Anti-HER3 / MUC1 antibodies and uses thereof

Abstract

The present disclosure relates to anti-HER3 / MUC1 antibodies, antibody drug conjugates derived therefrom, and uses thereof.

Description

[0001] priority

[0002] This application claims priority to PCT / CN2023 / 082375, filed on March 18, 2023, and PCT / CN2023 / 120377, filed on September 21, 2023. The above applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to multispecific anti-HER3 (human epidermal growth factor receptor 3) / MUC1 (mucin 1) antibodies (eg, bispecific antibodies or antigen-binding fragments thereof), and antibody-drug conjugates derived therefrom. Background Art

[0004] Bispecific antibodies are artificial 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 of 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) has become an important milestone in the development of bispecific antibodies.

[0005] Because bispecific antibodies have diverse applications, there is a continued need to develop a variety of bispecific antibody-based therapeutics. Summary of the Invention

[0006] The present disclosure relates to anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to HER3 and MUC1. In some embodiments, the antibodies or antigen-binding fragments thereof have the same light chain variable region. In some embodiments, the antibodies or antigen-binding fragments thereof have a common light chain. The present disclosure also relates to antibody-drug conjugates derived from these anti-HER3 / MUC1 antibodies.

[0007] In one aspect, the disclosure relates to an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to HER3; and a second antigen-binding domain that specifically binds to MUCl.

[0008] 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).

[0009] 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 a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; and

[0010] the first light chain variable region (VL1) comprising CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence,

[0011] wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences, the selected VL1 CDR 1, 2, and 3 amino acid sequences are one of the following:

[0012] (1) the selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively;

[0013] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively;

[0014] (3) the amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 7, 42, and 43, respectively, and the amino acid sequences of the selected VL1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively;

[0015] (4) the selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively;

[0016] (5) the selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively; and

[0017] (6) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 19, 20, and 44, respectively, and the amino acid sequences of the selected VL1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

[0018] In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 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

[0019] the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence,

[0020] wherein the selected VH2 CDR 1, 2, and 3 amino acid sequences, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are one of the following:

[0021] (1) the selected VH2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; and

[0022] (2) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL2 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively.

[0023] In some embodiments, (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 4 to 6, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1 to 3, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1 to 3, respectively;

[0024] (2) the selected VH1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively;

[0025] (3) the selected VH1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively;

[0026] (4) the selected VH1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively;

[0027] (5) the selected VH1 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 7, 42, and 43, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1 to 3, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1 to 3, respectively; or

[0028] (6) The selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, and 44, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VH2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively.

[0029] 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:26, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:25, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:28, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:25.

[0030] 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:27, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:25, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:28, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:25.

[0031] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:45, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:25, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:28, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:25.

[0032] In some embodiments, 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, wherein the selected VH sequence and the selected VL sequence are one of:

[0033] (1) the selected VH sequence is SEQ ID NO: 26, and the selected VL sequence is SEQ ID NO: 25;

[0034] (2) the selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 25; and

[0035] (3) The selected VH sequence is SEQ ID NO:45, and the selected VL sequence is SEQ ID NO:25.

[0036] In some embodiments, the VH1 comprises a VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL1 comprises a VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0037] (1) the selected VH sequence is SEQ ID NO: 26, and the selected VL sequence is SEQ ID NO: 25;

[0038] (2) the selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 25; and

[0039] (3) The selected VH sequence is SEQ ID NO:45, and the selected VL sequence is SEQ ID NO:25.

[0040] In some embodiments, 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, wherein the selected VH sequence is SEQ ID NO:28 and the selected VL sequence is SEQ ID NO:25.

[0041] In some embodiments, the VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO: 25.

[0042] In some embodiments, the VH1 comprises the sequence of SEQ ID NO:26, and the VL1 comprises the sequence of SEQ ID NO:25.

[0043] In some embodiments, the VH1 comprises the sequence of SEQ ID NO:27, and the VL1 comprises the sequence of SEQ ID NO:25.

[0044] In some embodiments, the VH1 comprises the sequence of SEQ ID NO:45, and the VL1 comprises the sequence of SEQ ID NO:25.

[0045] In some embodiments, the VH2 comprises the sequence of SEQ ID NO:28, and the VL2 comprises the sequence of SEQ ID NO:25.

[0046] In some embodiments, the first antigen binding domain specifically binds to human or monkey HER3; and / or the second antigen binding domain specifically binds to human or monkey MUC1.

[0047] In some embodiments, the first antigen binding domain is human or humanized; and / or the second antigen binding domain is human or humanized.

[0048] In some embodiments, the antibody is a multispecific antibody (eg, a bispecific antibody).

[0049] In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0050] In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0051] In one aspect, the disclosure relates to an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof that cross-compete with an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein.

[0052] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein.

[0053] In one aspect, the disclosure relates to a vector comprising a nucleic acid described herein.

[0054] In one aspect, the disclosure relates to a cell comprising a vector described herein.

[0055] In some embodiments, the cell is a CHO cell.

[0056] In one aspect, the disclosure relates to a cell comprising a nucleic acid described herein.

[0057] In one aspect, the present disclosure relates to a method of producing an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof, the method comprising:

[0058] (a) culturing the cell described herein under conditions sufficient for the cell to produce the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof; and

[0059] (b) collecting the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof produced by the cells.

[0060] In one aspect, the disclosure relates to an anti-HER3 / MUC1 antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein.

[0061] In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0062] In some embodiments, the therapeutic agent is MMAE or MMAF.

[0063] In some embodiments, the therapeutic agent is selected from:

[0064]

[0065] In some embodiments, the therapeutic agent is connected to the antibody or its antigen-binding fragment via a linker. In some embodiments, the linker has the following structure:

[0066]

[0067] In some embodiments, the antibody drug conjugate has the following structure:

[0068]

[0069] In some embodiments, n=1 to 8; in some embodiments, "Ab" represents the antibody or antigen-binding fragment thereof.

[0070] In some embodiments, the drug to antibody ratio (DAR) is about 4 or 8.

[0071] In one aspect, the disclosure relates to a method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein, or an anti-HER3 / MUC1 antibody drug conjugate described herein.

[0072] In some embodiments, the subject has a cancer that expresses HER3 and / or MUCl (eg, both HER3 and MUCl).

[0073] In some embodiments, the cancer is esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial cancer, lung cancer, melanoma, ovarian cancer, bladder cancer, stomach cancer, non-Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, lung adenocarcinoma, and cervical cancer.

[0074] In some embodiments, the subject is a human.

[0075] In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject.

[0076] In some embodiments, the method further comprises administering chemotherapy to the subject.

[0077] In one aspect, the disclosure relates to a method of reducing tumor growth rate, comprising contacting tumor cells with an effective amount of a composition comprising an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein, or an anti-HER3 / MUC1 antibody drug conjugate described herein.

[0078] In one aspect, the disclosure relates to a method of killing tumor cells, comprising contacting the tumor cells with an effective amount of a composition comprising an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein, or an anti-HER3 / MUC1 antibody-drug conjugate described herein.

[0079] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and

[0080] (a) an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof as described herein, and / or

[0081] (b) Anti-HER3 / MUC1 antibody drug conjugates described herein.

[0082] In one aspect, the present disclosure relates to an anti-HER3 / MUC1 antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to HER3; and a second antigen-binding domain that specifically binds to MUC1.

[0083] As used herein, the term "antigen binding domain" refers to one or more protein domains (e.g., formed by the amino acids of a single polypeptide, or formed by the amino acids of two or more polypeptides (e.g., the same or different polypeptides)) that are capable of specifically binding to one or more different antigens (e.g., effector antigens or control antigens). In some embodiments, the antigen binding domain can bind to an antigen or epitope with the specificity and affinity similar to a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or fragment thereof. One embodiment of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. In some embodiments, the antigen binding domain may include an alternative scaffold. In some embodiments, the antigen binding domain is a VHH. Non-limiting examples of antigen binding domains are described herein. Other embodiments of antigen binding domains are known in the art. In some embodiments, the antigen binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other embodiments, the antigen binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen).

[0084] As used herein, the term "antibody" is used in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen binding domains that specifically bind to an antigen or epitope. In particular, antibodies include, for example, complete antibodies (e.g., complete immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. One embodiment of an antibody is a protein complex comprising two heavy chains and two light chains. Other embodiments of antibodies are described herein.

[0085] As used herein, the term "multispecific antibody" is an antibody comprising two or more different antigen-binding domains, which specifically bind to two or more different epitopes together. The two or more different epitopes can be epitopes on the same antigen (such as a single polypeptide present on the surface of a cell), or epitopes on different antigens (such as different proteins present on the surface of the same cell or on the surface of different cells). In some aspects, multispecific antibodies bind to two different epitopes (i.e., "bispecific antibodies"). In some aspects, multispecific antibodies bind to three different epitopes (i.e., "trispecific antibodies"). In some aspects, multispecific antibodies bind to four different epitopes (i.e., "tetraspecific antibodies"). In some aspects, multispecific antibodies bind to five different epitopes (i.e., "pentaspecific antibodies"). Every kind of binding specificity can exist with any suitable valence. Non-limiting embodiments of multispecific antibodies are described herein.

[0086] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. These epitopes can be located on the same antigen or on different antigens.

[0087] As used herein, the term "common light chain" refers to a light chain that can interact with two or more different heavy chains to form different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. Similarly, the term "common light chain variable region" refers to a light chain variable region that can interact with two or more different heavy chain variable regions to form different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. In some embodiments, antibodies or antigen-binding fragments thereof may have a common light chain. In some embodiments, anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof may have a common light chain variable region.

[0088] As used herein, the term "anti-HER3 / MUCl antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that binds to both MUCl and HER3.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; in addition, other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to limit the present invention. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0090] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Shown are the mean tumor volumes of different groups of B-NDG mice injected with NUGC-4 cells and treated with phosphate-buffered saline (PBS) or ADC.

[0092] Figure 2 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived gastric tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0093] Figure 3 The heavy chain variable region CDR sequences of the anti-HER3 antigen binding domain (1B2, 3G6, and 3E1) and the anti-MUCl antigen binding domain (10D1) in the anti-HER3 / MUCl antibodies are listed, which are defined as per the Kabat definition.

[0094] Figure 4 The heavy chain variable region CDR sequences of the anti-HER3 antigen binding domain (1B2, 3G6, and 3E1) and the anti-MUCl antigen binding domain (10D1) in the anti-HER3 / MUCl antibodies are listed, as defined by the Chothia definition.

[0095] Figure 5 Certain amino acid sequences discussed in this disclosure are listed.

[0096] Figure 6 Shown are the mean tumor volumes of different groups of B-NDG mice injected with HCC70 cells and treated with PBS or ADC.

[0097] Figure 7 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived pancreatic tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0098] Figure 8Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived lung tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0099] FIG9A to FIG9C show the endocytosis rates of anti-HER3 antibody, anti-MUC1 antibody, or anti-HER3 / MUC1 bispecific antibody and ADC in NUGC-4 cells.

[0100] Figure 10 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived lung tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0101] Figure 11 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with NUGC-4 cells and treated with PBS or ADC.

[0102] Figure 12A Shown are the mean tumor volumes of different groups of B-NDG mice implanted with HCC70 cells and treated with PBS or ADC.

[0103] Figure 12B Shown are the average body weights of different groups of B-NDG mice implanted with HCC70 cells and treated with PBS or ADC.

[0104] Figure 13 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived colorectal tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0105] Figure 14 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived colorectal tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0106] Figure 15A Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived pancreatic tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0107] Figure 15B Shown are the mean body weights of different groups of B-NDG mice implanted with patient-derived pancreatic tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0108] Figure 16Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived mammary tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0109] Figures 17A-17B The binding activity of the anti-HER3 antibody, anti-MUC1 antibody, or anti-HER3 / MUC1 bispecific antibody to NUGC-4 cells and NCI-H226 cells was shown.

[0110] Figure 18 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived gastric tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0111] Figure 19 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived colorectal tumor fragments (2 mm x 2 mm x 2 mm) and treated with PBS or ADC.

[0112] Figure 20 Shown are the mean tumor volumes of different groups of BALB / c nude mice implanted with patient-derived lung tumor fragments (2 mm x 2 mm x 2 mm) and treated with 5% glucose or ADC.

[0113] Figure 21 Shown are the mean tumor volumes of different groups of BALB / c nude mice implanted with patient-derived ovarian tumor fragments (2 mm x 2 mm x 2 mm) and treated with 5% glucose or ADC. DETAILED DESCRIPTION

[0114] Bispecific antibodies or their antigen-binding fragments are artificial proteins that can simultaneously bind to two different epitopes (e.g., on two different antigens). In some embodiments, a bispecific antibody or its antigen-binding fragment can have two arms. Each arm can have a heavy chain variable region and a light chain variable region, which form an antigen-binding domain (or antigen-binding region). In some embodiments, a bispecific antibody has a common light chain.

[0115] The present disclosure relates to anti-HER3 / MUC1 antibodies (eg, bispecific antibodies or antigen-binding fragments thereof) that specifically bind to HER3 and MUC1; and antibody drug conjugates derived from these anti-HER3 / MUC1 antibodies.

[0116] Anti-HER3 / MUC1 antibodies

[0117] HER3 is a pseudokinase member of the EGFR family that plays a role in both tumor progression and drug resistance. HER3 is a unique EGFR family member that cannot cause cancer alone, but can collaborate with other receptors to induce tumor formation, metastasis, and drug resistance. HER3 is a highly sought-after target for cancer therapy. Unlike EGFR and HER2, which are widely targeted by TKIs, HER3, due to its extremely low kinase activity, is primarily targeted with monoclonal antibodies or bispecific antibodies, either by blocking ligand binding or by heterodimerization with other receptors.

[0118] HER3 expression is commonly detected in a variety of cancers, including breast, ovarian, colon, gastric, lung, skin, and pancreatic cancers. High HER3 expression is also associated with disease progression and / or poor prognosis in many cancer types. Although HER3 itself does not cause tumor formation, the HER2:HER3 heterodimer has the highest transforming ability among all possible EGFR family dimers. The remarkable oncogenic capacity of the dimer pair makes HER3 crucial for HER2-mediated tumor formation in a variety of tumor types. In breast cancer cell lines, the HER3 pair is crucial for maintaining cell viability, while EGFR is insignificant.

