibodies.

[0078] Anti-EGFR / MUC1 antibody The epidermal growth factor receptor (EGFR, ErbB1, or HER1) is a 170-kDa type 1 transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. It is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). Mutations affecting EGFR expression or activity can lead to cancer in many types of cancer. EGFR signaling is initiated by ligand binding, followed by conformational changes, homodimerization or heterodimerization of the receptor with other ErbB family members, and trans-autophosphorylation of the receptor, initiating a signal transduction cascade that ultimately affects different cellular functions, such as cell proliferation and survival. Increased expression or kinase activity of EGFR has been associated with various human cancers, making EGFR an attractive target for therapeutic intervention. Increased both EGFR gene copy number and protein expression are associated with a favorable response to the EGFR tyrosine kinase inhibitor, IRESSA® (gefitinib) in non-small cell lung cancer.

[0079] Binding of a ligand such as EGF (epidermal growth factor receptor) to EGFR stimulates receptor dimerization, autophosphorylation, activation of the receptor's internal cytoplasmic tyrosine kinase domain, and initiation of multiple signal transduction and transactivation pathways involved in regulating DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of EGFR signaling can lead to inhibition of one or more EGFRs. In some embodiments, EGFR ligands include EGF, TGFα, heparin-binding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI).

[0080] A detailed review of EGFR can be found in Sabbah, Dima A., Rima Hajjo, and Kamal Sweidan. “Review on epidermal growth factor receptor (EGFR) structure, signaling pathways, interactions, and recent updates of EGFR inhibitors.” Current Topics in Medicinal Chemistry (2020), which is incorporated herein by reference in its entirety.

[0081] Mucin 1 (also known as MUC1, episialin, PEM, H23Ag, EMA, CA15-3, and MCA) is a single-spanning type I membrane protein with a heavily glycosylated extracellular domain extending 200–500 nm from the cell surface. MUC1 is normally expressed in glandular or luminal epithelial cells of the breast, esophagus, stomach, duodenum, pancreas, uterus, prostate, and lung, and to a lesser extent in hematopoietic cells. It is absent in skin epithelium and mesenchymal cells. In healthy tissues, MUC1 provides protection to the underlying epithelium. The extended, negatively charged sugar branches of MUC1 create a physical barrier, endowing MUC1 with antiadhesive properties, limiting accessibility, and preventing pathogen colonization. The glycans oligomerize to form a mucus gel that lubricates the underlying epithelium and protects it from desiccation, pH changes, pollutants, and microorganisms. Abnormally glycosylated MUC1 is overexpressed in most human epithelial cancers and has been implicated as an oncogenic molecule.

[0082] MUC1 is overexpressed in cancer cells, and loss of cell polarity leads to the redistribution of TA-MUC1 to the cell surface and cytoplasm. The lack of cell polarity also leads to the redistribution of cell surface growth factors, which are normally restricted to the basolateral surface of epithelial cells. MUC1 and growth factors, in conjunction with intracellular kinases such as ZAP-70, PKC-g, GSK-3b, and c-Src, phosphorylate serine, tyrosine, and threonine residues on the MUC1 CT. Hypoglycosylation also exposes the peptide core of TA-MUC1, potentially allowing extracellular proteases to cleave and release MUC1-N. Release of MUC1-N induces a conformational change in MUC1-C, altering its ligand status and subsequently activating downstream cell signaling pathways, such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K / Akt), and Wingless (Wnt) pathways. Consequently, hyperactivation of these important signaling pathways is commonly observed in MUC1-positive pancreatic, breast, lung, and colon cancer cells. MUC1-C also associates with various transcription factors (STAT3, NF-kB, p53, and β-catenin) and binds to target gene promoters to promote their expression. Several studies have shown that MUC1 plays an important role in the transcriptional regulation of genes related to tumor invasion, metastasis, angiogenesis, proliferation, apoptosis, drug resistance, inflammation, and immune regulation.

[0083] For a detailed review of MUC1 and its function, see Nath, Sritama, and Pinku Mukherjee. “MUC1: a multifaceted oncoprotein with a key role in cancer progression.” Trends in molecular medicine 20.6(2014):332-342, which is incorporated by reference in its entirety.

[0084] In some embodiments, the bispecific anti-EGFR / MUC1 antibodies described herein can be designed to have an IgG1 subtype structure with knob-into-hole (KIH) mutations, which promote heterodimer formation and avoid mispairing between the two heavy chains. In some embodiments, the bispecific anti-EGFR / MUC1 antibodies have a higher endocytosis rate than the corresponding monoclonal antibody or a control bispecific antibody.

[0085] In some embodiments, the bispecific anti-EGFR / MUC1 antibodies described herein can be conjugated with a therapeutic agent to form an antibody-drug conjugate (ADC). In some embodiments, the drug-antibody ratio (DAR) of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, or about 3.6. to about 4.4, about 3.7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 to about 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.2, about 3.8 to about 4.2, about 3.9 to about 4.2, about 4.0 to about 4.2, about 4.1 to about 4.2, about 3.5 to about 4.1, about 3.6 to about 4.1, about 3.7 to about 4.1, about 3.8 to about 4.1, about 3.9 to about 4.1, about 4.0 to about 4.1, about 3.5 to about 4. 0, about 3.6 to about 4.0, about 3.7 to about 4.0, about 3.8 to about 4.0, about 3.9 to about 4.0, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7, or about 3.5 to about 3.6.In some embodiments, the DAR of the ADCs described herein is about 7.5 to about 8.5, about 7.6 to about 8.5, about 7.7 to about 8.5, about 7.8 to about 8.5, about 7.9 to about 8.5, about 8.0 to about 8.5, about 8.1 to about 8.5, about 8.2 to about 8.5, about 8.3 to about 8.5, about 8.4 to about 8.5, about 7.5 to about 8.4, about 7.6 to about 8.4, about 7.7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8.2 to about 8.3, about 7.5 to about 8.2, about 7.6 to about 8.2, about 7.7 to about 8.2, about 7.8 to about 8.2, about 7.9 to about 8.2, about 8.0 to about 8.2, about 8.1 to about 8.2, about 7.5 to about 8.1, about 7.6 to about 8.1, about 7.7 to about 8.1, about 7.8 to about 8.1, about 7.9 to about 8.1, about 8.0 to about 8.1, about 7.5 to about 8. 0, about 7.6 to about 8.0, about 7.7 to about 8.0, about 7.8 to about 8.0, about 7.9 to about 8.0, about 7.5 to about 7.9, about 7.6 to about 7.9, about 7.7 to about 7.9, about 7.8 to about 7.9, about 7.5 to about 7.8, about 7.6 to about 7.8, about 7.7 to about 7.8, about 7.5 to about 7.7, about 7.6 to about 7.7, or about 7.5 to about 7.6.

[0086] In some embodiments, the anti-EGFR / MUC1 ADCs described herein can effectively inhibit the growth of cancer cells in vitro at concentrations of less than 10 μg / mL, less than 3.33 μg / mL, less than 1.11 μg / mL, less than 0.37 μg / mL, less than 0.12 μg / mL, less than 0.04 μg / mL, or less than 0.01 μg / mL. In some embodiments, the anti-EGFR / MUC1 ADCs described herein can inhibit the growth of cancer cells (e.g., lung, gastric, breast, or pancreatic cancer) in vivo in a xenograft mouse model at dose levels of less than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg. In some embodiments, the anti-EGFR / MUC1 antibodies described herein share a common light chain. In some embodiments, the anti-EGFR / MUC1 antibodies share an anti-EGFR antigen-binding domain (e.g., E-6C4, E-9A6, E-9D2, E-9F3, or E-9H2) or an anti-MUC1 antigen-binding domain (e.g., 10M1 or M-11E8). In some embodiments, the anti-EGFR / MUC1 antibodies share an EGFR-targeting heavy chain variable region (e.g., any of the EGFR-targeting VHs described herein), a MUC1-targeting heavy chain variable region (e.g., any of the MUC1-targeting VHs described herein), and two identical common light chain variable regions.

[0087] The CDR sequences of the E-6C4 antigen-binding domain include the heavy chain variable domain CDRs SEQ ID NOS: 4-6 and the light chain variable domain CDRs SEQ ID NOS: 1-3, as defined by the Kabat definition. CDRs can also be defined by the Chothia definition. According to the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 25-27, and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 1-3. The human light chain variable region and human heavy chain variable region for E-6C4 are set forth in SEQ ID NOS: 46 and 47, respectively.

[0088] The CDR sequences of the E-9A6 antigen-binding domain include the heavy chain variable domain CDRs SEQ ID NOS: 7 to 9 and the light chain variable domain CDRs SEQ ID NOS: 1 to 3, as defined by the Kabat definition. According to the Chothia definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOS: 28 to 30, and the light chain variable domain CDR sequences are shown in SEQ ID NOS: 1 to 3. The human light chain variable region and human heavy chain variable region for E-9A6 are shown in SEQ ID NOS: 46 and 48, respectively.

[0089] The CDR sequences of the E-9D2 antigen-binding domain include the heavy chain variable domain CDRs defined by the Kabat definition, SEQ ID NOS: 10 to 12, and the light chain variable domain CDRs defined by the Chothia definition, SEQ ID NOS: 1 to 3. According to the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 31 to 33, and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 1 to 3. The human light chain variable region and human heavy chain variable region for E-9D2 are set forth in SEQ ID NOS: 46 and 49, respectively.

[0090] The CDR sequences of the E-9F3 antigen-binding domain include the heavy chain variable domain CDRs defined by the Kabat definition, SEQ ID NOS: 13 to 15, and the light chain variable domain CDRs defined by the Chothia definition, SEQ ID NOS: 1 to 3. According to the Chothia definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOS: 34 to 36, and the light chain variable domain CDR sequences are shown in SEQ ID NOS: 1 to 3. The human light chain variable region and human heavy chain variable region for E-9F3 are shown in SEQ ID NOS: 46 and 50, respectively.

[0091] The CDR sequences of the E-9H2 antigen-binding domain include the heavy chain variable domain CDRs defined by the Kabat definition, SEQ ID NOS: 16 to 18, and the light chain variable domain CDRs defined by the Kabat definition, SEQ ID NOS: 1 to 3. According to the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 37 to 39, and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 1 to 3. The human light chain variable region and human heavy chain variable region for E-9H2 are set forth in SEQ ID NOS: 46 and 51, respectively.

[0092] The CDR sequences of the 10M1 antigen-binding domain include the heavy chain variable domain CDRs defined by the Kabat definition, SEQ ID NOS: 19 to 21, and the light chain variable domain CDRs defined by the Kabat definition, SEQ ID NOS: 1 to 3. According to the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 40 to 42, and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 1 to 3. The human light chain variable region and human heavy chain variable region for 10M1 are set forth in SEQ ID NOS: 46 and 52, respectively.

[0093] The CDR sequences of the M-11E8 antigen-binding domain include the heavy chain variable domain CDRs SEQ ID NOS: 22 to 24 and the light chain variable domain CDRs SEQ ID NOS: 1 to 3, as defined by the Kabat definition. According to the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 43 to 45, and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 1 to 3. The human light chain variable region and human heavy chain variable region for M-11E8 are set forth in SEQ ID NOS: 46 and 53, respectively.