[0119] HER3 expression is a bypass mechanism for various targeted therapies, and an increase in HER3 signaling can lead to resistance to a variety of therapeutic agents. For example, since HER3 can dimerize with receptors other than EGFR (including HER2 and MET receptors), HER3 can produce resistance to EGFR targeted therapies via dimerization with partners other than EGFR. HER3 expression is also associated with resistance to hormone therapy. HER3 plays a crucial role in the phosphorylation of HER2 in breast cancer cells, and downregulating HER3 can reverse resistance to the anti-estrogen receptor (ER) tamoxifen in breast cancer cell lines.

[0120] Details on HER3 and its functions can be found in Haikala, Heidi M., and Pasi “ThirtyYears of HER3: From Basic Biology to Therapeutic Interventions 30 Years of HER3.” Clinical Cancer Research 27.13(2021):3528-3539; and Mishra, Rosalin, et al. “HER3 signaling and targeted therapy in cancer.” Oncology reviews 12.1(2018); Liu, Xiaolong, et al. “Development of effective therapeutics targeting HER3 for cancer treatment.” Biological procedures online 21.1(2019):1-10; each of which is incorporated by reference in its entirety.

[0121] Mucin 1 (MUC1; also known as episiialin, PEM, H23Ag, EMA, CA15-3, and MCA) is a unidirectional type I transmembrane protein with a heavily glycosylated extracellular domain extending up to 200 to 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 from skin epithelial cells and mesenchymal cells. In healthy tissue, MUC1 provides protection to the underlying epithelium. The negatively charged sugar branches extending from MUC1 form a physical barrier and confer anti-adhesion properties to MUC1, thereby limiting the entry of pathogens and preventing their colonization. Sugar chains oligomerize to form mucinous gel, which lubricates and protects the underlying epithelium from dryness, pH changes, pollutants, and microorganisms. Abnormally glycosylated MUC1 is overexpressed in most human epithelial cancers and has received widespread attention as an oncogenic molecule.

[0122] MUC1 is overexpressed in cancer cells, and the loss of cell polarity leads to a redistribution of TA-MUC1 on the cell surface and within the cytoplasm. Lack of cell polarity also leads to a redistribution of cell surface growth factors that are normally restricted to the basolateral surface of epithelial cells. Growth factors that juxtapose to MUC1 and intracellular kinases (e.g., ZAP-70, PKC-g, GSK-3b, and c-Src) phosphorylate serine, tyrosine, and threonine residues on MUC1 CT. Furthermore, hypoglycosylation is believed to unmask the peptide core of TA-MUC1, allowing MUC1-N to be cleaved and released by extracellular proteases. MUC1-N release induces conformational changes in MUC1-C, altering its ligand status and subsequently activating downstream cellular signaling pathways such as the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K / Akt), and wingless type (Wnt) pathways. Therefore, MUC1-positive pancreatic, breast, lung, and colon cancer cells often express hyperactivation of these key signaling pathways. MUC1-C also associates with various transcription factors (STAT3, NF-κB, p53, and β-catenin) and binds to target gene promoters to drive their expression. Multiple studies have shown that MUC1 plays a key role in the transcriptional regulation of genes related to tumor invasion, metastasis, angiogenesis, proliferation, apoptosis, drug resistance, inflammation, and immune regulation.

[0123] Details regarding MUC1 and its functions can be found in 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.

[0124] In some embodiments, the bispecific anti-HER3 / MUC1 antibodies described herein can be designed to have an IgG1 subtype structure with a knobs-into-holes (KIH) mutation, which promotes heterodimerization and avoids mispairing between the two heavy chains. In some embodiments, the bispecific anti-HER3 / MUC1 antibodies have a higher endocytosis rate than the corresponding cloned antibody or a control bispecific antibody.

[0125] In some embodiments, the bispecific anti-HER3 / 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 ADC 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 ADC 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, 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.

[0126] In some embodiments, the anti-HER3 / MUC1 ADC described herein can be effective in inhibiting cancer cell growth in vitro at a concentration 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-HER3 / MUC1 ADC described herein can be effective in inhibiting cancer cell growth in vivo (e.g., lung cancer, gastric cancer, or skin cancer) in a xenograft mouse model at a dose level 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.

[0127] In some embodiments, the anti-HER3 / MUC1 antibodies described herein have a common light chain. In some embodiments, the anti-HER3 / MUC1 antibodies comprise an anti-HER3 antigen binding domain (e.g., 1B2, 3G6, 3E1) or an anti-MUC1 antigen binding domain (e.g., 10D1). In some embodiments, the anti-HER3 / MUC1 antibodies have a heavy chain variable region that targets HER3 (e.g., any of the VHs targeting HER3 described herein), a heavy chain variable region that targets MUC1 (e.g., any of the VHs targeting MUC1 described herein), and two identical common light chain variable regions.

[0128] The CDR sequences of the 1B2 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 4 to 6) and the CDRs of the light chain variable domain (SEQ ID NOs: 1 to 3), which are defined according to the Kabat definition. CDRs can also be defined according to the Chothia definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 16 to 18, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 13 to 15. The human light chain variable region and human heavy chain variable region of 1B2 are shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0129] The CDR sequences of the 3E1 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 7 to 9) and the CDRs of the light chain variable domain (SEQ ID NOs: 1 to 3), which are defined according to the Kabat definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 19 to 21, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 13 to 15. The human light chain variable region and human heavy chain variable region of 3E1 are shown in SEQ ID NOs: 25 and 27, respectively.

[0130] The CDR sequences of the 3G6 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 7, 42, 43) and the CDRs of the light chain variable domain (SEQ ID NOs: 1 to 3), which are defined according to the Kabat definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 19, 20, 44, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 13 to 15. The human light chain variable region and human heavy chain variable region of 3E1 are shown in SEQ ID NOs: 25 and 45, respectively.

[0131] The CDR sequences of the 10D1 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 10 to 12) and the CDRs of the light chain variable domain (SEQ ID NOs: 1 to 3), which are defined according to the Kabat definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 22 to 24, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 13 to 15. The human light chain variable region and human heavy chain variable region of 3E1 are shown in SEQ ID NOs: 25 and 28, respectively.

[0132] In some embodiments, the anti-HER3 / MUC1 antibodies described herein may contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 4 to 6, SEQ ID NOs: 7 to 9, SEQ ID NOs: 10 to 12, SEQ ID NOs: 7, 42, 43, SEQ ID NOs: 16 to 18, SEQ ID NOs: 19 to 21, SEQ ID NOs: 19, 20, 44, and SEQ ID NOs: 22 to 24; and / or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1 to 3 and SEQ ID NOs: 13 to 15.

[0133] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof can have: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, and the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence. The CDR2 amino acid sequence has at least 80%, 85%, 90%, or 95% identity to the selected VL CDR3 amino acid sequence or consists of an amino acid sequence that has at least 80%, 85%, 90%, or 95% identity to the selected VH CDR 1, 2, 3 amino acid sequence and the selected VL CDR 1, 2, 3 amino acid sequence. Figure 3 and Figure 4 Displayed in.

[0134] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three 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.

[0135] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 16 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0136] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 7 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 9 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0137] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 19 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0138] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 7 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 43 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0139] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 19 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 44 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0140] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 10 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 12 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0141] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three 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.

[0142] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 1 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0143] In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three CDRs of: SEQ ID NO: 13 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 having zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0144] Insertions, deletions, and substitutions may be located within the CDR sequence, or at one or both ends of the CDR sequence.

[0145] In some embodiments, the anti-HER3 / MUC1 antibody comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 26, 27, 45, or 28, and the selected VL sequence is SEQ ID NO: 25.

[0146] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment may have three VH CDRs that are identical to the CDRs of any VH sequence as described herein. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment may have three VL CDRs that are identical to the CDRs of any VL sequence as described herein.

[0147] The present disclosure also provides nucleic acids comprising polynucleotides encoding anti-HER3 / MUC1 antibodies. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-HER3 / MUC1 antibody comprises Figure 3 、 Figure 4 or Figure 5 When the polypeptide is paired with a corresponding polypeptide (eg, a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to MUC1 and / or HER3.

[0148] Anti-HER3 / MUC1 antibodies can also be anti-HER3 / MUC1 antibody variants (including derivatives and conjugates) of anti-HER3 / MUC1 antibodies or antibody fragments. Other anti-HER3 / MUC1 antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies produced intracellularly (i.e., intrabodies), and antigen-binding fragments thereof. Anti-HER3 / MUC1 antibodies 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-HER3 / MUC1 antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or an antigen-binding fragment thereof.

[0149] As long as fragments of anti-HER3 / MUC1 antibodies retain the desired affinity and specificity for both MUC1 and HER3, they are suitable for use in the provided methods.Thus, fragments of anti-HER3 / MUC1 antibodies will retain the ability to bind to both MUC1 and HER3.

[0150] Antibodies and their antigen-binding fragments

[0151] In some embodiments, a multispecific anti-HER3 / MUC1 antibody (eg, a bispecific antibody) comprises an antigen-binding domain derived from an anti-HER3 antibody and an antigen-binding domain derived from an anti-MUC1 antibody. These anti-HER3 / MUC1 antibodies and antigen-binding fragments thereof can have various formats.

[0152] Generally speaking, antibodies (also known as immunoglobulins) can be composed of two types of polypeptide chains: light chains and heavy chains. The non-limiting anti-HER3 / MUC1 antibodies disclosed herein can be complete tetraimmunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chains of the anti-HER3 / MUC1 antibodies can be of any isotype (including IgM, IgG, IgE, IgA, or IgD) or subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chains can be kappa light chains or lambda light chains.

[0153] The hypervariable regions, called complementarity determining regions (CDRs), form loops that comprise the primary antigen-binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and contribute to the formation of the antigen-binding domain with the CDRs of the other chain.

[0154] Methods for identifying the CDR regions of antibodies by analyzing their amino acid sequences are well known, and various definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, while 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. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and 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 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

[0155] CDR pairs are crucial in recognizing antigenic epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by an antibody's antigen-binding domain. The minimum size of an epitope can be approximately three, four, five, six, or seven amino acids, but these amino acids do not necessarily represent a continuous linear sequence of the antigen's primary structure, as the epitope may depend on the antigen's three-dimensional configuration, which is based on the antigen's secondary and tertiary structures.

[0156] In some embodiments, the anti-HER3 / MUC1 antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved but differ in their constant regions, particularly in their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al. “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is herein incorporated by reference in its entirety.

[0157] The anti-HER3 / MUC1 antibody can also be an immunoglobulin molecule derived from any species (e.g., human, rodent, mouse, rat, camelid). An antigen-binding domain or antigen-binding fragment is a portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment may include, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide comprising a binding domain that is an antibody binding domain or is homologous thereto. Non-limiting examples of antigen binding domains include, e.g., 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 from the heavy or light chain of an intact antibody.

[0158] In some embodiments, the scFv in the anti-HER3 / MUC1 antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the anti-HER3 / MUC1 scFv has two antigen-binding regions (antigen-binding regions: A and B), and these two antigen-binding regions can bind to their respective target antigens with different affinities.

[0159] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof may comprise one, two, or three selected from Figure 3 and Figure 4 The heavy chain variable region CDRs of

[0160] In some embodiments, the anti-HER3 / MUC1 antibodies described herein can be conjugated to a therapeutic agent.Anti-HER3 / MUC1 antibody drug conjugates comprising an antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethylauristatin E, monomethylauristatin F, cytochalasin B, gramicidin D, ethidium bromide, cephalosporin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunomycin, dihydroxyanthracin, maytansinoids (e.g., DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and the like). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is coupled via a linker (e.g., a VC linker). Details of linkers used in ADCs can be found, for example, in Su, Z. et al., "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0161] In some embodiments, anti-HER3 / MUC1 antibody is a bispecific antibody. Bispecific antibodies can be manufactured by engineering the interface between a pair of antibody molecules, thereby maximizing the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (such as tyrosine or tryptophan). By using smaller amino acid side chains (such as alanine or threonine) to replace larger amino acid side chains, a compensatory "cavity" identical or similar to the larger side chain size is formed on the interface of the second antibody molecule. This provides a mechanism for increasing the output of heterodimers relative to other unwanted end products such as homodimers. This method is described in, for example, WO 96 / 27011, which is incorporated by reference in its entirety.

[0162] Any of the anti-HER3 / 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 of the anti-HER3 / MUC1 antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans), or prolong its biological activity in vitro or in vivo.

[0163] The anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof may also have various formats. Many different formats of bispecific antibodies or antigen-binding fragments thereof are well 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 herein by reference in its entirety.

[0164] In some embodiments, the anti-HER3 / MUCl antibody is a BiTe, (scFv)2, Nanobody, Nanobody-HSA, DART, TandAb, scdiabody, scdiabody-CH3, scFv-CH-CL-scFv, HSAbody, scdiabody-HSA, or tandem-scFv. In some embodiments, the anti-HER3 / MUCl antibody is a VHH-scAb, a VHH-Fab, a bis-scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knob-in-hole common light chain, a knob-in-hole assembly, a charge pair, a Fab-arm exchange, a SEEDbody, LUZ-Y, a Fcab, a kappa lambda-body, an orthogonal Fab, a DVD-IgG, an IgG(H)-scFv, a scFv-(H)IgG, an IgG(L)-scFv, a scFv-(L)IgG, an IgG(L,H)-Fv, an IgG(H)-V, a V(H)-IgG, an IgG(L)-V, a V(L)-IgG, a KIH IgG-scFab, a 2scFv-IgG, an IgG-2scFv, a scFv4-Ig, a Zybody, a DVI-IgG, a diabody-CH3, a triplet 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.

[0165] In some embodiments, the anti-HER3 / MUC1 antibody can be a TrioMab. In a TrioMab, the two heavy chains are derived from different species, where different sequences restrict heavy chain-light chain pairing.

[0166] In some embodiments, the anti-HER3 / MUC1 antibody has two different heavy chains and one common light chain. Heterodimerization of the heavy chains can be based on knobs-in-hole or some other heavy chain pairing technology.