[0094] In some embodiments, the anti-EGFR / MUC1 antibodies described herein may contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4 to 6, SEQ ID NOs: 7 to 9, SEQ ID NOs: 10 to 12, SEQ ID NOs: 13 to 15, SEQ ID NOs: 16 to 18, SEQ ID NOs: 19 to 21, SEQ ID NOs: 22 to 24, SEQ ID NOs: 25 to 27, SEQ ID NOs: 28 to 30, SEQ ID NOs: 31 to 33, SEQ ID NOs: 34 to 36, SEQ ID NOs: 37 to 39, SEQ ID NOs: 40 to 42, and SEQ ID NOs: 43 to 45, and / or one, two, or three light chain variable region CDRs set forth in SEQ ID NOs: 1 to 3.

[0095] In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VL CDR3. The light chain variable region (VL) may comprise or consist of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR1, the CDR2 region may comprise or consist of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VL CDR2, and the CDR3 region may comprise or consist of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3 and the selected VL CDR1, 2, and 3 are shown in Figures 6 to 8.

[0096] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:4 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:5 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO:6 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0097] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:7 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:8 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:9 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0098] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0099] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0100] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0101] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0102] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 24 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0103] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 27 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0104] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0105] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 33 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0106] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0107] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 39 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0108] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 42 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0109] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 45 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0110] In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0111] Insertions, deletions and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences.

[0112] In some embodiments, the anti-EGFR / MUC1 antibody comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO:47, 48, 49, 50, 51, 52, or 53, and the selected VL sequence is SEQ ID NO:46.

[0113] In some embodiments, an anti-EGFR / MUC1 antibody or antigen-binding fragment may have three VH CDRs that are identical to the CDRs of any VH sequence described herein. In some embodiments, an anti-EGFR / MUC1 antibody or antigen-binding fragment may have three VL CDRs that are identical to the CDRs of any VL sequence described herein.

[0114] The present invention also provides nucleic acids comprising polynucleotides encoding anti-EGFR / MUC1 antibodies. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-EGFR / MUC1 antibody comprises the CDRs shown in Figures 6 to 8. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to MUC1 and / or EGFR.

[0115] The anti-EGFR / MUC1 antibody can be an anti-EGFR / MUC1 antibody variant (including derivatives and conjugates) of the anti-EGFR / MUC1 antibody or antibody fragment. Additional anti-EGFR / MUC1 antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human, chimeric (e.g., human-mouse chimeras), single-chain, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The anti-EGFR / MUC1 antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or antigen-binding fragment thereof.

[0116] Fragments of anti-EGFR / MUC1 antibodies are suitable for use in the provided methods, so long as they retain the desired affinity and specificity for both MUC1 and EGFR. Thus, fragments of anti-EGFR / MUC1 antibodies retain the ability to bind to MUC1 and EGFR.

[0117] Antibodies and antigen-binding fragments thereof In some embodiments, a multispecific anti-EGFR / MUC1 antibody (e.g., a bispecific antibody) comprises an antigen-binding domain derived from an anti-EGFR antibody and an antigen-binding domain derived from an anti-MUC1 antibody. These anti-EGFR / MUC1 antibodies and antigen-binding fragments thereof can have a variety of forms.

[0118] Generally, antibodies (also called immunoglobulins) can be composed of two classes of polypeptide chains: light chains and heavy chains. Non-limiting anti-EGFR / MUC1 antibodies of the present disclosure can be intact four immunoglobulin chain antibodies, comprising two heavy chains and two light chains. The heavy chain of the anti-EGFR / MUC1 antibody can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

[0119] The hypervariable regions, known as complementarity-determining regions (CDRs), form the loops that comprise the principal antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form connecting loops that, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding domain.

[0120] Methods for identifying CDR regions of antibodies by analyzing their amino acid sequences are well known, and several definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, in Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422~439, Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al.,Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated by reference herein in its entirety.

[0121] CDRs are important for recognizing the epitope of an antigen. As used herein, "epitope" refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be approximately 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence in the primary structure of the antigen, as the epitope can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0122] In some embodiments, the anti-EGFR / MUC1 antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences between IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in Immunology 5 (2014); Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases," Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed., The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated by reference in its entirety herein.

[0123] Anti-EGFR / MUC1 antibodies can be immunoglobulin molecules derived from any species (e.g., human, rodent, mouse, rat, camelid). An antigen-binding domain or antigen-binding fragment is any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., that is capable of specifically binding to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof can include, for example, scFv, Fv, Fd, dAb, diabody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and polypeptides comprising a binding domain that is, or is homologous to, an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0124] In some embodiments, the scFv in the anti-EGFR / MUC1 antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the anti-EGFR / MUC1 scFv has two antigen-binding regions (antigen-binding regions: A and B), which can bind to their respective target antigens with different affinities.

[0125] In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof can comprise one, two, or three heavy chain variable region CDRs selected from FIGS.

[0126] In some embodiments, the anti-EGFR / MUC1 antibodies described herein may be conjugated to a therapeutic agent. The anti-EGFR / MUC1 antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently linked to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, e.g., a VC linker. Details of linkers used in ADCs are described, for example, in Su, Z. et al. “Antibody-drug conjugates: Recent advances in linker chemistry.” Acta Pharmaceutica Sinica B (2021), the entire contents of which are incorporated by reference.

[0127] In some embodiments, the anti-EGFR / MUC1 antibody is a bispecific antibody. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers. This method is described, for example, in WO 96 / 27011, incorporated by reference in its entirety.

[0128] Any of the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of the anti-EGFR / MUC1 antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0129] Anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof can also have a variety of forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art and are described, for example, in Suurs, et al., "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges," Pharmacology & Therapeutics (2019), which is incorporated by reference in its entirety.

[0130] In some embodiments, the anti-EGFR / MUC1 antibody is a BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. In some embodiments, the anti-EGFR / MUC1 antibody is selected from the group consisting of VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, diabody, crossMab, DAF(2in1), DAF(4in1), DutaMab, DT-IgG, knobs-in-hole common light chain, knobs-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0131] In some embodiments, the anti-EGFR / MUC1 antibody can be a TrioMab, in which the two heavy chains are derived from different species and different sequences restrict heavy-light chain pairing.

[0132] In some embodiments, the anti-EGFR / MUC1 antibodies have two different heavy chains and one common light chain. The heterodimerization of the heavy chains can be based on knobs-into-holes or some other heavy chain pairing technique.

[0133] In some embodiments, CrossMAb technology can be used to generate bispecific anti-EGFR / MUC1 antibodies. CrossMAb technology can be used to enhance correct light chain association in bispecific heterodimeric IgG antibodies, enabling the generation of a variety of bispecific antibody formats, including bivalent (1 + 1), trivalent (2 + 1), and tetravalent (2 + 2) bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab)-based antibodies. These formats can be derived from any existing antibody pair using domain crossover, without the need for identifying a common light chain, post-translational processing / ex vivo chemical assembly, or the introduction of a series of mutations that enhance correct light chain association. This method is described in Klein et al., "The use of CrossMAb technology for the generation of bi- and multispecific antibodies," MAbs. Vol. 8, No. 6, Taylor & Francis, 2016, incorporated by reference in its entirety. In some embodiments, the CH1 domain in the heavy chain and the CL domain in the light chain are swapped.

[0134] The anti-EGFR / MUC1 antibody can be a duobody. The Fab exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, a mechanism known as controlled Fab exchange. This format can ensure specific pairing between heavy and light chains.

[0135] In dual variable domain antibodies (DVD-Ig), an additional VH and variable light (VL) domain is added to each N-terminus for dual-specific targeting. This format is similar to IgG-scFv, but the additional binding domains are individually attached to the corresponding N-terminus of each heavy chain instead of to the N-terminus of the scFv.

[0136] In scFv-IgG, two scFvs are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is therefore also called tetravalent. In scFv-IgG, there is no problem with pairing of heavy and light chains.

[0137] In some embodiments, the anti-EGFR / MUC1 antibody can have an IgG-IgG format: two intact IgG antibodies are conjugated by chemically linking the C-termini of the heavy chains.

[0138] Anti-EGFR / MUC1 antibodies can also have a Fab-scFv-Fc format, in which a light chain, a heavy chain, and a third chain containing an Fc region and an scFv are assembled, which can ensure efficient production and purification.

[0139] In some embodiments, the anti-EGFR / MUC1 antibody can be TF. Three Fab fragments are linked by disulfide bridges. Two fragments target tumor-associated antigens (TAA) and one fragment targets a hapten. The TF format does not have an Fc region.

[0140] ADAPTIR has two scFvs attached to either side of an Fc region, which discards intact IgG as the basis for its construction but preserves the Fc region, extending half-life and facilitating purification.

[0141] Dual affinity retargeting (DART) has two peptide chains linking opposite fragments (i.e., VLA to VHB, VLB to VHA) and a sulfur bond fusing them together at the C-terminus. In DART, the sulfur bond can improve stability over BiTEs.

[0142] In DART-Fc, the Fc region is linked to a DART. This can be generated by assembling three chains (two via disulfide bonds, as in DART). One chain contains half of the Fc region, which dimerizes with the third chain, expressing only the Fc region. The addition of the Fc region extends half-life, provides a longer effective concentration, and avoids continuous IV administration.

[0143] In tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are created by half of the Fc region and dimerize. This format has bivalent binding to both targets, and therefore it is a tetravalent molecule.

[0144] Tandem diabodies (TandAbs) contain two diabodies, each consisting of a covalently associated VHA and VLB fragment and a VHA and VLB fragment. The two diabodies are linked by a peptide chain. This provides improved stability compared to diabodies consisting of two scFvs. They have two bivalent binding sites.

[0145] scFv-scFv-toxin comprises a toxin and two scFvs with stabilizing linkers, which can be used for specific delivery of payloads.

[0146] In some embodiments, the anti-EGFR / MUC1 antibody is a bispecific antibody. In some embodiments, the bispecific antibody of the present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce overall activity against cells with low levels of EGFR and MUC1 expression and increase binding activity against cells co-expressing EGFR and MUC1, thereby achieving improved targeting function.

[0147] In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof has a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54 and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 55 and 56.

[0148] In some embodiments, the anti-EGFR / MUC1 antibody comprises a KIH mutation. In some embodiments, the anti-EGFR / MUC1 antibody comprises a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MUC1. In some embodiments, the first antigen-binding domain comprises a heavy chain comprising one or more knob mutations (knob heavy chain), and the second antigen-binding domain comprises a heavy chain comprising one or more hole mutations (hole heavy chain). In some embodiments, the first antigen-binding domain comprises a heavy chain comprising one or more hole mutations (hole heavy chain), and the second antigen-binding domain comprises a heavy chain comprising one or more knob mutations (knob heavy chain). In some embodiments, the anti-EGFR / MUC1 antibody comprises a knob heavy chain comprising a constant region at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55. In some embodiments, the anti-EGFR / MUC1 antibody comprises a hole heavy chain comprising a constant region at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56.

[0149] Antibody characteristics The anti-EGFR / MUC1 antibody can comprise any anti-EGFR antigen binding domain and any anti-MUC1 antigen binding domain described herein.

[0150] The present disclosure provides anti-EGFR / MUC1 antibodies and antigen-binding fragments thereof capable of specifically binding to EGFR. These anti-EGFR / MUC1 antibodies can be agonists or antagonists. The anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein can bind to EGFR and block the binding between EGFR and its ligand. By blocking the binding between EGFR and its ligand, the anti-EGFR / MUC1 antibodies inhibit EGFR-associated signaling pathways and thus can treat cancer. In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof can initiate CMC or ADCC.

[0151] Common techniques that can be used to measure the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be estimated from the quotient of kinetic rate constants (KD=koff / k). In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof has a kinetic affinity of 0.1 s -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001ss -1 In some embodiments, the koff is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0152] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0153] In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is administered at a concentration of 1×10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 More than M or 1 x 10 -10 It's over M.

[0154] The anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof may further comprise an antigen-binding domain capable of specifically binding to MUC1. The anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein can block the binding between MUC1 and its ligand. In some embodiments, the anti-EGFR / MUC1 antibodies can also inhibit MUC1-associated signaling pathways by binding to MUC1, thereby inhibiting cell proliferation, differentiation, and / or metastasis. Thus, in some embodiments, the anti-EGFR / MUC1 antibodies described herein are MUC1 agonists. In some embodiments, the anti-EGFR / MUC1 antibodies are MUC1 antagonists.

[0155] In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is administered in a 0.1 s -1 Less than 0.01s -1Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 can bind to MUC1 (e.g., human MUC1, monkey MUC1, mouse MUC1, and / or chimeric MUC1) with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0156] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0157] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 More than M or 1 x 10 -10 It's over M.

[0158] Because anti-EGFR / MUC1 antibodies (e.g., bispecific antibodies) bind to both MUC1 and EGFR, for cells that express both MUC1 and EGFR, the antibodies have higher binding affinity to these cells. Avidity can be used to measure the binding affinity of the antibody to these cells. Avidity is the cumulative strength of the affinities of multiple individual non-covalent interactions.

[0159] Thermal stability can also be measured. The anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein can have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. Because IgG can be described as a multidomain protein, the melting curve sometimes shows two transitions, with the first denaturation temperature being Tm D1 and the second denaturation temperature being Tm D2. The presence of these two peaks often indicates the denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When two peaks are present, the Tm usually refers to Tm D2. Thus, in some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein have a Tm D1 of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein have a Tm D2 of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the Tm, Tm D1, Tm D2 is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0160] In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is capable of binding to human EGFR or monkey EGFR. In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is incapable of binding to human EGFR or monkey EGFR. In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is capable of binding to human MUC1 or monkey MUC1. In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is incapable of binding to human MUC1 or monkey MUC1.

[0161] In some embodiments, the purity of the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC is greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC. In some embodiments, the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC.

[0162] In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a purity of 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, or 98% or greater as determined by size exclusion chromatography (SEC). In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a hydrophobic interaction chromatography (HIC) retention time of 2 minutes or greater, 3 minutes or greater, 4 minutes or greater, or 5 minutes or greater. In some embodiments, the HIC retention time is less than 2 minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes, or less than 6 minutes.

[0163] In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a purity of 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater as determined by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).

[0164] In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a main peak that constitutes 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the total sample when measured by capillary isoelectric focusing (cIEF). In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has an acidic peak that constitutes less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, or less than 75% of the total sample when measured by capillary isoelectric focusing (cIEF).

[0165] In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. TGI (%) can be measured, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after initiation of treatment. As used herein, tumor growth inhibition rate or percentage (TGI %) is calculated using the following formula:

[0166] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100 Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0167] In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0168] In some embodiments, the anti-EGFR / MUC1 antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof described herein has an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).

[0169] Several other modifications to the Fc region can be made. For example, cysteine ​​residue(s) can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric fusion protein thus produced may optionally have increased in vitro and / or in vivo half-lives.

[0170] In some embodiments, the IgG4 has an S228P mutation (EU numbering), which prevents IgG4 Fab arm exchange in vivo and in vitro.

[0171] In some embodiments, an Fc Region is provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc Region. For example, the amount of fucose in such an Fc Region composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan located at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 within the Fc region (position 314 in the EU numbering of Fc region residues or Kabat numbering). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in the Fc region sequence. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region is further engineered to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0172] In some embodiments, the main HPLC-SEC peak represents at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the protein complex described herein after purification by Protein A-based affinity chromatography and / or size exclusion chromatography.

[0173] In some embodiments, the anti-EGFR / MUC1 ADCs described herein have an IC50 for in vitro killing of cancer cells of less than 5 μg / mL, less than 4.5 μg / mL, less than 4 μg / mL, less than 3.5 μg / mL, less than 3 μg / mL, less than 2.5 μg / mL, less than 2 μg / mL, less than 1.5 μg / mL, less than 1 μg / mL, less than 0.9 μg / mL, less than 0.8 μg / mL, less than 0.7 μg / mL, less than 0.6 μg / mL, less than 0.5 μg / mL, less than 0.4 μg / mL, less than 0.3 μg / mL, less than 0.2 μg / mL, less than 0.1 μg / mL, less than 0.05 μg / mL, less than 0.025 μg / mL, less than 0.0125 μg / mL, less than 0.005 μg / mL, or less than 0.0025 μg / mL.

[0174] In some embodiments, the bispecific anti-EGFR / MUC1 antibodies described herein have a higher endocytosis rate than the corresponding monoclonal antibodies and / or control antibodies described herein. In some embodiments, the anti-EGFR / MUC1 antibodies described herein have a higher endocytosis rate than cetuximab analogs and / or gatipotuzumab analogs. In some embodiments, the endocytosis rate can be measured by the percentage of positive population. In some embodiments, the bispecific anti-EGFR / MUC1 antibodies described herein have a percentage of positive population of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%.

[0175] In some embodiments, the bispecific anti-EGFR / MUC1 antibodies described herein have higher binding activity to cells (e.g., HCC827 cells, HCC70 cells, A431 cells, or ZR-75-1 cells) than the corresponding monoclonal antibodies and / or control bispecific antibodies described herein. In some embodiments, the binding activity to cells can be determined by EC50. In some embodiments, the bispecific anti-EGFR / MUC1 antibodies described herein have an EC50 of less than 30 μg / mL, less than 20 μg / mL, less than 10 μg / mL, less than 5 μg / mL, less than 4 μg / mL, less than 3 μg / mL, less than 2 μg / mL, less than 1 μg / mL, or less than 0.5 μg / mL.

[0176] Antibody Drug Conjugates (ADCs) The anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent (drug). The therapeutic agent can be covalently or non-covalently bound to the anti-EGFR / MUC1 antibody. In some embodiments, the anti-EGFR / MUC1 antibody is an anti-EGFR / MUC1 bispecific antibody. In some embodiments, the bispecific antibody shares a common light chain.

[0177] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs). Useful classes of cytotoxic, cytostatic, or immunoregulatory agents include, for example, antitubulin agents, DNA minor groove binding agents, DNA replication inhibitors, and alkylating agents.

[0178] In some embodiments, therapeutic agents may include, but are not limited to, cytotoxic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, etc.), antiviral agents, or antibacterial agents. In some embodiments, conjugable therapeutic agents may be selected from, but are not limited to, MMAE (monomethylauristatin E), MMAD (monomethylauristatin D), or MMAF (monomethylauristatin F).

[0179] Definitions of specific functional groups and chemical terms are described in detail below. For purposes of this invention, chemical elements are identified in accordance with the Periodic Table of the Elements, CAS Edition, Handbook of Chemistry and Physics, 75th Edition, inside cover, and specific functional groups are generally defined as described herein. Further, general principles of organic chemistry and specific functional groups and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley&Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0180] All ranges cited herein are inclusive unless expressly stated to the contrary. When a range of values ​​is listed, it is intended to encompass each value and subrange within that range. For example, "C 1-6 ” is C1, C2, C3, C4, C5, C6, C 1-6 , C1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 is intended to encompass:

[0181] A compound or any formula depicting and describing a compound of the present disclosure may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomers of a compound or any formula depicting and describing a compound of the present invention. Any asymmetric centers present in a compound or any formula depicting and describing a compound of the present invention can have either the (R) or (S) configuration, independently of one another. When a bond to a chiral carbon in a structural formula is depicted as a straight line, or when a compound name is written without an (R) or (S) chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of each chiral carbon, and therefore each enantiomer or diastereomer and mixtures thereof, are encompassed within the formula or name.

[0182] The present disclosure includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, e.g., mixtures of enantiomers and / or diastereomers, in any ratio. Accordingly, enantiomers are the subject of the present disclosure in enantiomerically pure form, both as levorotatory and dextrorotatory antipodes, in the form of racemates, and in the form of mixtures of the two enantiomers in any ratio. In the case of cis / trans isomerism, the present disclosure includes both cis and trans forms, as well as mixtures of these forms in any ratio. Preparation of individual stereoisomers can be carried out, if necessary, by separation of mixtures by conventional methods such as chromatography or crystallization, by using stereochemically uniform starting materials for synthesis, or by stereoselective synthesis. Optionally, derivatization can be carried out before separation of stereoisomers. Separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of the compound or on the final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereocenter of known configuration. Alternatively, absolute stereochemistry may be determined by vibrational circular dichroism (VCD) spectroscopy.

[0183] Unless otherwise stated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced with an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. These compounds are referred to as "isotopic variants." The present disclosure is intended to encompass all pharmaceutically acceptable isotopic variants of the compounds or any formula depicting and describing the compounds of the present invention. Examples of isotopes suitable for inclusion in the compounds of the present invention include: 2 H (i.e., D) and 3 Isotopes of hydrogen such as H, 11 C. 13 C, and 14 carbon isotopes such as C, 36 chlorine isotopes such as Cl, 18 fluorine isotopes such as F, 123I, and 125 isotopes of iodine, such as I, 13 N and 15 nitrogen isotopes such as N, 15 O. 17 O, and 18 isotopes of oxygen, such as O 32 Isotopes of phosphorus such as P, as well as 35 Specific isotopic variations of the compounds, or any formula depicting and describing compounds of the present disclosure, for example, incorporating a radioactive isotope, may be useful in drug and / or substrate tissue distribution studies. In particular, isotopes of hydrogen may be substituted with deuterium ( 2 Compounds having the depicted structures that differ only by substitution with heavier isotopes, such as substitution with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 120, 122, 130, 140, 141, 150, 162, 170, 182, 190, 191, 203, 210, 220, 230, 240,

[0184] The compounds provided herein are described with reference to both general formulas and specific compounds. Furthermore, the compounds of the present disclosure may exist in many different forms or derivatives, all within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0185] As used herein, the term "pharmaceutically acceptable salts" includes, unless otherwise specified, salts that retain the biological effectiveness of the free acid / base form of the particular compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include salts formed with inorganic and organic bases or acids. When compounds of the present disclosure contain one or more acidic or basic groups, the present disclosure also includes their corresponding pharmaceutically acceptable salts. Thus, compounds of the present invention that contain an acidic group, such as a carboxyl group, can exist in the form of a salt and be used in accordance with the present invention, for example, as an alkali metal salt, alkaline earth metal salt, aluminum salt, or ammonium salt. More non-limiting examples of these salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts are readily obtainable, for example, by reacting a compound having an acidic group with a suitable base, such as lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of the compounds of the present disclosure include, but are not limited to, copper(I), copper(II), iron(II), iron(III), manganese(II), and zinc salts. Compounds of the present disclosure that contain one or more basic groups, such as protonatable groups, can exist in the form of salts and can be used according to the present invention in the form of addition salts with inorganic or organic acids.Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids known to those skilled in the art. Salts formed include, among others, hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), tosylate, carbonate, bicarbonate, formate, acetate, sulfoacetate, triflate, oxalate, malonate, maleate, succinate, tartrate, malate, embonate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate, and glutamate. The stoichiometry of salts formed from compounds of the present disclosure may also be an integer or non-integer multiple of 1.