[0167] In some embodiments, CrossMAb technology can be used to generate bispecific anti-HER3 / MUC1 antibodies. CrossMAb technology can be used to enforce correct light chain association in bispecific heterodimeric IgG antibodies, allowing the generation of various bispecific antibody formats (including di(1+1), tri(2+1), and tetra(2+2) valence bispecific antibodies), as well as antibodies based on non-Fc tandem antigen-binding fragments (Fabs). These formats can be derived from any existing antibody pair using domain crossover, without the need to identify a common light chain, post-translational manipulation / in vitro chemical combination, or introduce a set of mutations that enforce 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. [Monoclonal Antibodies] Vol. 8, No. 6. Taylor & Francis, 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 domains in the heavy chain and the CL domains in the light chain are swapped.

[0168] Anti-HER3 / MUC1 antibodies can be duobodies. The Fab exchange mechanism naturally occurring in IgG4 antibodies is mimicked in an IgG1 antibody in a controlled manner, a mechanism known as controlled Fab exchange. This format ensures specific pairing between the heavy and light chains.

[0169] In dual variable domain antibodies (DVD-Ig), additional VH and variable light (VL) domains are added to each N-terminus to achieve bispecific targeting. This format is similar to IgG-scFv, but the added binding domains are individually bound to their respective N-termini, rather than scFv bound to each heavy chain N-terminus.

[0170] 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. The heavy and light chain pairing problem does not exist in scFv-IgG.

[0171] In some embodiments, the anti-HER3 / MUC1 antibody can have an IgG-IgG format, wherein two intact IgG antibodies are coupled by chemically linking the C-termini of the heavy chains.

[0172] Anti-HER3 / MUC1 antibodies can also be produced in a Fab-scFv-Fc format. The Fab-scFv-Fc format combines a light chain, a heavy chain, and a third chain containing an Fc region and scFv. This ensures efficient manufacturing and purification.

[0173] In some embodiments, the anti-HER3 / MUC1 antibody can be TF. Three Fab fragments are linked by disulfide bridges. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF format does not have an Fc region.

[0174] ADAPTIR has two scFvs attached to either side of the Fc region. It abandons the entire IgG as the basis of its construction, but retains the Fc region to extend half-life and facilitate purification.

[0175] Dual affinity retargeting (DART) has two peptide chains connected to opposite segments, so VLA is linked to VHB and VLB is linked to VHA, and they are fused together at their C-termini with a sulfide bond. In DART, the sulfide bond improves stability compared to BiTEs.

[0176] In DART-Fc, the Fc region is attached to the DART structure. It can be produced by combining three chains, two of which are linked by disulfide bonds, like DART. One chain contains half of the Fc region, which will dimerize with the third chain expressing only the Fc region. The addition of the Fc region increases the half-life, maintaining effective concentrations for longer periods of time and avoiding the need for continuous intravenous (IV) administration.

[0177] In a tetravalent DART, four peptide chains are combined. Essentially, two DART molecules are generated, each with half the Fc region, and they dimerize. This form has the ability to bind two targets bivalently, making it a tetravalent molecule.

[0178] Tandem diabodies (TandAbs) consist of two diabodies. Each diabody is composed of a VHA and VLB fragment and a VHA and VLB fragment, covalently linked. The two diabodies are linked by a peptide chain. This improves stability compared to diabodies composed of two scFvs. They possess two bivalent binding sites.

[0179] ScFv-scFv-toxins comprise a toxin and two scFvs with a stabilizing linker, which can be used for specific delivery of payloads.

[0180] In some embodiments, the anti-HER3 / MUC1 antibody is a bispecific antibody. In some embodiments, the bispecific antibodies disclosed herein are designed as 1+1 (monovalent for each target) and have an IgG1 subtype structure. These antibodies can reduce avidity for cells with low HER3 and MUC1 expression levels and increase avidity for cells co-expressing HER3 and MUC1, thereby achieving enhanced targeting.

[0181] In some embodiments, the anti-HER3 / MUCl 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:29; 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:30 and 31.

[0182] In some embodiments, the anti-HER3 / MUC1 antibody comprises a KIH mutation. In some embodiments, the anti-HER3 / MUC1 antibody comprises: a first antigen binding domain that specifically binds to HER3; 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-HER3 / MUCl antibody comprises a knob heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the anti-HER3 / MUCl antibody comprises a hole heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31.

[0183] Antibody characteristics

[0184] An anti-HER3 / MUCl antibody may comprise an anti-HER3 antigen binding domain as described herein and any anti-MUCl antigen binding domain.

[0185] The present disclosure provides anti-HER3 / MUC1 antibodies and antigen-binding fragments thereof that can specifically bind to HER3. These anti-HER3 / MUC1 antibodies can be agonists or antagonists. The anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof described herein can bind to HER3 and block the binding between HER3 and its ligand. By blocking the binding between HER3 and its ligand, the anti-HER3 / MUC1 antibodies can inhibit HER3-related signaling pathways and thereby treat cancer. In some embodiments, the anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof can induce CMC or ADCC.

[0186] The affinity of an antibody for an antigen can be measured using common techniques, including, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be derived from the quotient of kinetic rate constants (KD = koff / kon). In some embodiments, an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof can have a binding affinity of less than 0.1 s. -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 , or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s. -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 , or greater than 0.000001s -1 .

[0187] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms, or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms, or less than 1×10 7 / Ms.

[0188] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof may be less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, or less than 1×10 -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 greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, or greater than 1×10 -10 M.

[0189] The anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof may further comprise an antigen-binding domain that specifically binds to MUC1. The anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof described herein may block the binding between MUC1 and its ligand. In some embodiments, by binding to MUC1, the anti-HER3 / MUC1 antibodies may also inhibit MUC1-related signaling pathways, thereby inhibiting cell proliferation, differentiation, and / or metastasis. Thus, in some embodiments, the anti-HER3 / MUC1 antibodies described herein are MUC1 agonists. In some embodiments, the anti-HER3 / MUC1 antibodies are MUC1 antagonists.

[0190] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof can be less than 0.1s -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 , or less than 0.00001s -1 The dissociation rate (koff) of the antagonist is greater than 0.01 s. -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 , or greater than 0.000001s -1 .

[0191] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms, or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms, or less than 1×10 7 / Ms.

[0192] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, or less than 1×10 -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 greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, or greater than 1×10 -10 M.

[0193] Because anti-HER3 / MUC1 antibodies (e.g., bispecific antibodies) bind to both MUC1 and HER3, for cells expressing both MUC1 and HER3, the antibodies have a higher binding affinity for these cells. Avidity can be used to measure the binding affinity of an antibody to these cells. Avidity is the cumulative strength of the multiple affinities of individual non-covalent binding interactions.

[0194] Thermal stability can also be determined. The anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof described herein can have a Tm 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. Since IgG can be described as a multi-domain protein, a melting curve sometimes shows two transitions, a first denaturation temperature, Tm D1, and a second denaturation temperature, Tm D2. The appearance of these two peaks generally indicates denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When two peaks are present, Tm generally refers to Tm D2. Thus, in some embodiments, an anti-HER3 / MUCl antibody or antigen-binding fragment thereof as described herein has a Tm D1 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, an anti-HER3 / MUCl antibody or antigen-binding fragment thereof as described herein has a Tm D2 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, Tm, TmDl, TmD2 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.

[0195] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof can bind to human HER3 or monkey HER3. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof cannot bind to human HER3 or monkey HER3. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof can bind to human MUC1 or monkey MUC1. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof cannot bind to human MUC1 or monkey MUC1.

[0196] In some embodiments, the anti-HER3 / MUCl antibody, antigen-binding fragment, or ADC has a purity 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%, as measured, for example, 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%, as measured, for example, by HPLC.

[0197] In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has a purity greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, or greater than 98% as determined by size exclusion chromatography (SEC). In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has a hydrophobic interaction chromatography (HIC) retention time greater than 2 minutes, greater than 3 minutes, greater than 4 minutes, or greater than 5 minutes. 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.

[0198] In some embodiments, the anti-HER3 / MUCl antibody, antigen-binding fragment, or ADC has a purity of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% as determined by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).

[0199] In some embodiments, the anti-HER3 / MUCl antibody, antigen-binding fragment, or ADC has a major peak that is greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90% of the total sample as determined by capillary isoelectric focusing (cIEF). In some embodiments, the anti-HER3 / MUCl antibody, antigen-binding fragment, or ADC has an acidic peak that is 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 as determined by capillary isoelectric focusing (cIEF).

[0200] In some embodiments, the anti-HER3 / MUCl 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-HER3 / MUCl antibody, antigen-binding fragment, or ADC has a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. TGI (%) can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the start of treatment. As used herein, tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:

[0201] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0202] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0203] In some embodiments, the anti-HER3 / 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 cell-mediated 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.

[0204] In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof as 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 the LALA mutation (L234A and L235A mutations in EU numbering) or the LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0205] The Fc region can also be modified in some other ways. For example, one or more cysteine residues can be introduced into the Fc region to allow for the formation of interchain disulfide bonds in this region. The resulting homodimeric fusion protein may have an increased half-life in vitro and / or in vivo.

[0206] In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0207] In some embodiments, an Fc region is provided that lacks a carbohydrate structure that is fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an Fc region composition can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. For example, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in the Fc region sequence, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce polysaccharide heterogeneity, the Fc region may be further engineered to replace the asparagine at position 297 with an alanine (N297A).

[0208] In some embodiments, the major peak by HPLC-SEC after purification by Protein A-based affinity chromatography and / or size exclusion chromatography comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% of the protein complex described herein.

[0209] In some embodiments, the anti-HER3 / MUC1 ADCs described herein have an IC50 for killing cancer cells in vitro 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.

[0210] In some embodiments, the bispecific anti-HER3 / MUC1 antibodies described herein have a higher endocytosis rate than a corresponding monoclonal antibody and / or a control bispecific antibody described herein. In some embodiments, the anti-HER3 / MUC1 antibodies described herein have a higher endocytosis rate than a patritumab analog and / or a gatipotuzumab analog.

[0211] Antibody-drug conjugates (ADCs)

[0212] The anti-HER3 / 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-HER3 / MUC1 antibody. In some embodiments, the anti-HER3 / MUC1 antibody is an anti-HER3 / MUC1 bispecific antibody. In some embodiments, the bispecific antibody has a common light chain.

[0213] 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, cephalosporin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunomycin, dihydroxy anthracins, maytansinoids (e.g., DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and the like). Useful classes of cytotoxic, cytostatic, or immunomodulatory agents include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0214] In some embodiments, the therapeutic agent may include, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antimicrobial agent. In some embodiments, the therapeutic agent to be conjugated may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0215] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed., inside front cover, and general definitions of specific functional groups are as described therein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987.

[0216] Unless expressly stated otherwise, all ranges cited herein are inclusive. When a range of values is listed, it is intended to encompass every value and sub-range within that range. For example, "C 1-6 "Aims to cover C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、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 .

[0217] The compounds of the present disclosure or any formula depicting and describing the compounds may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the compounds of the present disclosure or any formula depicting and describing the compounds. Asymmetric centers present in the compounds of the present disclosure or any formula depicting and describing the compounds may each independently have the (R) or (S) configuration. When the bond to a chiral carbon is depicted as a straight line in a structural formula, or when a compound is mentioned in a name without the (R) or (S) chiral symbol for a chiral carbon, it is understood that the (R) and (S) configurations of each such chiral carbon, and therefore each enantiomer or diastereomer and mixtures thereof, are encompassed by the formula or name.

[0218] The present disclosure includes all possible enantiomers and diastereomers, and the mixture of two or more stereoisomers of all ratios, such as the mixture of enantiomers and / or diastereomers.Therefore, the enantiomer of the subject of the present disclosure can be the mixture form of two enantiomers of all ratios, such as enantiomers and / or diastereomers.Therefore, the enantiomer of the subject of the present disclosure can be enantiomerically pure (enantiomerically pure) form (comprising left-handed and right-handed enantiomers), racemate form and the mixture form of two enantiomers of all ratios.With regard to cis / trans isomerism, the present disclosure includes both cis-form and trans-form, and the mixture of these forms of all ratios.If necessary, single stereoisomer can be prepared by conventional method separation mixture, such as by chromatography or crystallization, by using stereochemically uniform synthesis starting material or by stereoselective synthesis.Optionally, derivatization can be carried out before stereoisomer separation. The separation of the mixture of stereoisomers can be carried out in the intermediate step during compound synthesis, and can also be carried out on final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates which have been derivatized, if necessary, with a reagent containing a stereocenter of known configuration. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0219] Unless otherwise indicated, structures depicted herein are also meant 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 by atoms having the same atomic number, but in which the atomic mass or mass number is different from the atomic mass or mass number predominantly found in nature. Such compounds are termed "isotopic variants." This disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds of the invention or any formula depicting and describing the compounds. Examples of isotopes suitable for inclusion in the compounds of the invention include, but are not limited to, isotopes of the following elements: hydrogen, for example2 H (ie D) and 3 H; carbon, e.g. 11 C. 13 C. and 14 C; chlorine, e.g. 36 Cl; fluorine, e.g. 18 F; iodine, e.g. 123 I and 125 I; nitrogen, e.g. 13 N and 15 N; oxygen, e.g. 15 O. 17 O, and 18 O; phosphorus, e.g. 32 P; and sulfur, e.g. 35 Certain isotopic variations of the compounds of the present disclosure or any formula depicting or describing the compounds, such as those incorporating radioactive isotopes, may be useful in drug and / or substrate tissue distribution studies. In particular, compounds having the depicted structures differ only in the substitution of heavier isotopes (e.g., by deuterium). 2 H, or D) instead of hydrogen), these compounds may have certain therapeutic advantages, such as better metabolic stability, increased half-life in vivo, or reduced dosage requirements, and therefore may be used in certain specific environments. Isotopic variations of the compounds disclosed herein or any formula depicting and describing the compounds can generally be prepared by techniques known to those skilled in the art, or by methods analogous to those described in the accompanying examples, and using appropriate isotopically labeled reagents to replace the non-labeled reagents previously employed for synthesis.

[0220] The compounds provided herein are described with reference to both general formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a variety of different forms or derivatives, all of which are within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvate forms, different crystal forms or polymorphs, and active metabolites, etc.