[0186] Compounds of the present disclosure that contain basic nitrogen-containing groups include C groups such as methyl, ethyl, isopropyl, and tert-butyl chlorides, bromides, and iodides. 1-4 Alkyl halides, dimethyl, diethyl, and diamyl sulfates, etc. 1-4 C alkyl sulfates, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides 10-18 Alkyl halides and aryl C such as benzyl chloride and phenethyl chloride 1-4 They can be quaternized using reagents such as alkyl halides.

[0187] When the compounds of the present disclosure contain both acidic and basic groups in the molecule, the present disclosure also encompasses internal salts or betaines (zwitterions) in addition to the aforementioned salt forms. The respective salts can be obtained by conventional methods known to those skilled in the art, such as by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion or cation exchange with other salts. The present disclosure also encompasses all salts of the compounds of the present disclosure that are not suitable for direct pharmaceutical use due to poor physiological compatibility, but can be used, for example, as intermediates in chemical reactions or to prepare pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0188] Any formula that describes and describes a compound or a compound of the present disclosure and its pharmaceutically acceptable salts can exist in unsolvated form and solvated form.As used herein, the term "solvate" refers to a molecular complex comprising a compound of formula (I) or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable solvent molecules.For example, when the solvent is water, the term "hydrate" is used.

[0189] Pharmaceutically acceptable solvates in accordance with the present disclosure include those wherein the solvent of crystallization may be isotopically substituted, eg, D2O, d6-acetone, d6-DMSO.

[0190] Linker (Binder Compound) In some embodiments, the therapeutic agent is conjugated via a linker (or binder compound). As used herein, the term "linker" or "linker compound" refers to a compound that can link a ligand (e.g., an antibody or antigen-binding fragment thereof described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) by reacting with groups on the ligand compound and therapeutic compound, respectively, e.g., by a coupling reaction, to form a ligand drug conjugate.

[0191] In some embodiments, a linker described herein is a compound having the formula:

[0192] [ka] or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein Q represents a junction that can be coupled to a ligand via a bond selected from the group consisting of a carbonyl, thioether, amide, disulfide, and hydrazone bond, and L represents a linker moiety that can attach Q to a therapeutic agent.

[0193] In some embodiments, the conjugation moiety (Q in formula (I)) has the structure:

[0194] [ka]

[0195] In some embodiments, the linker moiety (L in formula (I)) has the formula:

[0196] [ka] wherein L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, and contains at least one amino acid residue having a side chain carboxyl group, such as a glutamic acid residue or an aspartic acid residue, and wherein "-COOH" represents the carboxyl group of the C-terminal amino acid residue of the polypeptide residue; L2 is absent or is a monodentate, bidentate, or tridentate hydrophilic group attached to a side chain carboxyl group on an amino acid residue of the polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c ) structure, of which R L2a , R L2b , and R L2care each independently H, -(CHO)(CHCHO) m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d and R L2d is H or C optionally substituted with 1 to 6 hydroxy groups 1-6 alkyl, each m is independently an integer from 0 to 10, preferably 0 to 4, e.g., 0, 1, 2, 3, or 4, and particularly preferably m is 0, and each p is independently an integer from 1 to 4, e.g., 1, 2, 3, or 4; and [ka] indicates the N-terminal side of the polypeptide residue covalently attached to interface Q.

[0197] In some embodiments, the polypeptide residue L1 is NH -Glu-Val-Ala- COOH In some embodiments, the hydrophilic group L2 has the following structure:

[0198] [ka] Among them, "*" indicates a site covalently attached to the polypeptide residue L1, e.g. NH -Glu-Val-Ala- COOH The side chain of the Glu residue in

[0199] In some embodiments, a linker described herein is a compound having the following structure:

[0200] [ka] In some embodiments, the linker is a VC linker. Details of linkers used in ADCs are described, for example, in Su, Z. et al. "Antibody-drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), the entire contents of which are incorporated by reference.

[0201] therapeutic agent In some embodiments, therapeutic agents conjugated to the antibodies or antigen-binding fragments thereof described herein are considered as follows.

[0202] In some embodiments, the therapeutic agents described herein are cytotoxic agents. In some embodiments, the cytotoxic agent is a camptothecin compound, an analog, or a derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the following structure:

[0203] [ka] wherein X is selected from the group consisting of -CH2-, O, and S, and Y is selected from the group consisting of H, D, and F.

[0204] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione) (CPT-1). The structure of CPT-1 is shown below: [ka]

[0205] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(2H)-dione (CPT-2). The structure of CPT-2 is shown below: [ka]

[0206] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below: [ka]

[0207] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(2H)-dione (CPT-4). The structure of CPT-4 is shown below: [ka]

[0208] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structures of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957; International Patent Publication No. WO 02 / 088172, as well as U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5, Nos. 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein in its entirety for all purposes.

[0209] Auristatins have been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and have been shown to have anti-cancer activity. Auristatins can bind to tubulin and exert cytotoxic or cytostatic effects in cancer cells. Many different assays known in the art exist that can be used to determine whether an auristatin or the resulting antibody-drug conjugate exerts a cytostatic or cytotoxic effect in the desired cells.

[0210] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; chlorambucil, chlornaphazine, colofosfamide, estradiol, and estradiol. Nitrogen mustards such as mustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, and clomustine; Antibiotics such as momycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate and 5-fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridinene, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone;Antiadrenal agents such as aminoglutethimide, mitotein, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfomitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenazone Met; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) Squibb Oncology, Princeton, NJ), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition includes, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Also included are antihormonal agents that regulate or inhibit hormone action in tumors, such as antiestrogens, including pharmaceutically acceptable salts, acids, or derivatives of any of the above. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated by reference in its entirety.

[0211] Linker Therapeutic Compound In some embodiments, a linker (e.g., any of the linkers described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) can be linked to form a "linker-therapeutic agent" compound.

[0212] In some embodiments, the linker therapeutic compound has the structure:

[0213] [ka]

[0214] In some embodiments, the linker therapeutic compound has the structure:

[0215] [ka]

[0216] In some embodiments, an antibody ("Ab"), such as any of the antibodies or antigen-binding fragments thereof described herein, can be linked to a linker-therapeutic agent compound (e.g., any of the linker-therapeutic agent compounds described herein) to generate an antibody drug conjugate. In some embodiments, the antibody drug conjugate has the following structure:

[0217] [ka] wherein n=1 to 8. In some embodiments, n=1 to 8. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In some embodiments, n is about 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8. In some embodiments, n is an integer or non-integer multiple of 1.

[0218] In some embodiments, the anti-EGFR / MUC1 antibody is coupled to the drug via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0219] In some embodiments, the anti-EGFR / MUC1 antibody is coupled to the drug via a non-cleavable linker, such as an MCC linker formed using, for example, SMCC or sulfo-SMCC. The selection of an appropriate linker for a given ADC can be readily made by one of skill in the art, taking into account relevant factors such as the binding site on the anti-EGFR / MUC1 antibody, any structural restrictions of the drug, and the hydrophobicity of the drug (see, e.g., a review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). Numerous specific linker-toxin combinations have been described and can be used with the anti-EGFR / MUC1 antibodies or antigen-binding fragments thereof described herein to prepare ADCs in certain embodiments. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins, and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicin and doxorubicin; disulfide-based linkers with maytansinoids such as DM1 and DM4; and bismaleimide trioxyethylene glycol (BMPEO)-based linkers with the maytansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Publication No. U2015 / 0374847, and US20180193477A1, which are incorporated by reference herein in their entireties.

[0220] Depending on the desired drug and the selected linker, those skilled in the art can select a suitable method for coupling them to each other. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired drug-linker conjugate that still contains a reactive group for covalent conjugation to an anti-EGFR / MUC1 antibody or its antigen-binding fragment. In some embodiments, a drug-maleimide conjugate (i.e., a maleimide-linked drug) can be used for the payloads having a reactive group in the present disclosure. The most common reactive group that can be attached to a thiol group in ADC preparation is maleimide. In addition, organic bromides and iodides are also frequently used.

[0221] Anti-EGFR / MUC1 ADCs can be prepared by one of several art-known routes using organic chemistry reactions, conditions, and reagents well known to those skilled in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) reacting a nucleophilic or electrophilic group on an antibody with a bivalent linker reagent to form an antibody-linker intermediate Ab-L, followed by covalent reaction with an activated drug moiety D; or (2) covalently reacting a nucleophilic or electrophilic group on a drug moiety with a linker reagent, followed by covalent reaction with a nucleophilic or electrophilic group on the antibody to form a drug-linker intermediate DL. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the anti-EGFR / MUC1 ADCs described herein. The various linkers, linker components, and toxins prepared are either commercially available or can be prepared using standard synthetic organic chemistry techniques, as described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (GTHermanson, 2013, Academic Press); US20210379193A1, and US20180193477A1, which are incorporated by reference in their entireties. Additionally, preformed drug-linkers suitable for reaction with a selected anti-EGFR / MUC1 antibody or antigen-binding fragment are also commercially available; for example, linker-toxins including DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative BioLabs (Shirley, NY).

[0222] Some specific examples of methods for preparing anti-EGFR / MUC1 ADCs are known in the art and are described in U.S. Patent No. 8,624,003 (the Pott method), U.S. Patent No. 8,163,888 (one-step), and U.S. Patent No. 5,208,020 (a two-step method), as well as U.S. Patent No. 20180193477A1, which are incorporated by reference herein in their entireties. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0223] Drug loading is expressed by the number of drug moieties per antibody in an ADC molecule. For some antibody-drug conjugates, drug loading can be limited by the number of binding sites on the antibody. For example, when the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described herein, drug loading can range from 0 to 8 drug moieties per antibody. In certain embodiments, high drug loading, e.g., p≧5, can cause aggregation, insolubility, toxicity, or cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for anti-EGFR / MUC1 antibody-drug conjugates ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In fact, it has been shown that the optimal ratio of drug moieties per antibody for certain antibody-drug conjugates can be about 4. In some embodiments, the drug-antibody ratio (DAR) of an anti-EGFR / MUC1 ADC composition is about 1, 2, 3, 4, 5, 6, 7, or 8 or more. In some embodiments, the average DAR in an anti-EGFR / MUC1 ADC composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0224] In some embodiments, anti-EGFR / MUC1 antibody variants have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan located at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 within the Fc region (position 314 in the EU numbering of Fc region residues or Kabat numbering). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of an anti-EGFR / MUC1 antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0225] In some embodiments, to increase production efficiency by avoiding Fab arm exchange, the Fc region of the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof is further modified to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation," Journal of Biological Chemistry 290.9 (2015):5462-5469, which is incorporated by reference in its entirety.