[0221] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acid / base form of a particular compound and is not intended to be biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids. In the case where the compound of the present disclosure contains one or more acidic or basic groups, the disclosure also includes pharmaceutically acceptable salts corresponding to its compound. Therefore, the compound of the present invention containing an acidic group (e.g., a carboxyl group) may exist in salt form and may be used according to the present invention, for example, as an alkali metal salt, an alkaline earth metal salt, an aluminum salt, or as an ammonium salt. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts formed with ammonia or an organic amine (e.g., ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamic acid, or an amino acid). These salts can be easily obtained by reacting a compound with an acidic group with a suitable base (e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide). Other basic salts of the compounds disclosed herein include, but are not limited to, copper (I) salts, copper (II) salts, iron (II) salts, iron (III) salts, manganese (II) salts, and zinc salts. The compounds disclosed herein containing one or more basic groups (e.g., protonatable groups) can exist in salt form and can be used in the form of addition salts with inorganic or organic acids according to the present disclosure. 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, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, as well as others known to those skilled in the art.The salt formed includes but is not limited to: hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), toluenesulfonate, carbonate, bicarbonate, formates, acetate, sulfoacetate, trifluoromethanesulfonate, oxalate, malonate, maleate, succinate, tartrate, malate, embonate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate and glutamate. In addition, the stoichiometry of the salt formed by the compound of the present disclosure can be an integer multiple or non-integer multiple of one.

[0222] The compounds containing basic nitrogen groups disclosed herein can be used, for example: 1-4 Alkyl halides (such as methyl, ethyl, isopropyl, and tert-butyl chlorides, bromides, and iodides); di-C 1-4 Alkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, and diamyl sulfate); C 10-18 Alkyl halides (e.g., chlorides, bromides, and iodides of decyl, dodecyl, lauryl, myristyl, and stearyl); and aryl C 1-4 The quaternization is carried out using reagents that are alkyl halides such as phenyl chloride and phenethyl bromide.

[0223] If the compound of the present disclosure contains both acidic and basic groups in the molecule, then in addition to the salt forms mentioned, the present disclosure also includes inner salts or betaines (zwitterions). The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting such compounds with organic acids / bases or inorganic acids / bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present disclosure also includes all of the compounds of the present disclosure that are not suitable for direct use in medicine due to low physiological compatibility, but can be used as, for example, intermediates in chemical reactions or for preparing 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).

[0224] The compounds of the present disclosure or any formula depicting and describing the compounds, and pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. As used herein, the term "solvate" refers to a molecular complex comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules. For example, when the solvent is water, the term "hydrate" is used.

[0225] Pharmaceutically acceptable solvates according to the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, such as D2O, d6-acetone, d6-DMSO.

[0226] Linker (Linker Compound)

[0227] In some embodiments, the therapeutic agent is coupled via a linker (or linker 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) to a therapeutic agent (e.g., any of the therapeutic agents described herein), which reacts (e.g., via a coupling reaction) with groups of the ligand compound and the therapeutic compound, respectively, to form a ligand drug conjugate.

[0228] In some embodiments, the linker described herein is a compound having the formula:

[0229] QL

[0230] Formula (I),

[0231] or a pharmaceutically acceptable salt, solvate, stereoisomer or isotopic variant thereof, wherein Q represents a conjugating portion capable of being coupled to the ligand via a bond selected from the group consisting of a carbonyl bond, a thioether bond, an amide bond, a disulfide bond, and a hydrazone bond; and L represents a linker portion capable of connecting Q to a therapeutic agent.

[0232] In some embodiments, the binding moiety (Q in formula (I)) has the following structure:

[0233]

[0234] In some embodiments, the linker moiety (L in Formula (I)) has the formula:

[0235]

[0236] wherein L1 is a polypeptide residue consisting of three to eight amino acid residues, wherein the amino acid residues include at least one amino acid residue having a side chain carboxyl group, such as a glutamic acid residue or an aspartic acid residue, wherein "-COOH" represents the carboxyl group of the amino acid residue located at the C-terminus of the polypeptide residue;

[0237] L2 is absent or is a monodentate, bidentate, or tridentate hydrophilic group attached to the side chain carboxyl group of the amino acid residue of polypeptide residue L1, and L2 has -NHC(R L2a )(R L2b )(RL2c ) structure, where R L2a 、R L2b , and R L2c Each independently selected from H, -(CH2O)(CH2CH2O) m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d The group composed of R L2d is H or C optionally substituted with 1 to 6 hydroxyl groups 1-6 Alkyl, each m independently represents an integer from 0 to 10, preferably from 0 to 4 (e.g., 0, 1, 2, 3, or 4), particularly preferably m is 0, and each p independently represents an integer from 1 to 4 (e.g., 1, 2, 3, or 4); and

[0238] represents the N-terminal side of the polypeptide residue covalently attached to the binding moiety Q.

[0239] In some embodiments, polypeptide residue L1 is NH -Glu-Val-Ala- COOH In some embodiments, the hydrophilic group L2 has the following structure:

[0240]

[0241] Wherein "*" indicates the site of covalent attachment to polypeptide residue L1, e.g. NH -Glu-Val-Ala- COOH The side chain of the Glu residue in .

[0242] In some embodiments, the linker described herein is a compound having the following structure:

[0243]

[0244] In some embodiments, the linker is a VC linker. Details of linkers used in ADCs can be found, for example, in Su, Z. et al., "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0245] therapeutic agents

[0246] In some embodiments, the therapeutic agent conjugated to the antibodies or antigen-binding fragments thereof described herein is as discussed below.

[0247] In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, an analog or derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the following structure:

[0248]

[0249] 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.

[0250] 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:

[0251]

[0252] 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:

[0253]

[0254] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below:

[0255]

[0256] 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:

[0257]

[0258] 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 ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and architecture of exemplary auristatins are described in U.S. Patent Application Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957; International Patent Publication No. WO 02 / 088172; and 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, 4,816,444; and 4,486,414, each of which is incorporated herein by reference in its entirety and for all purposes.

[0259] Studies have shown that auristatins can interfere with microtubule dynamics, as well as nuclear and cell division, and have anticancer activity. Auristatins bind to tubulin and can produce cytotoxic or cytostatic effects on cancer cells. A variety of different assays are known in the art and can be used to determine whether auristatins or the resulting antibody-drug conjugates produce cytostatic or cytotoxic effects on the desired cells.

[0260] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN TM)); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trihydroxymethyl trimethylolomelamine; nitrogen mustards, for example, chiorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosoureas, for example, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomycins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopte rin), methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens (e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); antiadreners (e.g., aminoglutethimide, mitotane, trilostane); folic acid supplements (e.g., folinic acid); acetylgluconolactone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2',2',2'-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel (; Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France; chlorambucil; gemcitabine; 6-thioguanine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone one); teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any one of the foregoing. This definition also includes antihormonal agents for regulating or inhibiting the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibition 4 (5) -imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);And antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;With any of the above-mentioned pharmaceutically acceptable salts, acids or derivatives. A detailed description of chemotherapeutic agents can be found in, for example, US20180193477 A1, which is incorporated by reference in its entirety.

[0261] Linker therapeutic compounds

[0262] 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.

[0263] In some embodiments, the linker therapeutic compound has the structure:

[0264]

[0265] In some embodiments, the linker therapeutic compound has the structure:

[0266]

[0267] In some embodiments, an antibody ("Ab") (e.g., any of the antibodies or antigen-binding fragments thereof described herein) can be linked to a linker therapeutic compound (e.g., any of the linker therapeutic compounds described herein) to produce an antibody drug conjugate. In some embodiments, the antibody drug conjugate has the following structure:

[0268]

[0269] 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, 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 one.

[0270] In some embodiments, the anti-HER3 / MUC1 antibody is conjugated to the drug via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the anti-HER3 / MUC1 antibody is conjugated to the drug via a non-cleavable linker, such as an MCC linker formed using SMCC or sulfo-SMCC. One skilled in the art can readily select an appropriate linker for a given ADC, taking into account factors such as the site of attachment of the anti-HER3 / MUC1 antibody, any structural constraints on the drug, and the hydrophobicity of the drug (see, e.g., a review by Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer). In certain embodiments, a variety of specific linker-toxin combinations have been described and can be used with the anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof described herein to prepare ADCs. Examples include, but are not limited to, peptide-based cleavable linkers with auristatins (e.g., MMAE and MMAF), camptothecins (e.g., SN-38), duocarmycins, and PBD dimers; MC-based non-cleavable linkers with auristatins MMAF and MMAE; hydrazone-based acid-labile linkers with calicheamicin and doxorubicin; disulfide-based linkers with maytansines (e.g., DM1 and DM4); and bis-maleimide-trioxyethylene glycol (BMPEO)-based linkers with maytansine DM1. Some such therapeutic agents and linkers are described, for example, in Peters and Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Publication Nos. US2015 / 0374847 and US20180193477 A1; which are incorporated herein by reference in their entirety.

[0271] Depending on the desired drug and the selected linker, one skilled in the art can select an appropriate method to couple them together. For example, some conventional coupling methods (e.g., amine coupling methods) can be used to form the desired drug-linker complex, which still contains a reactive group for conjugating to an anti-HER3 / MUC1 antibody or antigen-binding fragment thereof via a covalent bond. In some embodiments, a drug-maleimide complex (i.e., a maleimide-linked drug) can be used for payloads with reactive groups as disclosed herein. The most common reactive group that can be bonded to a thiol group in ADC preparation is maleimide. In addition, organic bromides and iodides are also frequently used.

[0272] Anti-HER3 / MUC1 ADCs can be prepared by one of several routes known in the art using organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, e.g., Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). For example, conjugation can be achieved by: (1) reacting a nucleophilic or electrophilic group of an antibody with a divalent linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, which is then reacted with an activated drug moiety D; or (2) reacting a nucleophilic or electrophilic group of a drug moiety with a linker reagent to form a drug-linker intermediate DL via a covalent bond, which is then reacted with a nucleophilic or electrophilic group of an antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the anti-HER3 / MUC1 ADCs described herein. Various prepared linkers, linker compounds, and toxins are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith and March, 2006, 6th ed., Wiley); Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press); US20210379193 A1 and US20180193477 A1, which are incorporated herein by reference in their entirety. In addition, a variety of preformed drug-linkers suitable for reaction with the selected anti-HER3 / MUC1 antibody or antigen-binding fragment are also commercially available. For example, linker-toxins comprising DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative BioLabs (Shirley, NY).

[0273] Several specific examples of methods for preparing anti-HER3 / MUC1 ADCs are known in the art and are described in U.S. Pat. No. 8,624,003 (pot method), U.S. Pat. No. 8,163,888 (one-step method), and U.S. Pat. No. 5,208,020 (two-step method), and US20180193477 A1, which are incorporated herein by reference in their entirety. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer.

[0274] Drug loading is represented by the number of drug moieties per antibody in the ADC molecule. For some antibody drug conjugates, drug loading may be limited by the number of attachment sites on the antibody. For example, when the attachment moiety is cysteine thiol, as in certain exemplary embodiments described herein, the drug loading per antibody may range from 0 to 8 drug moieties. In certain embodiments, higher drug loadings, such as p≥5, may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates. In certain embodiments, the average drug loading of anti-HER3 / 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, studies have shown that for certain antibody drug conjugates, the most preferred ratio of drug moieties per antibody is about 4. In some embodiments, the DAR of the anti-HER3 / MUC1 ADC composition is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the anti-HER3 / MUCl 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.

[0275] In some embodiments, provided anti-HER3 / MUC1 antibody variants have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. For example, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain located at Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce polysaccharide heterogeneity, the Fc region of the anti-HER3 / MUC1 antibody may be further engineered to replace the asparagine at position 297 with an alanine (N297A).

[0276] In some embodiments, to improve production efficiency by avoiding Fab arm exchange, the Fc region of the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). For a detailed description of the S228 mutation, see, for example, 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.

[0277] In some embodiments, the methods described herein are designed to manufacture bispecific anti-HER3 / MUC1 antibodies. Bispecific anti-HER3 / MUC1 antibodies can be manufactured by engineering the interface between a pair of antibody molecules, thereby maximizing the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is formed on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products such as homodimers. This method is described in, for example, WO 96 / 27011, which is incorporated by reference in its entirety.

[0278] In some embodiments, knob-into-hole (KIH) technology can be used, which involves engineering the CH3 domain to create a "knob" or "hole" 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, which is 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 following substitutions: Y349C and T366W (EU numbering). The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A, and Y407V (EU numbering). In addition, a substitution (-ppcpScp-->-ppcpPcp-) may be introduced in the hinge region of both substituted IgGs.

[0279] recombinant vector

[0280] The present disclosure also provides: a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein); a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains the polynucleotide and / or a vector comprising the polynucleotide); and producing the anti-HER3 / MUC1 antibody polypeptide or fragment thereof by recombinant technology.

[0281] 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 one or more polynucleotides of interest and expressing them as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is operably linked to a regulatory element (e.g., a promoter, enhancer, and / or poly-A tail) at or near or flanking the integration site (integration site) of the polynucleotide of interest within the vector or in the host cell genome, thereby being positioned in the vector for expression, thereby causing the polynucleotide of interest to be translated in the host cell into which the expression vector has been introduced.

[0282] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Therefore, non-limiting examples of vectors include: viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0283] 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., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses. In the latter case, viral propagation typically occurs only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld 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. The 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 can be efficiently transported into cells.

[0284] To facilitate expression, a DNA insert comprising a polynucleotide encoding a polypeptide disclosed herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage λ PL promoter, E. coli lac, trp, and tac promoters, SV40 early-onset and late-onset promoters, and promoters of retroviral LTRs. Other suitable promoters are well known to those skilled in the art. The expression construct may further contain sites for transcription initiation and termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation initiation site at the beginning of the polypeptide to be translated, and a stop codon (UAA, UGA, or UAG) at an appropriate position at the end.

[0285] As described above, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for culture in eukaryotic cells, and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative embodiments of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as fruit fly S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK293 cells; and plant cells. Culture media and conditions suitable for the host cells described herein are well known in the art.

[0286] Non-limiting bacterial vectors 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, 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 skilled in the art.

[0287] Non-limiting bacterial promoters suitable for use include: E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters, and trp promoter. Suitable eukaryotic promoters include: CMV immediate early-onset promoter, HSV thymidine kinase promoter, early-onset and late-onset SV40 promoters, promoters of retroviral LTRs (e.g., Rous sarcoma virus (RSV) promoter), and metallothionein promoters (e.g., mouse metallothionein-I promoter).

[0288] In Saccharomyces cerevisiae, a variety of vectors containing constitutive or inducible promoters (e.g., alpha factor, alcohol oxidase, and PGH) are available. For details, 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).

[0289] 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. Such methods are described in many standard laboratory manuals, for example, Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0290] Transcription of the DNA encoding the anti-HER3 / MUC1 antibodies of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, typically about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include: the SV40 enhancer located on the late side of the replication origin, between base pairs 100 and 270; the cytomegalovirus early-acting promoter enhancer; the polyoma enhancer located on the late side of the replication origin; and adenovirus enhancers.

[0291] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide, or such signal may be a heterologous signal.

[0292] Polypeptides (e.g., anti-HER3 / MUC1 antibodies) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion protein) or with a histidine tag, and can include not only a secretion signal 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 the polypeptide to improve stability and persistence in the host cell during purification or subsequent processing and storage. In addition, a peptide portion can be added to the polypeptide to facilitate purification. Such a region can be removed prior to the final preparation of the polypeptide. Adding a peptide portion to a polypeptide to induce secretion or excretion, thereby improving stability and facilitating purification, etc., are all conventional techniques well known in the art.

[0293] 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. The present invention also provides the amino acid sequences of the present invention, and 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.

[0294] 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. The present invention also provides the amino acid sequence of 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% homologous to any of the amino acid sequences described herein.

[0295] 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 as 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.

[0296] 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.

[0297] 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.

[0298] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for most preferably comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid / nucleic acid sequences for most preferably alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When 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 identical at that position (as used herein, "identity" of amino acids or nucleic acids is equivalent to "homology" of amino acids or nucleic acids). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percent identity between the two sequences can be accomplished using the Blossum 62 scoring matrix, where the gap penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5.

[0299] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. Methods for determining the percentage of sequence homology are well known in the art. In some embodiments, conserved amino acid residues (e.g., leucine and isoleucine) with similar physicochemical properties (homology percentage) 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), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, aspartyl acid, glutamic acid, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0300] The present disclosure provides one or more nucleic acids encoding any one of the polypeptides as described herein. In some embodiments, nucleic acids (e.g., cDNA) comprise polynucleotides encoding heavy chain polypeptides as described herein. In some embodiments, nucleic acids comprise polynucleotides encoding light chain polypeptides as described herein. In some embodiments, nucleic acids comprise polynucleotides encoding scFv polypeptides as described herein.

[0301] In some embodiments, a vector may have two of the nucleic acids described herein, wherein the vectors encode a VL region and a VH region that together bind to HER3. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, wherein the pair of vectors together encode a VL region and a VH region that together bind to HER3.

[0302] 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 together bind to MUC1. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, wherein the pair of vectors together encode a VL region and a VH region that together bind to MUC1.

[0303] Treatment

[0304] The methods described herein include methods for treating cancer-related conditions. Generally, these methods comprise administering to a subject in need or identified as needing such treatment a therapeutically effective amount of an anti-HER3 / MUC1 antibody or anti-HER3 / MUC1 antibody drug conjugate as described herein.

[0305] As used herein, "treat" refers to ameliorating at least one symptom of a cancer-related condition. Cancer typically results in death, and thus, treatment can result in an increase in life expectancy (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years). Administration of a therapeutically effective amount of an agent described herein for treating a cancer-related condition will result in a decrease in the number of cancer cells and / or alleviation of symptoms.

[0306] As used herein, the term "cancer" refers to cells with the ability to grow autonomously, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. This term is meant to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasion stage. As used herein, the term "tumor" refers to a cancer cell, such as a group of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include: malignant tumors of various organ systems (e.g., affecting the lungs, breasts, thyroid, lymphatics, gastrointestinal tract, and genitourinary tract); and adenocarcinomas including malignant tumors (e.g., most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell carcinomas of the lungs, cancers of the small intestine, and cancers of the esophagus). In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. This term also includes carcinosarcoma, which, for example, includes a malignant tumor composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue, or a cancer in which tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from the stroma.

[0307] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0308] In one aspect, the present disclosure also provides methods for treating cancer in a subject, methods for reducing the rate at which a subject's tumor volume increases over time, methods for reducing the risk of developing metastasis, or methods for reducing the risk of a subject developing other metastases. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a decrease in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0309] In one aspect, the disclosure features methods comprising administering a therapeutically effective amount of an anti-HER3 / MUC1 antibody or anti-HER3 / MUC1 antibody drug conjugate disclosed herein to a subject in need thereof, e.g., having, or identified or diagnosed as having, a cancer, e.g., 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 oesophageal cancer. In some embodiments, the cancer is esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial cancer, lung cancer, melanoma, ovarian cancer, bladder cancer, gastric cancer, non-Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, and cervical cancer.

[0310] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal (human or non-human) that is treated according to the methods of the present invention. The present invention contemplates both veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., people under the age of 18). In addition to humans, patients also include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Including, for example: non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcine (e.g., pigs, miniature pigs), equines, canines, felines, bovines, and other domesticated animals, farm animals, and zoo animals.

[0311] 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 using various methods known in the art.

[0312] As used herein, "effective amount" refers to an amount or dosage sufficient to produce a beneficial or desired result, including stopping, slowing, delaying, or inhibiting the progression of a disease (e.g., cancer). The effective amount will vary depending on factors such as the age and weight of the subject to whom the anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, anti-HER3 / MUC1 antibody-drug conjugate, polynucleotide encoding an anti-HER3 / MUC1 antibody, vector comprising the polynucleotide, and / or composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus can be determined based on the subject's condition.

[0313] An effective amount can be administered in one or more administrations. For example, an effective amount of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 antibody drug conjugate is an amount sufficient to improve, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to improve, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As understood in the art, the effective amount of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 antibody drug conjugate can vary depending on factors including, but not limited to, the patient's medical history and other factors, such as the type (and / or dose) of the agent used.

[0314] The effective amount and time course of administration of the anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments thereof, polynucleotides encoding anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antibody drug conjugates, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. One skilled in the art will appreciate that the dosage that must be administered will vary depending on, for example: the mammal to which the anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments thereof, polynucleotides encoding anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antibody drug conjugates, and / or compositions disclosed herein will be administered; the route of administration; the specific type of agent or composition disclosed herein used; and other drugs administered to the mammal.

[0315] A typical daily dose of an effective amount of an anti-HER3 / MUC1 antibody or anti-HER3 / MUC1 ADC is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may 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 may 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.

[0316] In any of the methods described herein, at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment thereof, anti-HER3 / MUC1 antibody drug conjugate, or pharmaceutical composition (e.g., comprising any of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 ADC) and optionally at least one additional therapeutic agent can be administered to the subject (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0317] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, anti-HER3 / MUC1 antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 ADC). In some embodiments, one or more additional therapeutic agents are administered to a subject along with at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 antibody-drug conjugate, such that the period of biological activity of the one or more additional therapeutic agents and the at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 ADC in the subject overlaps.

[0318] In some embodiments, at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, anti-HER3 / MUC1 antibody drug conjugate, or pharmaceutical composition (e.g., comprising any of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 ADC) may be administered to a subject 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 a treatment cycle using any of the methods described herein to diagnose or monitor the effectiveness of treatment (e.g., to observe at least one symptom of cancer). As described herein, a skilled medical professional can also change the identity and amount (e.g., increase or decrease) of the anti-HER3 / MUC1 antibody, or anti-HER3 / MUC1 antigen-binding antibody fragment, anti-HER3 / MUC1 antibody drug conjugate (and / or one or more other therapeutic agents) administered to a subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art), and can also adjust (e.g., increase or decrease) the dose or frequency of at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 ADC (and / or one or more other therapeutic agents) administered to a subject.

[0319] In some embodiments, one or more other therapeutic agents may be administered to the subject. The other therapeutic agent may include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, HER3, MEK, ERK, K-Ras, c-Met, MUC1, anaplastic lymphoma kinase (ALK), phosphatidylinositol 3-kinase (PI3K), Akt, mTOR, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK), and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the other therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0320] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of activated hedgehog signaling pathways, and agents that selectively degrade estrogen receptors.

[0321] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, nab-paclitaxel ... afenib), Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0322] In some embodiments, the other therapeutic agents may include one or more therapeutic agents selected from the group consisting of adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0323] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0324] 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-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0325] Pharmaceutical compositions and routes of administration

[0326] Also provided herein are pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) anti-HER3 / MUC1 antibody (e.g., a bispecific antibody), anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 antibody-drug conjugate described herein. The pharmaceutical compositions can be formulated by any method known in the art.

[0327] The pharmaceutical composition is formulated to be compatible with the intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may contain: a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial or antifungal agent (e.g., benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.); an antioxidant (e.g., ascorbic acid or sodium bisulfite); a chelating agent (e.g., ethylenediaminetetraacetic acid); a buffer (e.g., acetate, citrate, or phosphate); and an isotonic agent. agent) (e.g., a sugar (e.g., dextrose), a polyol (e.g., mannitol or sorbitol); or a salt (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 formulations of the composition can be formulated and packaged in ampoules, disposable syringes, or multiple-dose vials. When necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin) or a surfactant. The absorption of the anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment thereof, or anti-HER3 / MUC1 ADC can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled 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 [Alza Corporation] and Nova Pharmaceuticals, Inc. [Nova Pharmaceuticals Co., Ltd.]).

[0328] Compositions containing one or more of any of the anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments, and anti-HER3 / 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 to ensure uniformity of administration and dosage).

[0329] The toxicity and therapeutic efficacy of the 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 the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. When an agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0330] Data obtained from cell culture assays and animal studies can be used to formulate an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of an anti-HER3 / MUC1 antibody, an anti-HER3 / MUC1 antigen-binding fragment thereof, or an anti-HER3 / MUC1 ADC is an amount that treats the disease (e.g., kills cancer cells) in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who previously developed cancer but is now cured), and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The effectiveness and dosing of any of the anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments thereof, or anti-HER3 / MUC1 ADCs described herein can be determined by a healthcare professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors may affect the dose and duration required to effectively treat a subject (e.g., the severity of the disease or condition, previous treatments, the overall health and / or age of the subject, and the presence of other diseases).

[0331] Exemplary dosages include milligram or microgram amounts of any of the anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments thereof, or anti-HER3 / 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 dosages encompass a broad range, those skilled in the art will understand that the potency of therapeutic agents varies and that effective amounts can be determined by methods known in the art. Generally, a relatively low dose is administered initially, and the attending medical professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development phase) can then gradually increase the dose until an appropriate response is obtained. Furthermore, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including: the activity of the specific compound employed, the age, weight, general health, sex, and diet of the subject, the time of administration, route of administration, rate of excretion, and in vivo half-life of the therapeutic agent.

[0332] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments thereof, or anti-HER3 / MUC1 ADCs for the various uses as described herein.

[0333] Example

[0334] The present invention is further illustrated in the following examples, which do not limit the scope of the invention described in the claims.

[0335] Example 1. Preparation of anti-HER3 / MUC1 bispecific antibody

[0336] Provided herein are bispecific antigen-binding molecules that target HER3 and MUC1. Hereinafter, these antigen-binding molecules are referred to as anti-HER3 / MUC1 bispecific antibodies.

[0337] Anti-HER3 antibodies (1B2, VH SEQ ID NO: 26, VL SEQ ID NO: 25; 3E1, VH SEQ ID NO: 27, VL SEQ ID NO: 25; and 3G6, VH SEQ ID NO: 45, VL SEQ ID NO: 25) and anti-MUC1 antibodies (10D1, VH SEQ ID NO: 28, VL SEQ ID NO: 25) can be paired to form bispecific antibodies. Vectors encoding the light and heavy chains of the antibodies were constructed. CHO-S cells were co-transfected with three vectors, including a first vector encoding the heavy chain of the anti-HER3 antibody, a second vector encoding the heavy chain of the anti-MUC1 antibody, and a third vector encoding the common light chain. After 14 days of culture, the cell supernatant was collected and purified by protein A affinity chromatography. Exemplary bispecific antibodies obtained include 3E1-10D1, 3G6-10D1, and 1B2-10D1.

[0338] To verify the binding affinity of the bispecific antibodies, an anti-HER3 control bispecific antibody and an anti-MUC1 control bispecific antibody were also generated, where one arm of the control bispecific antibody would recognize HER3 or MUC1 and the other arm would recognize CD28. These control bispecific antibodies were generated using similar methods, for example, by incorporating RenLite TM Mice were immunized to obtain VH sequences. Exemplary control bispecific antibodies were designated 3E1-CD28, 3G6-CD28, 1B2-CD28, and 10D1-CD28.

[0339] A variety of methods can be used to reduce the chance of mispairing between the two heavy chains. For example, a knob-to-hole mutation is introduced into the Fc region of the anti-HER3 arm heavy chain, the anti-MUC1 arm heavy chain, and the anti-CD28 arm heavy chain. For example, in 3E1-10D1, the heavy chain constant region of 3E1 includes a knob mutation, and the heavy chain constant region of 10D1 includes a hole mutation. In 3E1-CD28, the heavy chain constant region of 3E1 includes a knob mutation, and the heavy chain constant region of CD28 includes a hole mutation.

[0340] The sequences of the light chain constant region, the heavy chain constant region with a knob mutation, and the heavy chain constant region with a hole mutation are shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[0341] Example 2. Species Cross-Binding Activity of Anti-HER3 / MUC1 Bispecific Antibodies

[0342] CHO-hHER3 cells, CHO-fasHER3 cells, H293F-hMUC1 cells, CHO-fasMUC1 cells, NUGC-4 cells (Cobioer, catalog number: CBP60493), or HCC827 cells (ATCC, catalog number: CRL-2868) were cultured at a medium temperature of 5×10 4 Cells were transferred to a 96-well plate at a density of 100 cells / well. Anti-HER3 / MUC1 bispecific antibodies were added to the 96-well plate and incubated at 4°C for 30 minutes. The cells were then incubated with a secondary antibody, anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170), at 4°C in the dark for 15 minutes before flow cytometric analysis.