[0226] In some embodiments, the methods described herein are designed to generate bispecific anti-EGFR / MUC1 antibodies. Bispecific anti-EGFR / MUC1 antibodies can be generated by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers. This method is described, for example, in WO 96 / 27011, incorporated by reference in its entirety.

[0227] In some embodiments, knobs-into-holes (KIH) technology can be used, which involves recombining the CH3 domains to create either "knobs" or "holes" in each heavy chain to promote heterodimerization. KIH technology is described, for example, in Xu, Yiren, et al., "Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system." MAbs. Vol. 7. No. 1. Taylor & Francis, 2015, incorporated by reference in its entirety. In some embodiments, one heavy chain has T366W and / or S354C (knob) substitutions (EU numbering), and the other heavy chain has Y349C, T366S, L368A, and / or Y407V (hole) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the substitutions Y349C and T366W (EU numbering). The other heavy chain may have one or more of the substitutions E356C, T366S, L368A, and Y407V (EU numbering). Furthermore, substitutions (-ppcpScp-->-ppcpPcp-) may be introduced in the hinge region of both substituted IgGs.

[0228] Recombinant vector The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors containing the polynucleotides), and the production of anti-EGFR / MUC1 antibody polypeptides or fragments thereof by recombinant techniques.

[0229] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as a promoter, enhancer, and / or polyA tail, at, near, or adjacent to the integration site of the polynucleotide of interest, either within the vector or in the genome of the host cell, such that the polynucleotide of interest is translated in a host cell into which the expression vector is introduced.

[0230] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0231] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may employ a replication-defective virus, in which case viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321;Flexner et al.,1989,Ann.NYAcad Sci.569:86-103;Flexner et al. al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; 91 / 02805;Berkner-Biotechniques,6:616-627,1988;Rosenfeld et al. Suitable systems are disclosed in Kolls et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be enhanced by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0232] For expression, a DNA insert containing a polynucleotide encoding a polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral long terminal repeats, to name a few. Other suitable promoters are known to those of skill in the art. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) positioned approximately at the end of the polypeptide to be translated.

[0233] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0234] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0235] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0236] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0237] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0238] Transcription of DNA encoding the anti-EGFR / MUC1 antibodies of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that serve to increase transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0239] For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0240] Polypeptides (e.g., anti-EGFR / MUC1 antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in host cells during purification or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to effect secretion or excretion, improve stability, and facilitate purification, among other things, are well-known and routine techniques in the art.

[0241] The present disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein, and Also provided are amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the amino acid sequences described herein.

[0242] The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the nucleotide sequences described herein; Also provided are amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the amino acid sequences described herein.

[0243] In some embodiments, the disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0244] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0245] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0246] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are considered identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. For example, sequence comparison and percent identity determination between two sequences can be performed using the Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0247] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be measured. Methods for measuring percentage of sequence homology are well known in the art. In some embodiments, conserved amino acid residues with similar physicochemical properties (% homology), e.g., leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology will be higher than the percent identity.

[0248] The present disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a heavy chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a light chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding an scFv polypeptide described herein.

[0249] In some embodiments, a vector can have two of the nucleic acids described herein, where the vectors encode a VL region and a VH region that both bind to EGFR. In some embodiments, a pair of vectors is provided, where each vector comprises one of the nucleic acids described herein, and the pair of vectors together encode a VL region and a VH region that both bind to EGFR.

[0250] In some embodiments, a vector comprises two of the nucleic acids described herein, wherein the vectors encode a VL region and a VH region that both bind to MUC 1. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, and the pair of vectors together encode a VL region and a VH region that both bind to MUC 1.

[0251] Treatment method The methods described herein include methods for treating disorders related to cancer. Generally, the methods involve administering a therapeutically effective amount of an anti-EGFR / MUC1 antibody or anti-EGFR / MUC1 antibody-drug conjugate described herein to a subject in need of, or determined to be in need of, such treatment.

[0252] As used in this context, "treatment" means alleviating at least one symptom of a disorder associated with cancer. Cancer often results in death. Thus, treatment can result in an increase in life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). Administering a therapeutically effective amount of an agent described herein for treating a condition associated with cancer results in a reduction in the number of cancer cells and / or alleviation of symptoms.

[0253] As used herein, the term "cancer" refers to an abnormal state or condition characterized by cells capable of autonomous proliferation, i.e., rapidly proliferating cell proliferation. This term is intended to include any type of cancerous growth or oncogenic process, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. As used herein, the term "tumor" refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including malignancies such as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular cancer, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose carcinoma in a subject. The term "carcinoma" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory system cancer, digestive system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal derivation. In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0254] In one aspect, the present disclosure also provides methods of treating cancer in a subject, methods of reducing the rate of growth of tumor volume in a subject over time, methods of reducing the risk of developing metastases, or methods of reducing the risk of developing further metastases in a subject. In some embodiments, treatment can halt, slow, prevent, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0255] In one aspect, the disclosure features a method that includes administering a therapeutically effective amount of an anti-EGFR / MUC1 antibody or anti-EGFR / MUC1 antibody drug conjugate disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, cancer, such as a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung carcinoma), gastric cancer (e.g., gastric carcinoma), skin cancer (e.g., skin carcinoma), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, brain cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, cervical cancer, or esophageal cancer. In some embodiments, the cancer is lung cancer, head and neck cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, glioma, esophageal cancer, gastric cancer, stomach cancer, prostate cancer, cervical cancer, multiple myeloma, or non-Hodgkin's lymphoma.

[0256] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to whom treatment according to the methods of the invention is provided. Veterinary and non-veterinary uses are contemplated by the present invention. A human patient can be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0257] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified by a variety of methods known in the art.

[0258] As used herein, "effective amount" means an amount or dosage sufficient to bring about beneficial or desired results, including halting, slowing, preventing, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding fragment, anti-EGFR / MUC1 antibody-drug conjugate, polynucleotide encoding the anti-EGFR / MUC1 antibody, vector comprising the polynucleotide, and / or composition thereof is administered, the severity of symptoms, and the route of administration, and therefore administration can be determined individually.

[0259] An effective amount can be administered in one or more administrations. For example, an effective amount of an anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding fragment, or anti-EGFR / MUC1 antibody-drug conjugate is an amount sufficient to palliate, arrest, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or to palliate, arrest, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective amount of an anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding fragment, or anti-EGFR / MUC1 antibody-drug conjugate can vary depending on other factors, such as the patient's medical history and the type (and / or dosage) of drug used, among other factors.

[0260] Effective amounts and schedules for administering the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antigen-binding fragments thereof, polynucleotides encoding the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antibody-drug conjugates, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending, for example, on the mammal receiving the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antigen-binding fragments thereof, polynucleotides encoding the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antibody-drug conjugates, and / or compositions disclosed herein, the route of administration, the particular type of agent or composition disclosed herein used, and other agents administered to the mammal.

[0261] A typical daily dose of an effective amount of an anti-EGFR / MUC1 antibody or anti-EGFR / MUC1 ADC is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0262] In any of the methods described herein, at least one anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding fragment thereof, anti-EGFR / MUC1 antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of an anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, or anti-EGFR / MUC1 ADC), and optionally at least one additional therapeutic agent, can be administered to a subject (e.g., once weekly, twice weekly, three times weekly, four times weekly, once daily, twice daily, or three times daily).

[0263] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, anti-EGFR / MUC1 antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of an anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, or anti-EGFR / MUC1 ADC). In some embodiments, the one or more additional therapeutic agents and at least one anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, or anti-EGFR / MUC1 antibody-drug conjugate are administered to a subject such that the periods of biological activity of the one or more additional therapeutic agents and the at least one anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding fragment, or anti-EGFR / MUC1 ADC in the subject overlap.

[0264] In some embodiments, a subject can be administered at least one anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, anti-EGFR / MUC1 antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of an anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, or anti-EGFR / MUC1 ADC) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to monitor at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of anti-EGFR / MUC1 antibodies or anti-EGFR / MUC1 antigen-binding antibody fragments, anti-EGFR / MUC1 antibody drug conjugates (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dose or frequency of administration of at least one anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding antibody fragment, or anti-EGFR / MUC1 ADC (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0265] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, MUC1 inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0266] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of inhibitors of EGFR, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0267] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 The present invention may include one or more therapeutic agents selected from the group consisting of 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0268] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0269] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0270] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0271] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the anti-EGFR / MUC1 antibodies (e.g., bispecific antibodies), anti-EGFR / MUC1 antigen-binding fragments, or anti-EGFR / MUC1 antibody-drug conjugates described herein. Pharmaceutical compositions can be formulated in any manner known in the art.

[0272] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol, methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The composition preparation can be formulated and enclosed in ampoules, disposable syringes, or multiple-dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of the anti-EGFR / MUC1 antibody, its anti-EGFR / MUC1 antigen-binding fragment, or anti-EGFR / MUC1 ADC can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0273] Compositions containing any one or more of the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antigen-binding fragments, and anti-EGFR / MUC1 antibody-drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound(s) for ease of administration and uniformity of dosage).

[0274] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0275] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of an anti-EGFR / MUC1 antibody, anti-EGFR / MUC1 antigen-binding fragment thereof, or anti-EGFR / MUC1 ADC is an amount that treats the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as at risk for developing the disease (e.g., a subject who previously developed cancer but has now been cured), (e.g., kills cancer cells), or reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antigen-binding fragments thereof, or anti-EGFR / MUC1 ADCs described herein can be determined by a medical or veterinary professional using methods well known in the art and by observation of one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and the presence of other diseases).

[0276] Exemplary doses include milligram or microgram amounts of any of the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antigen-binding fragments thereof, or anti-EGFR / MUC1 ADCs described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg). While these doses cover a wide range, those of skill in the art will understand that therapeutic agents can be administered at their efficacy and effective amounts by methods well known in the art. Typically, a relatively low dose is administered initially, and the dose can be subsequently and gradually increased by the attending health care professional or veterinary professional (for therapeutic uses) or by a researcher (if still working in the development phase) until an appropriate response is obtained. It is further understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life of the therapeutic agent in the body.

[0277] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The present invention also provides methods for producing the anti-EGFR / MUC1 antibodies, anti-EGFR / MUC1 antigen-binding fragments thereof, or anti-EGFR / MUC1 ADCs for the various uses described herein.

[0278] Example The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0279] Example 1. Preparation and analysis of anti-EGFR / MUC1 bispecific antibodies Preparation of anti-EGFR / MUC1 bispecific antibodies Anti-EGFR antibodies (E-6C4, E-9A6, E-9D2, E-9F3, and E-9H2) and anti-MUC1 antibodies (10M1 and M-11E8) were paired to form various anti-EGFR / MUC1 bispecific antibodies. Specifically, vectors encoding the light and heavy chains of the antibodies were constructed. CHO-S cells were cotransfected with three vectors: the first vector encoding the heavy chain of the anti-EGFR antibody, the second vector encoding the heavy chain of the anti-MUC1 antibody, and the third vector encoding the common light chain. After 14 days of culture, the cell supernatant was collected and purified by protein A affinity chromatography.