[0343] CHO-hHER3 cells, CHO-fasHER3 cells, and CHO-fasMUC1 cells were generated by transfecting CHO-S cells with vectors expressing human HER3 (hHER3, SEQ ID NO: 32), monkey (Macaca fascicularis) HER3 (fasHER3, SEQ ID NO: 33), and monkey (Macaca fascicularis) MUC1 (fasMUC1, SEQ ID NO: 35), respectively. H293F-hMUC1 cells were generated by transfecting H293F cells with a vector expressing human MUC1 (hMUC1, positions 961 to 1152 of SEQ ID NO: 34). RNA sequencing analysis showed that the expression levels of HER3 and MUC1 in NUGC-4 cells were 155 and 79, respectively, while their expression levels in HCC827 cells were 27 and 41, respectively.

[0344] Gatuzumab is a glycoengineered, humanized monoclonal antibody that recognizes a tumor-associated epitope on MUC1. It is currently in Phase II clinical development at Glycotope for the treatment of recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer. The heavy and light chain variable regions of Gatuzumab are set forth in SEQ ID NO:36 and SEQ ID NO:37, respectively.

[0345] The test results are shown in the table below: 3E1-10D1, 3G6-10D1, and 1B2-10D1 can bind to human HER3, monkey HER3, human MUC1, and monkey MUC1.

[0346] Table 1

[0347]

[0348]

[0349] In another experiment, the binding activity of anti-HER3 / MUC1 bispecific antibody to tumor cells NUGC-4 or NCI-H226 was measured by flow cytometry. The secondary antibodies used in the experiment were: Alexa 647 anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170). Human IgG1 was used as an isotype control (ISO). Mean fluorescence intensity (MFI) was determined using serially diluted sample antibodies (maximum concentration: 30 μg / mL, 3-fold dilution). A fitted curve was obtained using concentration (μg / mL) as the X-axis and MFI as the Y-axis. The results are shown in Figures 17A to 17B.

[0350] Example 3. Internalization of anti-HER3 / MUC1 bispecific antibodies

[0351] Anti-HER3 antibodies, anti-MUC1 antibodies, anti-HER3 / MUC1 bispecific antibodies, anti-HER3 / CD28 bispecific antibodies, or anti-MUC1 / CD28 bispecific antibodies were added to HCC70 cells (ATCC, catalog number: CRL-2315) along with pHAb-AffiniPure Fab goat anti-human IgG secondary antibody and incubated for 6 hours. The cells were centrifuged and washed with FACS buffer. The MFI was measured using a flow cytometer. The endocytosis rate of the antibodies was calculated. Human IgG1 protein was used as an isotype control (ISO). The results are shown in the table below.

[0352] RNA sequencing analysis showed that the expression levels of HER3 and MUC1 in HCC70 cells were 19 and 110, respectively.

[0353] Pertratuzumab is an anti-HER3 fully human monoclonal antibody developed by Daiichi Sankyo Co., Ltd., and its heavy chain variable region and light chain variable region are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0354] Table 2

[0355]

[0356] The results showed that the endocytosis rates of the bispecific antibodies 3E1-10D1, 3G6-10D1, and 1B2-10D1 in HCC70 cells were equal to or higher than those of the corresponding monoclonal antibodies 3E1, 3G6, 1B2, or 10D1.

[0357] Example 4. Binding activity of anti-HER3 / MUC1 bispecific antibodies

[0358] Using Biacore TM The binding activity of the anti-HER3 / MUC1 bispecific antibody to human HER3, human MUC1, monkey HER3, and monkey MUC1 was verified by surface plasmon resonance (SPR) (Biacore, Piscataway, NJ). The 8K biosensor was equipped with a pre-immobilized Protein A sensor chip.

[0359] Specifically, hHER3-His (ACRO Biosystems Inc., catalog number: ER3-H5223), hMUC1 (24-1158)-His (positions 14 to 1158 of SEQ ID NO: 34), hMUC1 (961-1152)-His (positions 961 to 1152 of SEQ ID NO: 34), fasHER3-His (Sino Biological, Inc., catalog number: 90043-K08H), and fasMUC1-His (SEQ ID NO: 35) were diluted to 200 nM using 1× HBS-EP+ buffer (pH 7.4) and then injected into the Biacore at 10 μL / min. TM The 8K biosensor was titrated for approximately 50 seconds to reach the desired protein density (e.g., approximately 200 response units (RU)). Subsequently, purified antibody at a concentration of 2 μg / ml 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 using glycine solution (pH 1.5) at 30 μL / min for 30 seconds.

[0360] Using Biacore TM 8K Evaluation Software 3.0 fitted the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110), thereby simultaneously obtaining kinetic association rates (k) and dissociation rates (k). Affinity was derived from the quotient of the kinetic rate constants (K = k / k).

[0361] As will be understood by those skilled in the art, the same method is performed for each antibody tested, with appropriate adjustments made to parameters (eg, antibody concentration).

[0362] The results for the tested antibodies are summarized in the table below, which shows that the anti-HER3 / MUCl bispecific antibodies 3E1-10D1, 3G6-10D1, and 1B2-10D1 have good binding affinity to human HER3, monkey HER3, human MUCl, and monkey MUCl.

[0363] Table 3

[0364]

[0365] Example 5. Stability of anti-HER3 / MUC1 bispecific antibody

[0366] The anti-HER3 / MUC1 bispecific antibodies 3E1-10D1 and 1B2-10D1 were buffer-exchanged to pH 6.0 (3 mg / ml histidine, 80 mg / ml sucrose, and 0.2 mg / ml Tween 80). The antibodies were placed in sealed Eppendorf tubes and stored at 40 ± 2 ° C, 60% ± 5% RH, and 4 ± 3 ° C for 7 days, and their thermal stability was evaluated. Alternatively, the bispecific antibodies were loaded onto a protein A column and washed with buffer (0.1 mol / L HAc) at pH 3.5. Half of the antibody was immediately added to 2M Tris buffer to bring the pH to 7.5. The remaining half was stored at pH 3.5 for 6 hours, and then the pH was adjusted to 7.5. The diluted antibodies were placed in sealed Eppendorf tubes and stored at pH 3.5±0.1, 25±2°C (hereinafter referred to as pH 3.5) for 6 hours or 24 hours to test the stability at low pH.

[0367] After the above treatment, the following tests were performed: (1) the purity of the antibody was measured by size exclusion high performance liquid chromatography (SEC-HPLC) (expressed as the percentage of the main peak area to the total area of all peaks (purity, %)); (2) the hydrophobicity of the antibody was measured by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (expressed as the retention time of the main peak (HIC, min)); (3) the pI (isoelectric point) and charge variant of the antibody were measured by capillary isoelectric focusing (cIEF) (expressed as the percentage of the main component, acidic component, and basic component); (4) the purity change of the antibody was measured by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method (CE-SDS (NR)) under non-reducing conditions (expressed as the percentage of the main peak area to the total area of all peaks (purity, %)); (5) whether there was the appearance and presence of visible insoluble particles.

[0368] In the SEC-HPLC experiment, the antibody sample was diluted to 1 mg / mL with purified water and HPLC was performed using an Agilent 1290 chromatography system (connected to an XBridge Protein BEH SEC column ( Waters Corporation). The following parameters were used: mobile phase: 0.1 M phosphate buffer (PB) + 10% ACN, pH 7.4; flow rate: 1.8 ml / min; column temperature: 25° C.; detection wavelengths: 280 nm, 220 nm; injection volume: 10 μL; sample plate temperature: approximately 8° C.; and run time: 7 minutes.

[0369] In the HIC-HPLC experiments, an Agilent 1260 chromatography system (connected to a ProPac HIC-10 column (4.6×250 mm, Thermo Scientific) was used, and the samples were diluted 10-fold by using mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile, pH 6.5; mobile phase B: 0.1 M phosphate buffer (PB), 10% acetonitrile, pH 6.5; flow rate: 0.8 ml / min; gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30° C.; detection wavelengths: 280 nm, 220 nm; injection volume: 10 μg; sample plate temperature: approximately 10° C.; and run time: 50 min.

[0370] In the cIEF experiment, the Maurice cIEF Method Development Kit (ProteinSimple, catalog number: PS-MDK01-C) was used for sample preparation. Specifically, 8 μL of 30 ug protein sample was mixed with the following reagents in the kit: 1 μL of Maurice cIEF pI Marker-7.05, 1 μL of Maurice cIEF pI Marker-10.10, 35 μL of 1% methylcellulose solution, 2 μL of Maurice cIEF 500 mM arginine, 1.33 μL of ampholytes (Pharmalyte pH range 3 to 10), 6.66 μL of ampholytes (Pharmalyte pH range 8 to 10.5), and water (added to make the final volume 100 μL). Imaging capillary isoelectric focusing spectra were generated on a Maurice analyzer (Protein Simple, Santa Clara, CA) using Maurice cIEF Cartridges (PS-MC02-C). The sample was focused for a total of 10 minutes. The absorbance of the focused protein at 280 nm was integrated using the analysis software installed on the instrument.

[0371] In CE-SDS (NR) experiments, Maurice (Protein simple, Maurice TM ) and Maurice CE-SDS Size Application kit (Protein simple, catalog number: PS-MAK02-S). In CE-SDS (NR), 30 μL sample buffer, 30 μL 30ug antibody sample, 1.5 μL 25x internal standard, 3 μL 250nM iodoacetamide (SIGMA, catalog number: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 min and heating in a 70°C water bath for 10 min. The sample was then cooled to room temperature and then centrifuged at 10000 rpm for 3 minutes. The supernatant sample preparation was then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage: 4.6 kV; injection time: 20 sec; separation voltage: 5.75 kV; and separation time: 40 min.

[0372] The detailed results of the anti-HER3 / MUC1 bispecific antibodies are shown in the table below. The results show that 3E1-10D1 and 1B2-10D1 have good stability and physicochemical properties.

[0373] Table 4

[0374]

[0375]

[0376] Example 6. Preparation of anti-HER3 / MUC1 antibody drug conjugate (ADC)

[0377] Each purified antibody (3E1, 3G6, 1B2, 10D1, 3E1-10D1, 3G6-10D1, or 1B2-10D1) was coupled to MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0378] In the designation of antibody-drug conjugates, "ADC" is added directly after the antibody name. For example, if 3E1-10D1, which has an IgG1 constant region, is coupled to MMAE, it is designated 3E1-10D1-ADC. Antibody-drug conjugates produced by similar methods also include pertrastuzumab-ADC, gatuzumab-ADC, and 1H7-ADC. Human IgG1 is coupled to MMAE to form an ISO-ADC, which serves as an isotype control.

[0379] 1H7 is a murine 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 chain variable region and light chain variable region of 1H7 are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

[0380] HIC-HPLC was used to detect the coupling of antibody and drug molecules. In the HIC-HPLC experiment, an Agilent 1260 chromatography system (connected to ProPac TMA HIC-10 column (4.6×250 mm, Thermo Scientific) was used, and the sample was diluted to 0.5 mg / mL using mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile, pH 6.5; mobile phase B: 0.1 M PB, 10% acetonitrile, pH 6.5; flow rate: 0.8 mL / min; gradient: 100% A at 0 min, 100% A at 2 min, 100% B at 32 min, 100% B at 34 min, 100% A at 35 min, and 100% A at 45 min; column temperature: 30° C.; detection wavelength: 280 nm; injection volume: 10 μL; sample plate temperature: approximately 6° C.; and run time: 45 minutes.

[0381] HIC-HPLC assay results showed that the drug-to-antibody ratio (DAR) of each ADC was approximately 4.

[0382] Example 7. Antitumor activity in NUGC-4 xenograft model

[0383] The effect of ADC on tumor growth in vivo was tested in a NUGC-4 gastric cancer xenograft model. The expression levels of HER3 and MUC1 in NUGC-4 cells were 154.99 and 79.44, respectively, as determined by RNAseq. 6 NUGC-4 cells were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., catalog number: B-CM-002). When the tumor volume of the mice reached approximately 200 mm 3 At 4 hr, mice were randomly divided into different groups based on tumor volume. Next, mice were injected with phosphate-buffered saline (PBS), pertrastuzumab-ADC, 1H7-ADC, 3E1-10D1-ADC, 1B2-10D1-ADC, 3E1-ADC, 1B2-ADC, or 10D1-ADC via intravenous (iv) injection. Administration was once weekly (two administrations total). Details are shown in the table below.

[0384] Table 5

[0385]

[0386] The tumor volume was measured twice a week and the weight of the mice was recorded. 3 Euthanasia was performed.

[0387] The lengths of the long and short axes of the tumor were measured, and the volume of the tumor 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 average tumor volume of the treatment group on day i. T0 is the average tumor volume of the treatment group on day zero. Vi is the average tumor volume of the control group on day i. V0 is the average tumor volume of the control group on day zero. T test was performed for statistical analysis. P < 0.05 was the threshold value indicating a significant difference.

[0388] There was no significant difference in body weight among the groups during the experimental period.

[0389] The following table summarizes the results of this experiment, including tumor volume on the day of grouping (Day 0), 11 days after grouping (Day 11), 21 days after grouping (Day 21), and 32 days after grouping (Day 32); TGI (%); and statistical differences (P values) in body weight and tumor volume between the treatment group and the control group on Day 21.

[0390] Table 6

[0391]

[0392] The tumor volumes in the ADC-treated groups were as follows: Figure 1 As shown in the figure, the treatment groups exhibited different tumor inhibitory effects. In a gastric cancer model, the anti-HER3 / MUC1 bispecific antibody ADCs 3E1-10D1-ADC (G4) and 1B2-10D1-ADC (G5) showed superior anti-tumor activity compared to monoclonal ADCs (G6, G7, and G8) and positive controls (G2 and G3).

[0393] Example 8. Antitumor activity in a gastric cancer patient-derived xenograft model

[0394] The effect of ADC on in vivo tumor growth was tested in a xenograft model derived from a gastric cancer patient. Immunofluorescence staining was performed on patient-derived gastric tumor fragments, and the images were analyzed by HALO version 3.2. The results showed that HER3-positive cells accounted for 60.23% of the total cell number in human gastric tumor tissue, and MUC1-positive cells accounted for 91.30% of the total cell number. Specifically, patient-derived gastric tumor fragments (2mm×2mm×2mm) were implanted into the right flank of B-NDG mice. When the tumor volume of the mice reached approximately 250 to 300mm 3 At 4 hr, mice were randomly divided into different groups based on tumor volume. Mice were then injected with PBS, pertrastuzumab-ADC, 1H7-ADC, gatuzumab-ADC, 3E1-10D1-ADC, or 1B2-10D1-ADC. Details are shown in the table below.

[0395] Table 7

[0396]

[0397]

[0398] Body weights were measured twice weekly. During the experimental period, there were no significant differences in body weights between groups, indicating that the mice tolerated the tested ADC well without significant toxicity.

[0399] The tumor volumes of mice in different groups were Figure 2 As shown. Compared with the PBS group (G1), the treatment groups (G2 to G6) showed more tumor inhibition. In addition, both 1B2-10D1-ADC (G6) and 3E1-10D1-ADC (G5) showed higher TGI% (50.1% and 69.1%) on day 20 compared with the positive control patrastuzumab-ADC (G2, 18.9%), 1H7-ADC (G3, 24.8%), and gamazumab-ADC (G4, 19.2%). After day 20, the tumor volume and survival rate of the mice were also continuously monitored. When the tumor volume of the mice reached 3000mm 3 At the end of the experiment (day 34), 1B2-10D1-ADC (G6, TGI% = 52.7%) and 3E1-10D1-ADC (G5, TGI% = 37.8%) maintained superior anti-tumor effects compared to the positive controls, pertrastuzumab-ADC (G2, TGI% = 10.4%), 1H7-ADC (G3, TGI% = 28.4%), and gatuzumab-ADC (G4, TGI% = 24.9%).

[0400] Example 9. Antitumor activity in HCC70 xenograft model

[0401] The effect of ADC on tumor growth in vivo was tested in a breast ductal carcinoma HCC70 cell xenograft model. 7 HCC70 cells were subcutaneously injected into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 4 hr, mice were randomly divided into different groups based on tumor volume. Mice were then injected intravenously (iv) with PBS, pertuzumab-ADC, 1H7-ADC, 3E1-10D1-ADC, 3G6-10D1-ADC, or 1B2-10D1-ADC. Administration was once weekly (two administrations total). Details are shown in the table below.

[0402] Table 8

[0403]

[0404]

[0405] The tumor volume was measured twice a week and the weight of the mice was recorded. 3 Euthanasia was performed.

[0406] Mice in all groups gained weight. On day 0, the average body weight ranged from 21.6 g to 22.0 g across all groups. By the end of the experiment (day 39), the average body weight ranged from 24.3 g to 26.6 g across all groups, with a mean body weight gain of 112.5% to 121.2%. These results demonstrate that the ADC was well tolerated by mice, with no significant toxicity.

[0407] The following table summarizes the results of this experiment, including the tumor volume on the day of grouping (day 0), 18 days after grouping (day 18), 28 days after grouping (day 28), and 39 days after grouping (day 39); the survival rate of mice; TGI (%); and the statistical differences (P values) in body weight and tumor volume between the treatment group and the control group.

[0408] Table 9

[0409]

[0410] The tumor volumes in the ADC-treated groups were as follows: Figure 6 In a breast ductal carcinoma model, 3E1-10D1-ADC (G5), 3G6-10D1-ADC (G6), and 1B2-10D1-ADC (G7) showed superior anti-tumor activity compared to the positive controls, pertrastuzumab-ADC (G2), 1H7-ADC (G3), and gatuzumab-ADC (G4).

[0411] Example 10. Antitumor activity in a pancreatic cancer patient-derived xenograft model

[0412] The effect of ADC on in vivo tumor growth was tested in a pancreatic cancer patient-derived xenograft model. Immunofluorescence staining was performed on patient-derived pancreatic tumor fragments, and the images were analyzed by HALO version 3.2. The results showed that HER3-positive cells accounted for 26.78% of the total cell number in human pancreatic tumor tissue, and MUC1-positive cells accounted for 64.77% of the total cell number. Specifically, patient-derived pancreatic tumor fragments (2mm×2mm×2mm) were implanted into the right flank of B-NDG mice. When the tumor volume of the mice reached approximately 250 to 300mm 3At 4 hr, mice were randomly divided into different groups (5 mice per group) based on tumor volume. Mice were then injected with PBS (G1), pertrastuzumab-ADC (G2), 1H7-ADC (G3), gatuzumab-ADC (G4), 3E1-10D1-ADC (G5), or 1B2-10D1-ADC (G6) at 3 mg / kg by intravenous (iv) injection. The frequency of administration was once a week (2 administrations in total).

[0413] The tumor volumes of mice in different groups are shown in Table 10 and Figure 7 As shown. Compared with the PBS group (G1), the treatment groups (G2 to G6) showed significant tumor inhibition. In addition, 3E1-10D1-ADC (G5) and 1B2-10D1-ADC (G6) showed higher TGI% on day 56 compared with the positive control patrastuzumab-ADC (G2), 1H7-ADC (G3), and gatuzumab-ADC (G4). The anti-HER3 / MUC1 bispecific ADC showed robust and sustained anti-tumor effects in the pancreatic cancer model.

[0414] Table 10

[0415]

[0416] Example 11. Antitumor activity in a lung cancer patient-derived xenograft model

[0417] The effect of ADC on in vivo tumor growth was tested in a xenograft model derived from lung cancer patients. Immunofluorescence staining was performed on patient-derived lung tumor fragments, and the images were analyzed by HALO version 3.2. The results showed that HER3-positive cells accounted for 22.84% of the total cell number in human lung tumor tissue, and MUC1-positive cells accounted for 82.88% of the total cell number. Specifically, patient-derived lung tumor fragments (2mm×2mm×2mm) were implanted into the right flank of B-NDG mice. When the tumor volume of the mice reached approximately 250 to 300mm 3 At 4 hr, mice were randomly divided into different groups (5 mice per group) based on tumor volume. Mice were then injected with PBS, pertrastuzumab-ADC, 1H7-ADC, gatuzumab-ADC, 3E1-10D1-ADC, 1B2-10D1-ADC, 3E1-ADC, 1B2-ADC, or 10D1-ADC via intravenous (iv) injection. Details are shown in the table below.

[0418] Table 11

[0419] Group Number of mice Antibody dose way frequency Total number of applications G1 5 PBS - iv QW 2 G2 5 Paclitaxel-ADC 3 mg / kg iv QW 2 G3 5 1H7-ADC 3 mg / kg iv QW 2 G4 5 Gatuzumab-ADC 3 mg / kg iv QW 2 G5 5 3E1-10D1-ADC 3 mg / kg iv QW 2 G6 5 1B2-10D1-ADC 3 mg / kg iv QW 2 G7 5 3E1-10D1-ADC 6 mg / kg iv QW 2 G8 5 1B2-10D1-ADC 6 mg / kg iv QW 2 G9 5 3E1-ADC 3 mg / kg iv QW 2 G10 5 1B2-ADC 3 mg / kg iv QW 2 G11 5 10D1-ADC 3 mg / kg iv QW 2

[0420] The tumor volumes of mice in different groups were Figure 8Since all mice in groups G1, G2, G3, and G4 reached the euthanasia criteria before day 39 after grouping, the tumor size on day 39 could not be obtained.

[0421] 3E1-10D1-ADC and 1B2-10D1-ADC demonstrated optimal tumor growth inhibition at doses of 3 mg / kg and 6 mg / kg (G5 to G8). Furthermore, 3E1-ADC, 1B2-ADC, and 10D1-ADC (G9 to G11) achieved superior efficacy compared to the positive controls, pertrastuzumab-ADC, 1H7-ADC, and gatuzumab-ADC (G2 to G4).

[0422] Example 12. Internalization of anti-HER3 / MUC1 bispecific ADC

[0423] NUGC-4 cells cultured in cell culture dishes were treated with anti-HER3 antibodies, anti-MUC1 antibodies, or anti-HER3 / MUC1 bispecific antibodies and ADCs and cultured using IncuCyte (Sartorius AG, S3) After 15 to 24 hours of incubation, internalization activity was measured. The results are shown in Figures 9A to 9C.

[0424] The data showed that the endocytic activity of 3E1-10D1 and 1B2-10D1 was unchanged after conjugation with MMAE. In addition, 3E1-10D1 and 1B2-10D1 exhibited enhanced internalization compared to the corresponding parental monoclonal antibodies (3E1, 1B2, and 10D1) and positive controls (pertrastuzumab analogs, 1H7 analogs, and gatuzumab analogs).

[0425] Example 13. Antibody Drug Conjugates

[0426] Purified antibodies (1B2, 3E1, 10D1, 1B2-10D1, 3E1-10D1, 1H7 analogs, and gatuzumab analogs) are conjugated to CPT-1, CPT-2, CPT-3, or CPT-4 via a CPT-L linker. For the designation of the antibody-drug conjugate, CPTx (x = 1, 2, 3, or 4) is added directly after the antibody name. For example, when 1B2 is conjugated to CPT-1, it is designated as 1B2-CPT1. Or, for example, when 1B2-10D1 is conjugated to CPT-2, it is designated as 1B2-10D1-CPT2. Exemplary ADCs obtained by this method include 1B2-10D1-CPT2, 3E1-10D1-CPT2, 1H7-CPT2, and gatuzumab-CPT2.

[0427] For comparison purposes, pertratuzumab analogs and gatuzumab analogs were also conjugated to Dxd via a GGFG linker, and the resulting ADCs were named pertratuzumab-Dxd and gatuzumab-Dxd, respectively.

[0428] IgG1 monoclonal antibodies targeting unrelated targets were coupled to CPT-2 via a CPT-L linker and to Dxd via a GGFG linker, respectively. This resulted in isotype-CPT2 (ISO-CPT2) and isotype-Dxd (ISO-Dxd), which served as isotype controls.

[0429] Mass spectrometry (MS) was used to detect the coupling of the antibody and drug molecule. The MS test results showed that the drug-to-antibody ratio (DAR) of the ADC was approximately 4 or 8. All DARs of the controls (including 1H7-CPT2, Gatuzumab-CPT2, ISO-CPT2, Gatuzumab-Dxd, Pertratuzumab-Dxd, and ISO-Dxd) were 8. With respect to the designations of 1B2-10D1-CPT2 and 3E1-10D1-CPT2, if the DAR was approximately 4, the ADCs were designated as 1B2-10D1-CPT2 (DAR4) and 3E1-10D1-CPT2 (DAR4). If the DAR was approximately 8, the ADCs were designated as 1B2-10D1-CPT2 (DAR8) and 3E1-10D1-CPT2 (DAR8).

[0430] Example 14. Antitumor activity in lung cancer PDX models

[0431] The in vivo antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested in a HER3-low / MUC1-high lung cancer PDX model. Specifically, patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of B-NDG mice. When the mice had tumors that reached a volume of approximately 200 to 300 mm 3 At 4 hr, mice were randomly divided into different groups (5 mice per group) based on tumor volume and then injected with PBS, 1B2-10D1-CPT2, or 3E1-10D1-CPT2 by iv injection. Tumor volume was measured twice a week.

[0432] like Figure 10As shown, the tumor volumes of all treatment groups (G2 to G13) were smaller than those of the control group (G1). The results also showed that 1B2-10D1-CPT2 and 3E1-10D1-CPT2 (DAR4 and DAR8) had different tumor inhibitory effects and were dose-dependent. When administered at dose levels of 3 mg / kg or higher (G7, G9, G10, G11, G12, and G13), 1B2-10D1-CPT2 and 3E1-10D1-CPT2 could significantly inhibit tumor growth with a TGI% greater than 100% (e.g., on day 24).

[0433] After day 24, the tumor volume and survival of the mice were also continuously monitored.

[0434] On day 28, all mice in group G1 had died, while groups G2 to G13 had a superior survival rate. By the end of the experiment on day 49, all mice in groups G1 to G5 had died; only one mouse had died in groups G6, G8, G9, and G11; and all mice in groups G7, G10, G12, and G13 had survived, demonstrating that 1B2-10D1-CPT2 and 3E1-10D1-CPT2 have excellent tumor suppressive effects and a favorable safety profile.

[0435] Example 15. Antitumor activity in NUGC-4 xenograft model

[0436] The effect of ADC on tumor growth in vivo was tested in a NUGC-4 gastric cancer xenograft model. 5 NUGC-4 cells were subcutaneously injected into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 4 hr, mice were randomly divided into different groups based on tumor volume and then injected with PBS, Pertratuzumab-Dxd, Gatuzumab-Dxd, 1B2-10D1-CPT2, or 3E1-10D1-CPT2 by iv injection. The frequency of administration was once a week (2 times in total). Tumor volume was measured twice a week, and the results were shown in Table 1. Figure 11 shown.

[0437] The results showed that 1B2-10D1-CPT2 and 3E1-10D1-CPT2 (DAR4 and DAR8) inhibited tumor growth with a higher TGI% (e.g., on day 35) than the positive control (G10 and G11). For example, at a dose level of 6 mg / kg, the TGI% for G6 to G9 was above 90%, while the TGI% for G10 and G11 were 50.7% and 40.4%, respectively. 1B2-10D1-CPT2 and 3E1-10D1-CPT2 exhibited a potent tumor suppressive effect in a gastric cancer model.

[0438] Example 16. Antitumor activity in HCC70 xenograft model

[0439] The effect of ADC on tumor growth in vivo was tested in a breast ductal carcinoma HCC70 xenograft model. 7 HCC70 cells were subcutaneously injected into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 4 hr, mice were randomly divided into different groups based on tumor volume and then injected with PBS, pertrastuzumab-Dxd, gatuzumab-Dxd, ISO-Dxd, 1B2-10D1-CPT2, or 3E1-10D1-CPT2 by IV injection. Tumor volume was measured twice a week, and the body weight of the mice was recorded. Figure 12A and Figure 12B shown.

[0440] Figure 12A Tumor volume measurements were presented, with 1B2-10D1-CPT2 and 3E1-10D1-CPT2 (DAR4 and DAR8) exhibiting different tumor suppressive effects, and exhibiting a dose-dependent pattern. At a dose level of 3 mg / kg, the TGI% (e.g., on day 38) in the G6 to G9 groups was higher than that in the control groups G10 to G12. Specifically, on day 38, the TGI% in the G6 to G9 groups were 89.1%, 106.1%, 100.0%, and 106.3%, respectively; while the lower values were 68.5% in the G10 group, 52.1% in the G11 group, and 19.7% in the G12 group, indicating that 1B2-10D1-CPT2 and 3E1-10D1-CPT2 have good tumor suppressive effects in breast cancer models.