[0280] Various methods can be used to reduce the possibility of incorrect pairing between the two heavy chains. For example, knob-into-hole mutations were introduced into the Fc regions of the anti-EGFR arm heavy chain and the anti-MUC1 arm heavy chain. The resulting exemplary bispecific antibodies included E-6C4-10M1, E-9A6-10M1, E-9D2-10M1, E-9F3-10M1, E-9H2-10M1, E-6C4-M-11E8, E-9A6-M-11E8, E-9D2-M-11E8, E-9F3-M-11E8, and E-9H2-M-11E8. To verify the binding affinity of the bispecific antibodies, anti-EGFR or anti-MUC1 control bispecific antibodies were also generated, in which one arm of the control bispecific antibody recognized EGFR or MUC1 and the other arm recognized CD28. To generate these control bispecific antibodies, use a similar method (e.g., RenLite TM (VH sequences were obtained by immunizing mice with the antibody. Exemplary control bispecific antibodies are designated E-6C4-CD28, E-9A6-CD28, E-9D2-CD28, E-9F3-CD28, E-9H2-CD28, CD28-E-6C4, CD28-E-9A6, CD28-E-9D2, CD28-E-9F3, CD28-E-9H2, 10M1-CD28, M-11E8-CD28, CD28-10M1, and CD28-10M1.

[0281] Knob-into-hole mutations were introduced into all bispecific antibodies. For example, E-6C4-10M1 contains knob mutations in the heavy chain constant region of E-6C4 and hole mutations in the heavy chain constant region of 10M1. E-6C4-CD28 contains knob mutations in the heavy chain constant region of E-6C4 and hole mutations in the heavy chain constant region of CD28. Exemplary antibody structures are shown in Figure 1, in which target 1 and target 2 can be (1) EGFR and MUC1, respectively; (2) MUC1 and EGFR, respectively; (3) EGFR and CD28, respectively; (4) CD28 and EGFR, respectively; (5) MUC1 and CD28, respectively; or (6) CD28 and MUC1, respectively.

[0282] The sequences of the light chain constant region, the heavy chain constant region with the knob mutation, and the heavy chain constant region with the hole mutation are shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively.

[0283] The VH and VL CDR sequences of E-6C4, E-9A6, E-9D2, E-9F3, E-9H2, 10M1 and M-11E8 are shown in Figures 6, 7 and 8, and the sequences of their VH and VL regions are shown in Figure 9.

[0284] Cross-species binding of anti-EGFR / MUC1 bispecific antibodies CHO-S-hMUC1(484AA) cells, CHO-S-fasMUC1 cells, CHO-S-hEGFR cells, or CHO-S-fasEGFR cells were cultured at 1 × 10 5 The cells were transferred to a 96-well plate at a density of 1000 cells / well. Anti-EGFR / MUC1 bispecific antibody (5 μg / mL) was added to the 96-well plate and incubated at 4°C for 30 minutes. The cells were then incubated with the secondary antibody, anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Catalog No. 109-606-170), in the dark for 15 minutes at 4°C, followed by flow cytometry analysis. In the negative control (NC), only cells were added, but no antibody was added.

[0285] CHO-S-hEGFR cells or CHO-S-fasEGFR cells were obtained by transfecting CHO-S cells with vectors expressing human EGFR (hEGFR, SEQ ID NO: 57) or monkey EGFR (fasEGFR, SEQ ID NO: 58), respectively. CHO-S-hMUC1(484AA) cells were obtained by transfecting CHO cells with a vector expressing the 484 amino acid sequence of human MUC1 (hMUC1(484AA), SEQ ID NO: 67). CHO-S-fasMUC1 cells were obtained by transfecting CHO cells with a vector expressing monkey (Macaca) MUC1 (fasMUC1, SEQ ID NO: 60). For isotype control (ISO), an antibody targeting an irrelevant target protein was used.

[0286] The test results are shown in the table below. All of the anti-EGFR / MUC1 bispecific antibodies E-6C4-10M1, E-9A6-10M1, E-9D2-10M1, E-9F3-10M1, E-9H2-10M1, E-6C4-M-11E8, E-9A6-M-11E8, E-9D2-M-11E8, E-9F3-M-11E8, and E-9H2-M-11E8 are capable of binding to human MUC1, monkey MUC1, human EGFR, and monkey EGFR.

[0287] [Table 1-1] [Table 1-2]

[0288] Binding affinity of anti-EGFR / MUC1 bispecific antibody The binding affinity of the anti-EGFR / MUC1 bispecific antibodies to His-tagged human EGFR protein (hEGFR-His, ACROBiosystems Inc., Catalog No. EGR-H5222), His-tagged monkey EGFR protein (fasEGFR-His, ACROBiosystems Inc., Catalog No. EGR-C52H1), His-tagged human MUC1 protein (hMUC1(24-1158)-His, containing positions 24 to 1158 of human MUC1 (SEQ ID NO: 59)), His-tagged human MUC1 protein (hMUC1(961-1152)-His, containing positions 961 to 1152 of human MUC1 (SEQ ID NO: 59)), and His-tagged monkey MUC1 (fasMUC1-His, SEQ ID NO: 60) was measured using a Biacore™ system equipped with a pre-immobilized Protein A sensor chip. TM (Biacore, Inc., Piscataway NJ) Validation was performed by surface plasmon resonance (SPR) using an 8K biosensor.

[0289] Specifically, the his-tagged protein was diluted to 200 nM in 1x HBS-EP+ buffer (pH 7.4) and then analyzed by Biacore at 10 μL / min for approximately 50 seconds. TM The 8K biosensor was injected to achieve the desired protein density (e.g., approximately 100 response units (RU)). Next, 1 μg / mL of purified antibody in 1× HBS-EP+ buffer (pH 7.4) was injected at 10 μL / min for 50 seconds. Dissociation was monitored for 400 seconds. After the last injection of each titration, the chip was regenerated with glycine solution (pH 1.5) at 30 μL / min for 30 seconds.

[0290] Biacore TMThe kinetic association rate (k) and dissociation rate (k) were obtained simultaneously by fitting the entire data set to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0291] The same method was performed for each test antibody, with appropriate adjustments to parameters (e.g., antibody concentration), as would be understood by one of skill in the art. The results for the test antibodies are summarized in the table below.

[0292] [Table 2-1] [Table 2-2]

[0293] The results showed that the anti-EGFR / MUC1 bispecific antibodies E-6C4-10M1, E-9A6-10M1, E-9D2-10M1, E-9F3-10M1, E-9H2-10M1, E-6C4-M-11E8, E-9A6-M-11E8, E-9D2-M-11E8, E-9F3-M-11E8, and E-9H2-M-11E8 were all capable of binding to human EGFR, human MUC1, monkey EGFR, and monkey MUC1.

[0294] Internalization of antibodies targeting EGFR and / or MUC1 Anti-EGFR antibody, anti-MUC1 antibody, anti-EGFR / MUC1 bispecific antibody, anti-EGFR / CD28 bispecific antibody, or anti-MUC1 / CD28 bispecific antibody (2.5 μg / mL) was added together with pHAb-goat anti-human IgG secondary antibody to NUCG-4 cells (Cobioer, Reference No. CBP60493), Panc 02.03 cells (ATCC, Reference No. CRL-2553), or HCC70 cells (ATCC, Reference No. CRL-2315), respectively, and incubated for 6 hours. Cells were centrifuged and washed with FACS buffer. MFI was measured using a flow cytometer. The endocytosis rate of the antibody was calculated. An antibody targeting an irrelevant target protein was used as an isotype control (ISO). The results are shown in the table below.

[0295] [Table 3]

[0296] Cetuximab is an EGFR-targeting chimeric monoclonal IgG1 antibody originally developed by ImClone Systems and marketed in Switzerland in 2003 by Merck KGaA as a monotherapy and in combination with irinotecan for the treatment of irinotecan-refractory metastatic colorectal cancer under the name Erbitux. TM The heavy and light chain sequences of the cetuximab analog are shown in SEQ ID NO: 61 and SEQ ID NO: 62, respectively.

[0297] 1H7 is a mouse monoclonal IgG antibody targeting the extracellular region of the human MUC1 C-terminal subunit, developed by Chungbuk National University and Peptron Co. Ltd. The heavy and light chain variable regions of 1H7 are set forth in SEQ ID NO: 63 and SEQ ID NO: 64, respectively.

[0298] Gatipotuzumab is a glycoengineered humanized monoclonal antibody that recognizes a tumor-associated epitope of MUC1 and is currently in Phase II clinical development at Glycotope, Inc. as a treatment for recurrent epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. The heavy and light chain variable regions of gatipotuzumab are set forth in SEQ ID NOs: 65 and 66, respectively.

[0299] Data showed that in NUCG-4 cells, E-6C4-10M1, E-9A6-10M1, E-9D2-10M1, E-9F3-10M1, E-9H2-10M1, E-6C4-M-11E8, E-9A6-M-11E8, E-9D2-M-11E8, E-9F3-M-11E8, and E-9H2-M-11E8 had higher cellular endocytosis rates compared to the positive controls cetuximab analogs, 1H7 analogs, and gatipotuzumab analogs. E-6C4-10M1 and E-6C4-M-11E8 had higher cell endocytosis rates compared to the corresponding monoclonal antibodies E-6C4, 10M1, and M-11E8, and the corresponding monovalent antibodies CD28-E-6C4, 10M1-CD28, and M-11E8-CD28.

[0300] Verification of the binding activity of antibodies targeting EGFR and / or MUC1 The binding activity of anti-EGFR antibodies, anti-MUC1 antibodies, anti-EGFR / MUC1 bispecific antibodies, anti-EGFR / CD28 bispecific antibodies, or anti-MUC1 / CD28 bispecific antibodies to HCC827 cells (ATCC, Reference Number: CRL-2868), HCC70 cells, A431 cells (ATCC, Reference Number: CRL-1555), and ZR-75-1 cells (ATCC, Reference Number: CRL-1500) was verified by flow cytometry. The secondary antibody used in the experiment was Alexa Fluor® 647 anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc., Catalog Number: 109-606-170). EC50 values ​​were determined using serially diluted sample antibodies (maximum concentration: 20 μg / mL, 2-fold dilutions, 11 gradients). The results are shown in the table below.

[0301] [Table 4]

[0302] Example 2. Antibody Drug Conjugates (ADCs) After Protein A purification, antibodies E-6C4-10M1, E-9A6-10M1, E-9D2-10M1, E-9F3-10M1, E-6C4, E-9A6, E-9D2, E-9F3, and 10M1 were dialyzed and concentrated by ultrafiltration into PBS buffer. Concentrations were determined by UV absorption. These antibodies were used in subsequent antibody-drug coupling reactions.

[0303] Coupling of antibodies with drug molecules Each purified antibody was coupled to MMAE (monomethylauristatin E) or MMAF (monomethylauristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0304] For the name of the antibody drug conjugate, "ADC" is added immediately after the antibody name. For example, E-6C4-10M1 coupled to MMAE would be named E-6C4-10M1-ADC.