[0441] The weight of mice in different groups increased (e.g. Figure 12B ), indicating that all anti-HER3 / MUC1 ADCs were well tolerated by mice without toxicity.

[0442] Example 17. Antitumor activity in colorectal cancer PDX models

[0443] The in vivo antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested in a HER3-high / MUC1-low colorectal cancer PDX model. Specifically, patient-derived colorectal tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of B-NDG mice. When the mice had tumors that reached a volume of approximately 200 to 300 mm 3At 4 hr, mice were randomly divided into different groups (5 mice per group) based on tumor volume and then administered PBS, 1B2-10D1-CPT2, 3E1-10D1-CPT2, 1H7-CPT2, or Pertrastuzumab-Dxd by iv injection. Tumor volume was measured twice a week.

[0444] like Figure 13 As shown, 1B2-10D1-CPT2 and 3E1-10D1-CPT2 (DAR4 and DAR8) inhibited tumor growth with a higher TGI % (eg, on day 31) compared to the positive controls 1H7-CPT2 and Pertratuzumab-Dxd.

[0445] In a similar experiment, colorectal cancer PDX model mice were injected with PBS, ISO-CPT2, 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR8), 1H7-CPT2, or patrastuzumab-Dxd by IV injection. Tumor volume was measured twice a week. Figure 14 As shown in the results, 1B2-10D1-CPT2(DAR8) and 3E1-10D1-CPT2(DAR8) exhibited better anti-tumor activity than the positive controls 1H7-CPT2 and patrastuzumab-Dxd.

[0446] In another similar experiment, colorectal cancer PDX model mice were injected with PBS (G1), ISO-CPT2 (G2), 3E1-10D1-CPT2 (DAR8) (G3 and G4), a combination of 3E1-CPT2 (DAR8) and 10D1-CPT2 (DAR8) (G5), ISO-Dxd (G6), Pertratuzumab-Dxd (G7), Gatuzumab-Dxd (G8), or 3E1-10D1-Dxd (DAR8) (G9) by iv injection. The administration frequency was once a week (a total of 2 administrations). Tumor volume was measured twice a week, and the results were as follows. Figure 19 As shown in Figure 3, 3E1-10D1-CPT2(DAR8) (G3 and G4) exhibited the most potent tumor suppression effect compared to the positive control (G7 and G8) and the combination therapy (G5), in a dose-dependent manner. Furthermore, 3E1-10D1-Dxd (G9) exhibited superior efficacy in inhibiting tumor growth compared to the positive control (G7 and G8).

[0447] Example 18. Antitumor activity in pancreatic cancer PDX models

[0448] The in vivo antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested using a HER3 / MUC1 co-overexpressing pancreatic cancer PDX model. Patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of B-NDG mice. When the tumor volume of the mice reached approximately 200 to 300 mm 3 At 4 hr, mice were randomly divided into different groups (5 mice per group) based on tumor volume and then administered PBS, 1B2-10D1-CPT2 (DAR8), 3E1-10D1-CPT2 (DAR8), 1H7-CPT2, Gatuzumab-CPT2, or Pertrastuzumab-Dxd by iv injection. Tumor volume was measured twice a week, and the body weight of the mice was recorded.

[0449] like Figure 15A As shown, 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR8) induced significant tumor growth inhibition at 3 mg / kg compared to the positive control 1H7-CPT2, Gatuzumab-CPT2, or Pertratuzumab-Dxd with a higher TGI% (e.g., on day 31).

[0450] like Figure 15B As shown, mice in the 1B2-10D1-CPT2 (DAR8) and 3E1-10D1-CPT2 (DAR8)-treated groups (G3 and G4) experienced increased body weight. On the day of grouping, the average body weights of the G3 and G4 groups were 21.8 g and 21.6 g, respectively. Thirty-one days after grouping, the average body weights of the G3 and G4 groups were 24.8 g and 24.4 g, respectively, representing body weight changes of 114.4% and 113.1%, respectively. However, body weights of the 1H7-CPT2, Gatuzumab-CPT2, and Pertrastuzumab-Dxd-treated groups remained constant or decreased slightly. These results indicate that both 1B2-10D1-CPT2 and 3E1-10D1-CPT2 were well tolerated by mice and did not cause any toxicity.

[0451] Example 19. Antitumor activity in breast cancer PDX models

[0452] The in vivo antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested using a breast cancer PDX model with low HER3 expression and high MUC1 expression. Patient-derived breast tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of B-NDG mice. Tumor volume was measured twice weekly. Figure 16 shown.

[0453] 1B2-10D1-CPT2 and 3E1-10D1-CPT2 (DAR4 and DAR8) significantly inhibited tumor growth with higher TGI% (e.g., on day 16) compared to the positive controls, Gatuzumab-CPT2 and Pertratuzumab-Dxd. This suggests that anti-HER3 / MUC1 ADCs have the potential to treat breast cancer.

[0454] Example 20. Antitumor activity in gastric cancer PDX

[0455] The effect of ADCs on tumor growth in vivo was tested in a gastric cancer patient-derived xenograft model. Specifically, patient-derived gastric tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of B-NDG mice. When the tumor volume reached approximately 250 mm 3 At the time of the study, mice were randomly divided into different groups based on tumor volume. Mice were then administered PBS (G1), ISO-CPT2 (G2), 3E1-10D1-CPT2 (DAR8) (G3 and G4), a combination of 3E1-CPT2 (DAR8) and 10D1-CPT2 (DAR8) (G5), Pertratuzumab-Dxd (G6), Gatuzumab-Dxd (G7), ISO-ADC (G8), or 3E1-10D1-ADC (G9) by intravenous (iv) injection. The administration frequency was once a week (2 administrations in total).

[0456] Tumor volume was measured twice a week. Figure 18 As shown in the figure, the treatment groups (G2 to G9) showed different tumor suppression compared to the PBS group (G1). 3E1-10D1-CPT2(DAR8) (G3 and G4) demonstrated a dose-dependent and superior tumor suppression compared to the positive control (G6 and G7) and the combination therapy (G5). Furthermore, 3E1-10D1-ADC (G9) inhibited tumor growth with superior efficacy compared to the positive control (G6 and G7).

[0457] Example 21. Antitumor activity in lung cancer PDX models

[0458] The effect of 3E1-10D1-CPT2 (DAR8) on in vivo tumor growth was tested in a lung cancer patient-derived xenograft model. Immunohistochemistry (IHC) staining of patient-derived tumor tissues revealed that the histochemical scores (H-scores) for HER3 and MUC1 expression levels in patient-derived tumor tissues were 104.68 and 89.9, respectively. Patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right flank of BALB / c nude mice. When the tumor volume reached approximately 200 mm 3At 4 hr, mice were randomized into different groups based on tumor volume. Mice were then administered 5% glucose (G1) or 3 mg / kg 3E1-10D1-CPT2(DAR8) (G2) by intravenous (iv) injection.

[0459] Tumor volume was measured twice a week. Figure 20 As shown, 3E1-10D1-ADC (DAR8) showed good tumor growth inhibition effect in a lung cancer model.

[0460] Example 22. Antitumor activity in ovarian cancer PDX models

[0461] The effect of 3E1-10D1-CPT2 (DAR8) on tumor growth was tested in a patient-derived xenograft model of ovarian cancer. IHC staining of patient-derived tumor tissues showed that the H-scores for HER3 and MUC1 expression in patient-derived tumor tissues were 245.87 and 112.00, respectively. When the tumor volume reached approximately 200 mm 3 At 4 hr, mice were randomized into different groups based on tumor volume. Mice were then administered 5% glucose (G1) or 6 mg / kg 3E1-10D1-CPT2 (DAR8) (G2) by iv injection.

[0462] Tumor volume was measured twice a week, and the results were Figure 21 As shown in the results, 3E1-10D1-ADC (DAR8) also showed good tumor growth inhibition effect in the ovarian cancer model.

[0463] Example 23. Toxicological Evaluation

[0464] In preliminary experiments, to investigate safety and toxicokinetic (TK) properties, 1B2-10D1-CPT2(DAR4), 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR4), or 3E1-10D1-CPT2(DAR8) were administered to cynomolgus macaques via three intravenous injections, each administered three weeks apart (day 1, day 22, and day 43). The dose formulations are shown in the table below. The animals were then sacrificed on day 50 for gross and histopathological examinations. Mortality / moribundity, general observations, body weight, food consumption, clinical pathology (hematology, blood coagulation, serum chemistry, and urinalysis), and gross lesions were assessed. Blood samples were also collected for TK analysis, and key TK parameters such as payload, total antibody, and Tmax, Cmax, and AUC of ADC were calculated. (0-t)The results showed that T1B2-10D1-CPT2(DAR4), 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR4) and 3E1-10D1-CPT2(DAR8) all had favorable safety profiles.

[0465] Table 12

[0466]

[0467] Other implementations

[0468] It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate the invention and not to limit its scope, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to HER3; and a second antigen-binding domain that specifically binds to MUC1.

2. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of claim 1, wherein 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).

3. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to claim 2, wherein 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 a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprising CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences, the selected VL1 CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively; (3) the selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively; and (4) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL1 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively.

4. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the second heavy chain variable region (VH2) comprises CDRs 1, 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 CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, and 3 amino acid sequences, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; and (2) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL2 CDRs 1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively.

5. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, wherein (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) the selected VH1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively; (3) the selected VH1 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 7 to 9, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1 to 3, respectively, and the selected VH2 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10 to 12, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1 to 3, respectively; or (4) The selected VH1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VH2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22 to 24, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively.

6. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 5, wherein the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 26, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 28, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

25.

7. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 5, wherein the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 27, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 28, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

25.

8. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 7, 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, wherein the selected VH sequence and the selected VL sequence are one of: (1) the selected VH sequence is SEQ ID NO: 26, and the selected VL sequence is SEQ ID NO: 25; and (2) The selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO:

25.

9. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 8, wherein the VH1 comprises a VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL1 comprises a VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 26, and the selected VL sequence is SEQ ID NO: 25; and (2) The selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO:

25.

10. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 9, wherein the VH2 comprises an amino acid sequence at least 90% identical to a selected VH sequence, and the VL2 comprises an amino acid sequence at least 90% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO:

25.

11. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 10, wherein the VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO:

25.

12. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 11, wherein the VH1 comprises the sequence of SEQ ID NO: 26, and the VL1 comprises the sequence of SEQ ID NO:

25.

13. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 12, wherein the VH1 comprises the sequence of SEQ ID NO: 27, and the VL1 comprises the sequence of SEQ ID NO:

25.

14. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 13, wherein the VH2 comprises the sequence of SEQ ID NO: 28, and the VL2 comprises the sequence of SEQ ID NO:

25.

15. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the first antigen-binding domain specifically binds to human or monkey HER3; and / or the second antigen-binding domain specifically binds to human or monkey MUC1.

16. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 16, wherein the first antigen-binding domain is human or humanized; and / or the second antigen-binding domain is human or humanized.

17. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 16, wherein the antibody is a multispecific antibody (eg, a bispecific antibody).

18. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 17, wherein the first antigen-binding domain is a single-chain variable fragment (scFv); and / or the second antigen-binding domain is a scFv.

19. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 2 to 18, wherein the first light chain variable region and the second light chain variable region are identical.

20. An anti-HER3 / MUC1 antibody or antigen-binding fragment thereof that cross-competes with the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19.

21. A nucleic acid comprising a polynucleotide encoding the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 20. A vector comprising the nucleic acid of claim 21 .

23. A cell comprising the vector of claim 22.

24. The cell of claim 23, wherein the cell is a CHO cell.

25. A cell comprising the nucleic acid of claim 21.

26. A method for producing an anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cell under conditions sufficient for the cell to produce the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 23 to 25; and (b) collecting the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof produced by the cells.

27. An anti-HER3 / MUC1 antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 20.

28. The anti-HER3 / MUC1 antibody drug conjugate of claim 27, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

29. The anti-HER3 / MUC1 antibody drug conjugate of claim 27 or 28, wherein the therapeutic agent is MMAE or MMAF.

30. The antibody drug conjugate of claim 27, wherein the therapeutic agent is selected from:

31. The antibody drug conjugate of claim 27 or 30, wherein the therapeutic agent is linked to the antibody or antigen-binding fragment thereof, or antigen-binding protein construct via a linker.

32. The antibody drug conjugate of claim 31, wherein the linker has the following structure:

33. The antibody drug conjugate of any one of claims 27 and 30 to 32, wherein the antibody drug conjugate has the following structure: wherein n=1 to 8; wherein "Ab" represents the antibody or antigen-binding fragment thereof, or antigen-binding protein construct.

34. 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-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 20, or the anti-HER3 / MUC1 antibody drug conjugate of any one of claims 27 to 33.

35. The method of claim 34, wherein the subject has a cancer that expresses HER3 and / or MUCl (eg, both HER3 and MUCl).

36. The method of claim 34 or claim 35, wherein the cancer is esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial cancer, lung cancer, melanoma, ovarian cancer, bladder cancer, stomach cancer, non-Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, lung adenocarcinoma, and cervical cancer.

37. The method of any one of claims 34 to 36, wherein the subject is a human.

38. The method of any one of claims 34 to 37, wherein the method further comprises administering an anti-PD1 antibody to the subject.

39. The method of any one of claims 34 to 38, wherein the method further comprises administering chemotherapy to the subject.

40. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 20, or the anti-HER3 / MUC1 antibody drug conjugate of any one of claims 27 to 33.

41. A method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, or the anti-HER3 / MUC1 antibody-drug conjugate according to any one of claims 27 to 33.

42. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and (a) the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, and / or (b) the anti-HER3 / MUC1 antibody-drug conjugate according to any one of claims 27 to 33.

43. An anti-HER3 / MUC1 antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to HER3; and a second antigen-binding domain that specifically binds to MUC1.

44. The anti-HER3 / MUCl ADC of any one of claims 27 to 33 and 43, wherein the drug to antibody ratio (DAR) is about 4 or 8.

Images