[0305] Coupling of the antibody with the drug molecule was detected using HIC-HPLC. The HIC-HPLC detection results showed that the drug-antibody ratio (DAR) of the ADC was approximately 4. For the isotype control, a human IgG1 isotype control was coupled to MMAE to form an isotype ADC (ISO-ADC).

[0306] In vitro killing activity Different concentrations of ADC (10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, 0.123 μg / mL, 0.041 μg / mL, 0.013 μg / mL, and 0.004 μg / mL) were used to culture human lung cancer cell line NCI-H1975 (5 × 10 cells / mL) in cell culture plates.3 ) or pancreatic cancer cell line Panc.02.03 (5 × 10 3 ) and incubated for 72 hours in an IncuCyte (Sartorius AG, IncuCyte® S3) to detect killing activity. The results are shown in the table below.

[0307] [Table 5]

[0308] The results showed that the cytotoxic potency of the monovalent ADC (CD28-E-6C4-ADC) was reduced compared to the monoclonal anti-EGFR ADC (E-6C4-ADC) (the killing activity of CD28-E-6C4-ADC was 8-12-fold lower than that of the anti-EGFR ADC in Panc.02.03 and NCI-H1975 cells), suggesting that E-6C4-10M1-ADC may enhance tumor cell specificity and minimize skin toxicity due to ubiquitous basal EGFR expression.

[0309] Example 3. Antitumor activity in the NUGC-4 xenograft model The ADC was tested for its effect on tumor growth in vivo in a model of gastric cancer. 6 NUGC-4 cells were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Catalog No. B-CM-002). When tumor size reached approximately 200 mm 3 At the time of tumor size, mice were randomly assigned to treatment or control groups based on tumor size. Mice were then injected intravenously (iv) with phosphate buffered saline (PBS) or ADC. The dosing frequency was once a week (total of one dose). Details are shown in the table below.

[0310] [Table 6]

[0311] The lengths of the long and short axes of the tumor were measured, and the tumor volume was calculated as 0.5 × (long axis) × (short axis). 2 Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0. T-test was performed for statistical analysis. A TGI of more than 60% indicates a clear inhibition of tumor growth. P < 0.05 is the threshold for indicating a significant difference.

[0312] There was little difference in the body weight of mice in different groups during the experimental period. The table below lists the results of this experiment, including tumor volume on the day of grouping (day 0), 7 days after grouping (day 7), and at the end of the experiment (day 14); mouse survival rate; TGI (%); tumor volume and statistical difference (P value) between the treatment group and the control group.

[0313] [Table 7]

[0314] The treatment groups demonstrated varying tumor-inhibitory effects. Overall, the antitumor activity of the bispecific antibody ADCs (G5–G6) was more potent than the corresponding monoclonal antibody ADCs (G7–G9) and the positive control (G2–G4).

[0315] In another similar experiment, the injection volume was calculated based on the mouse's weight to arrive at a dose of 1.5 mg / kg, 3 mg / kg, or 5 mg / kg. The administration frequency was once a week (two administrations in total). Details and results are shown in the table below.

[0316] [Table 8]

[0317] Table 9 lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21); mouse survival rate; TGI (%); and statistical differences (P values) in tumor volumes between treatment and control groups.

[0318] [Table 9]

[0319] The treatment groups (G5-G19) showed superior tumor inhibition effects compared to the control groups (G1-G4) treated with PBS or ISO-ADC. The anti-EGFR / MUC1 bispecific antibody ADC (G8-G19) showed superior tumor inhibition effects compared to the positive control groups (G5-G7) at the same dose. Furthermore, the treatment groups (G8-G19) showed dose-dependent antitumor activity.

[0320] Example 4. Antitumor activity in HCC70 xenograft model The ADC was tested for its effect on tumor growth in vivo in a model of breast cancer. 7 HCC70 cells were injected subcutaneously into each B-NDG mouse. Tumors in the mice were approximately 200 mm 3 When the tumor volume reached 100 μg / ml, the mice were randomly assigned to different groups (5 mice per group) based on tumor volume. Then, the mice were injected intravenously (iv) with phosphate buffered saline (PBS) or ADC. The administration frequency was once a week (total of 2 administrations). Details are shown in the table below.

[0321] [Table 10]

[0322] The weight of the mice was also measured twice a week. On the day of grouping (day 0), the mean weight of each group ranged from 22.6 g to 24.2 g. At the end of the experiment (day 25), the mean weight of each group ranged from 23.4 g to 25.2 g. Therefore, the mean weight change of each group ranged from 100.9% to 107%. The weights of all mice in the different groups increased. The results showed that the tested ADCs were well tolerated and had no obvious toxicity to mice.

[0323] The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and at the end of the experiment (day 25); mouse survival rate; TGI (%); tumor volumes and body weights between the treatment and control groups, and statistical differences (P values).

[0324] [Table 11]

[0325] The tumor volumes of different groups of mice treated with ADC or PBS are shown in Figure 2. The treatment groups (G3–G9) showed superior tumor inhibition effects compared to the control groups (G1–G2) treated with PBS or ISO-ADC. All four anti-EGFR / MUC1 bispecific antibody ADCs (G6–G9) showed sustained and potent tumor-inhibitory effects with high TGI at a dose of 3 mg / kg. The TGI values ​​of all tested EGFR / MUC1 bispecific antibody ADCs (G6–G9) were higher than those of the positive controls cetuximab analog ADC (G3), 1H7 analog ADC (G4), and gatipotuzumab analog ADC (G5).

[0326] Example 5. Antitumor activity Panc 02.03 xenograft model The ADC was tested for its effect on tumor growth in vivo in a xenograft model of pancreatic adenocarcinoma. 6 Pancreatic adenocarcinoma epithelial cells, Panc 02.03, were subcutaneously injected into each B-NDG mouse. Tumors in the mice reached approximately 200 mm 3When the tumor volume reached 100 μg / ml, the mice were randomly assigned to different groups based on tumor volume. Then, the mice were intravenously (iv) injected with PBS or ADC. The administration frequency was once a week (total of 2 administrations). Details are shown in the table below.

[0327] [Table 12]

[0328] There was little difference in the body weight of mice in different groups during the experimental period. The table below lists the results of this experiment, including tumor volume on the day of grouping (day 0), 17 days after grouping (day 17), and at the end of the experiment (day 27); mouse survival rate; TGI (%); body weight and tumor volume between the treatment group and the control group, and statistical difference (P value).

[0329] [Table 13]

[0330] The tumor sizes in the ADC-treated groups are shown in Figure 3. The treatment groups (G3 to G9) exhibited various tumor-inhibitory effects. Overall, the ADCs (G6 to G9) exhibited superior antitumor activity at a dose of 3 mg / kg compared to the positive control group (G3 to G5).

[0331] Example 6. Antitumor activity in the NCI-H1975 xenograft model The ADC was tested for its effect on tumor growth in vivo in a model of lung adenocarcinoma. 6 NCI-H1975 cells were injected subcutaneously into each B-NDG mouse. Tumors in the mice were approximately 200 mm 3 When the tumor volume reached 100 μg / ml, the mice were randomly assigned to different groups (5 mice per group) based on the tumor volume. Then, the mice were intravenously (iv) injected with PBS or ADC. The administration frequency was once a week (total of 2 administrations). Details are shown in the table below.

[0332] [Table 14]

[0333] The table below lists the results of this experiment, including tumor volumes on the day of grouping (day 0), 10 days after grouping (day 10), and at the end of the experiment (day 21); TGI (%); and statistical differences (P values) in tumor volumes between the treatment and control groups.

[0334] [Table 15]

[0335] The tumor volumes of different groups of mice treated with ADC or PBS are shown in Figure 4. The treatment groups (G2-G8) showed superior tumor-inhibiting effects compared to the PBS-treated control group (G1). All four anti-EGFR / MUC1 bispecific antibody ADCs (G5-G8) showed sustained and potent tumor-inhibiting effects at a dose of 3 mg / kg with TGIs higher than those of the positive control (G2-G4).

[0336] Example 7. Antitumor activity in the NCI-H1650 xenograft model The ADC was tested for its effect on tumor growth in vivo in a model of lung cancer. 7 NCI-H1650 cells were injected subcutaneously into each B-NDG mouse. Tumors in the mice were approximately 200 mm 3 When the tumor volume reached 100 μg / ml, the mice were randomly placed into different groups based on tumor volume. The mice were then intravenously (iv) injected with PBS, antibody, or ADC. The administration frequency was once a week (two administrations in total). Details are shown in the table below.

[0337] [Table 16]

[0338] There was little difference in the body weight of mice in different groups during the experimental period. The table below lists the results of this experiment, including tumor volumes on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21); mouse survival rate; TGI (%); and statistical difference (P value) of tumor volumes between the treatment group and the control group.

[0339] [Table 17]

[0340] Tumor sizes in the antibody-treated groups are shown in Figure 5. The treatment groups exhibited various tumor-inhibitory effects. Overall, the antitumor activity of the bispecific antibody ADCs (G4-G5) was more potent than that of the corresponding monoclonal antibody ADCs (G6-G8) and the positive control (G2-G3).

[0341] Example 8. Antitumor activity in a patient-derived lung cancer xenograft model The ADC was tested for its effect on tumor growth in vivo in a lung cancer xenograft model. Specifically, tumor fragments from lung cancer patients were subcutaneously inoculated into B-NDG mice. Tumors in the mice grew to approximately 250-300 mm. 3 When the tumor volume reached 100 μg / ml, the mice were randomly assigned to different groups based on tumor volume. Then, the mice were intravenously (iv) injected with PBS or ADC. The administration frequency was once a week (total of 2 administrations). Details are shown in the table below.

[0342] [Table 18]

[0343] All mice in all treatment groups (G2-G5) gained weight. The results showed that the tested antibodies were well tolerated and had no apparent toxicity to the mice.

[0344] The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 10 days after grouping (day 10), and at the end of the experiment (day 21); mouse survival rate; TGI (%); tumor volumes and statistical differences (P values) between treatment and control groups.

[0345] [Table 19]

[0346] The results showed that the antitumor activity of the bispecific antibody ADCs (G4-G5) at a dose of 3 mg / kg was more potent than that of the positive control group (G2-G3).

[0347] Example 9. In vivo efficacy in a human ampullary carcinoma PDX model The efficacy of ADC was tested in a human papillary carcinoma PDX model. Immunofluorescence staining of patient-derived papillary tumor sections was performed, and images were analyzed using HALO 3.2. The results showed that EGFR-positive cells and MUC1-positive cells were 94.03% and 81.20%, respectively.

[0348] Patient-derived papillary tumor fragments (2 mm x 2 mm x 2 mm) were implanted into the right flank of B-NDG mice. Tumors in the mice grew to approximately 250–300 mm. 3 When the tumor volume reached 100 μg / ml, the mice were randomly placed into different groups based on tumor volume. The mice were then injected intravenously (iv) with PBS or ADC. The details of the administration scheme are shown in the table below.

[0349] [Table 20]

[0350] The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), 28 days after grouping (day 28), and 42 days after grouping (day 42); TGI (%); and statistical differences (P values) in tumor volumes between treatment and control groups.

[0351] [Table 21]

[0352] The tumor volumes of mice in different groups are shown in Figure 11. The results showed that the antitumor activity of E-6C4-10M1-ADC (G6) at a dose of 3 mg / kg was stronger than that of the positive control groups (G3 to G5). Furthermore, the experiment was continued until day 60 after grouping, and E-6C4-10M1-ADC (G6) showed a superior tumor inhibitory effect to the positive control groups (G3 to G5), although all mice in the 1H7 analog ADC and gatipotuzumab analog ADC treatment groups (G3 to G4) died.

[0353] Example 10. Internalization of anti-EGFR / MUC1 bispecific antibodies and ADCs NUGC-4 cells, HCC70 cells, or Panc 02.03 cells cultured in cell culture plates were treated with anti-EGFR antibodies, anti-MUC1 antibodies, anti-EGFR / MUC1 bispecific antibodies, or anti-EGFR / MUC1 bispecific ADCs, and internalization activity was detected after 8 or 24 hours of incubation in an IncuCyte (Sartorius AG, IncuCyte® S3) tube. The results are shown in Figures 12A-12B and 13A-13B.

[0354] As shown in Figures 12A-12B, E-6C4-10M1 exhibited superior internalization efficacy to the corresponding parent monoclonal antibodies (E-6C4 and 10M1) in NUGC-4 and HCC70 cells. Figures 13A-13B showed that the endocytic activity of E-6C4-10M1-ADC was unchanged after MMAE conjugation.

[0355] Example 11. Antibody drug conjugates Coupling of antibodies with drug molecules The purified antibody was coupled to CPT-1, CPT-2, CPT-3, or CPT-4 via a CPT-L linker. The antibody-drug conjugate is named by adding CPTx (x=1, 2, 3, or 4) immediately after the antibody name. For example, when E-6C4-10M1 is coupled to CPT-1, it is named E-6C4-10M1-CPT1. As another example, when E-6C4-10M1 is coupled to CPT-2, it is named E-6C4-10M1-CPT2. Exemplary ADCs obtained by this method included E-6C4-CPT1, 10M1-CPT1, E-6C4-10M1-CPT1, E-6C4-CPT2, 10M1-CPT2, and E-6C4-10M1-CPT2.

[0356] As positive controls, 1H7 and gatipotuzumab analogs were coupled to CPT-2 via the CPT-L linker to obtain 1H7-CPT2 and gatipotuzumab-CPT2, respectively. Gatipotuzumab analogs were also coupled to GGFG-Dxd (Deruxtecan, MedChemExpress, catalog number: HY-13631E) to form gatipotuzumab-Dxd.

[0357] The coupling between the antibody and the drug molecule was detected using MS (mass spectrometry), and the MS detection results showed that the DAR of the ADC was approximately 8.

[0358] Antitumor activity in patient-derived pancreatic cancer xenograft models Tumor tissue fragments (2 mm × 2 mm × 2 mm) derived from pancreatic cancer patients were transplanted into the right flank of B-NDG mice. Immunofluorescence staining showed that EGFR-positive cells and MUC1-positive cells in the pancreatic tumor fragments were 60.62% and 64.77%, respectively. Tumors in mice were approximately 200–300 mm 3When the tumor volume reached 100 μg / ml, the mice were randomly placed into different groups based on tumor volume. The mice were then injected intravenously with PBS or ADC. Details are shown in the table below.

[0359] [Table 22]

[0360] The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and 39 days after grouping (day 39); TGI (%); and statistical differences (P values) in tumor volumes between treatment and control groups.

[0361] [Table 23]

[0362] The tumor volumes of mice in different groups are shown in Figure 14. The results showed that E-6C4-10M1-CPT2 exhibited a dose-dependent tumor-inhibiting effect, and demonstrated superior tumor-inhibiting effects to those of the parent monoclonal ADCs E-6C4-CPT2 and 10M1-CPT2. Furthermore, E-6C4-10M1-CPT2 exhibited superior tumor-inhibiting effects to those of the positive controls 1H7-CPT2, gatipotuzumab-CPT2, and gatipotuzumab-Dxd.

[0363] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An anti-EGFR / MUC1 antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MUC1; An anti-EGFR / MUC1 antibody or an antigen-binding fragment thereof.

2. the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to claim 1.

3. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to claim 2, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR3; and the first light chain variable region (VL1) comprises CDR1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL1 CDR3; the selected VH1 CDR1, 2, and 3 amino acid sequences, and the selected VL1 CDR1, 2, and 3 amino acid sequences, are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (4) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (5) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (6) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (7) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (8) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (9) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are set forth in SEQ ID NOs: 34 to 36, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively; and (10) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

4. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to claim 2 or 3, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH2 CDR3; and the second light chain variable region (VL2) comprises CDR1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL2 CDR3; the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (2) the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are set forth in SEQ ID NOs: 40 to 42, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively; and (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

5. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (4) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (5) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (6) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (7) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (8) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (9) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (10) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (11) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (12) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (13) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (14) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 34 to 36, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (15) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (16) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (17) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (18) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (19) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are set forth in SEQ ID NOs: 34 to 36, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are set forth in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively; or (20) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

6. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:47; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:52; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

7. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:48; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:52; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

8. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:49; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:52; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

46. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

9. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:50; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:52; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

10. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:51; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:52; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

11. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:47; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:53; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

12. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:48; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

13. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:49; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:53; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

46. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

14. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:50; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:53; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

15. the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:51; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:53; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5.

16. 16. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 15, wherein the VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:47 and the selected VL sequence is SEQ ID NO:46; (2) the selected VH sequence is SEQ ID NO: 48 and the selected VL sequence is SEQ ID NO: 46; (3) the selected VH sequence is SEQ ID NO:49 and the selected VL sequence is SEQ ID NO:46; (4) the selected VH sequence is SEQ ID NO: 50 and the selected VL sequence is SEQ ID NO: 46; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO:

46.

17. 17. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 16, wherein the VH1 comprises VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; the VL1 comprises VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence; and the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:47 and the selected VL sequence is SEQ ID NO:46; (2) the selected VH sequence is SEQ ID NO: 48 and the selected VL sequence is SEQ ID NO: 46; (3) the selected VH sequence is SEQ ID NO:49 and the selected VL sequence is SEQ ID NO:46; (4) the selected VH sequence is SEQ ID NO: 50 and the selected VL sequence is SEQ ID NO: 46; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO:

46.

18. 18. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 17, wherein the VH2 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 46; and (2) The selected VH sequence is SEQ ID NO: 53 and the selected VL sequence is SEQ ID NO:

46.

19. 19. The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 18, wherein the VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; the VL2 comprises VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence; and the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 46; and (2) The selected VH sequence is SEQ ID NO: 53 and the selected VL sequence is SEQ ID NO:

46.

20. said VH1 comprising the sequence of SEQ ID NO: 47 and said VL1 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 19.

21. said VH1 comprising the sequence of SEQ ID NO: 48 and said VL1 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 19.

22. said VH1 comprising the sequence of SEQ ID NO: 49 and said VL1 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 19.

23. said VH1 comprising the sequence of SEQ ID NO: 50 and said VL1 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 19.

24. said VH1 comprising the sequence of SEQ ID NO: 51 and said VL1 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 19.

25. said VH2 comprising the sequence of SEQ ID NO: 52 and said VL2 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 24.

26. said VH2 comprising the sequence of SEQ ID NO: 53 and said VL2 comprising the sequence of SEQ ID NO: 46; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 24.

27. the first antigen-binding domain specifically binds to human EGFR or monkey EGFR, and / or the second antigen-binding domain specifically binds to human MUC1 or monkey MUC1; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 26.

28. the first antigen-binding domain is human or humanized, and / or the second antigen-binding domain is human or humanized; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 27.

29. the antibody is a bispecific antibody; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 28.

30. the first antigen-binding domain is a single-chain variable fragment (scFv), and / or the second antigen-binding domain is an scFv; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 29.

31. the first light chain variable region and the second light chain variable region are identical; The anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 30.

32. An anti-EGFR / MUC1 antibody or antigen-binding fragment thereof that cross-competes with the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 31.

33. A nucleic acid comprising a polynucleotide encoding the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 32.

34. A vector comprising the nucleic acid of claim 33.

35. A cell comprising the vector of claim 34.

36. 36. The cell of claim 35, wherein the cell is a CHO cell.

37. A cell comprising the nucleic acid of claim 33.

38. A method for producing an anti-EGFR / MUC1 antibody or an antigen-binding fragment thereof. (a) culturing the cell of any one of claims 35 to 37 under conditions sufficient for the cell to produce the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof; (b) recovering the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof produced by the cells.

39. 33. An anti-EGFR / MUC1 antibody drug conjugate (ADC) comprising a therapeutic agent covalently attached to the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 32.

40. the therapeutic agent is a cytotoxic or cytostatic agent; 40. The anti-EGFR / MUC1 antibody drug conjugate of claim 39.

41. The therapeutic agent is MMAE or MMAF.

41. The anti-EGFR / MUC1 antibody drug conjugate of claim 39 or 40.

42. 40. The antibody drug conjugate of claim 39, wherein the therapeutic agent is selected from the following: 【Chemistry 1】

43. the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker; 43. The antibody drug conjugate of claim 39 or 42.

44. 44. The antibody drug conjugate of claim 43, wherein the linker has the structure: 【Chemistry 2】

45. 45. The antibody drug conjugate of any one of claims 39 and 42 to 44, wherein the antibody drug conjugate has the following structure: 【Transformation 3】 【Chemistry 4】 where n=1 to 8 and "Ab" represents an antibody or antigen-binding fragment thereof.

46. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 32, or the anti-EGFR / MUC1 antibody-drug conjugate of any one of claims 39 to 45. method.

47. the subject has a cancer that expresses EGFR and / or MUC1 (e.g., both EGFR and MUC1); 47. The method of claim 46.

48. The cancer is a solid tumor, lung cancer, head and neck cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, glioma, esophageal cancer, gastric cancer, prostate cancer, cervical cancer, multiple myeloma, or non-Hodgkin's lymphoma; 48. The method of claim 46 or 47.

49. The method of any one of claims 46 to 48, wherein the subject is a human.

50. The method of any one of claims 46 to 49, further comprising administering an anti-PD1 antibody to the subject.

51. 51. The method of any one of claims 46 to 50, wherein the method further comprises administering chemotherapy to the subject.

52. 10. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 32, or a composition comprising an anti-EGFR / MUC1 antibody-drug conjugate of any one of claims 39 to 45. method.

53. 1. A method of killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising the anti-EGFR / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 32, or the anti-EGFR / MUC1 antibody-drug conjugate of any one of claims 39 to 45. method.

54. a pharmaceutically acceptable carrier; and (a) an anti-EGFR / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 32, and / or (b) an anti-EGFR / MUC1 antibody-drug conjugate according to any one of claims 39 to 45; and a pharmaceutical composition comprising:

55. The drug-antibody ratio (DAR) is about 4 or 8; The anti-EGFR / MUC1 ADC of any one of claims 39 to 45.

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