Anti-HER3 / MUC1 antibodies and their use

Abstract

This disclosure relates to anti-HER3 / MUC1 antibodies, antibody-drug conjugates derived therefrom, and their uses.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to international application PCT / CN2023 / 082375 filed on 18 March 2023 and international application PCT / CN2023 / 120377 filed on 21 September 2023. The entire contents of the above applications are incorporated herein by reference.

[0002] This disclosure relates to a multispecific anti-HER3 (human epidermal growth factor receptor 3) / MUC1 (mucin 1) antibody (e.g., a bispecific antibody or its antigen-binding fragment) and antibody-drug conjugates derived therefrom. [Background technology]

[0003] Bispecific antibodies are artificial proteins that can simultaneously bind to two different types of antigens or two different epitopes. This bispecificity enables a wide range of applications, including the induction of T cells into tumor cells, dual targeting of different disease mediators, and delivery of payloads to target sites. The approval of catumakisomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) represents a significant milestone in the development of bispecific antibodies.

[0004] Because bispecific antibodies have a wide range of applications, it is necessary to continue developing various therapeutic drugs using bispecific antibodies in the future. [Overview of the project] [Means for solving the problem]

[0005] This disclosure relates to anti-HER3 / MUC1 antibodies or their antigen-binding fragments, wherein the antibody or its antigen-binding fragment specifically binds to HER3 and MUC1. In some embodiments, the antibody or its antigen-binding fragment has the same light chain variable region. In some embodiments, the antibody or its antigen-binding fragment has a common light chain. This disclosure also relates to antibody-drug conjugates obtained from these anti-HER3 / MUC1 antibodies.

[0006] In one embodiment, the present 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 MUC1.

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

[0008] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence. The first light chain variable region (VL1) includes CDR1, 2, and 3, the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence. The selected VH1 CDR1, 2, and 3 amino acid sequences, and the selected VL1 CDR1, 2, and 3 amino acid sequences, (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 4-6, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1-3, respectively. (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 7-9, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1-3, respectively. (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 7, 42, and 43, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1 to 3, respectively. (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 16-18, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively. (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 19-21, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively, and (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 19, 20, and 44, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1 to 3, respectively. It is one of the following.

[0009] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence. The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region includes an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region includes an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region includes an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence. The selected VH2 CDR1, 2, and 3 amino acid sequences, and the selected VL2 CDR1, 2, and 3 amino acid sequences, (1) The selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 10 to 12, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1 to 3, respectively, and (2) The selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 22-24, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively. It is one of the following.

[0010] In some embodiments, (1) the selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 4-6, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1-3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 10-12, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1-3, respectively. (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 16-18, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 22-24, respectively; the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively. (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 7-9, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1-3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 10-12, respectively; the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1-3, respectively. (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 19-21, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 22-24, respectively; the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13-15, respectively. (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 7, 42, and 43, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 10 to 12, respectively; the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 1 to 3, respectively, or (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 19, 20, and 44, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13 to 15, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 22 to 24, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs. 13 to 15, respectively.

[0011] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 26, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 25, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 28, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 25.

[0012] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 27, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 25, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 28, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 25.

[0013] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 45, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 25, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 28, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 25.

[0014] In some embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 90% identical to the selected VL sequence, where the selected VH sequence and the selected VL sequence are (1) The selected VH sequence is sequence number 26, and the selected VL sequence is sequence number 25. (2) The selected VH sequence is sequence number 27, and the selected VL sequence is sequence number 25, and (3) The selected VH sequence is sequence number 45, and the selected VL sequence is sequence number 25. It is one of the following.

[0015] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, where the selected VH sequence and the selected VL sequence are (1) The selected VH sequence is sequence number 26, and the selected VL sequence is sequence number 25. (2) The selected VH sequence is sequence number 27, and the selected VL sequence is sequence number 25, and (3) The selected VH sequence is sequence number 45, and the selected VL sequence is sequence number 25. It is one of the following.

[0016] In some embodiments, VH2 comprises an amino acid sequence that is at least 90% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 90% identical to the selected VL sequence, where the selected VH sequence is sequence number 28 and the selected VL sequence is sequence number 25.

[0017] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, where the selected VH sequence is sequence number 28 and the selected VL sequence is sequence number 25.

[0018] In some embodiments, VH1 includes the sequence of sequence number 26, and VL1 includes the sequence of sequence number 25.

[0019] In some embodiments, VH1 includes the sequence of sequence number 27, and VL1 includes the sequence of sequence number 25.

[0020] In some embodiments, VH1 includes the sequence of sequence number 45, and VL1 includes the sequence of sequence number 25.

[0021] In some embodiments, VH2 includes the sequence of sequence number 28, and VL2 includes the sequence of sequence number 25.

[0022] 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.

[0023] In some embodiments, the first antigen-binding domain is a human-derived or humanized domain, and / or the second antigen-binding domain is a human-derived or humanized domain.

[0024] In some embodiments, the antibody is a multispecific antibody (e.g., a bispecific antibody).

[0025] In some embodiments, the first antigen-binding domain is a single-chain variable fragment (scFv), and / or the second antigen-binding domain is an scFv.

[0026] In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0027] In one embodiment, this disclosure relates to an anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof that cross-competes with the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof described herein.

[0028] In one embodiment, this disclosure relates to nucleic acids comprising polynucleotides encoding an anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof as described herein.

[0029] In one embodiment, this disclosure relates to a vector comprising nucleic acids as described herein.

[0030] In one embodiment, this disclosure relates to cells, including the vector described herein.

[0031] In some embodiments, the cells are CHO cells.

[0032] In one embodiment, this disclosure relates to cells comprising nucleic acids as described herein.

[0033] In one embodiment, the present disclosure relates to a method for producing an anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof, the method being (a) Culturing the cells described herein under conditions sufficient to produce an anti-HER3 / MUC1 antibody or its antigen-binding fragment, (b) Recovering anti-HER3 / MUC1 antibodies or their antigen-binding fragments produced by cells. Includes.

[0034] In one embodiment, the present disclosure relates to an anti-HER3 / MUC1 antibody-drug conjugate (ADC) comprising an anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof as described herein, covalently bound to a therapeutic agent.

[0035] In some embodiments, the therapeutic agent is a cytotoxic agent or a cell quiescent agent.

[0036] In some embodiments, the therapeutic agent is MMAE or MMAF.

[0037] In some embodiments, the therapeutic agent [ka] Selected from.

[0038] In some embodiments, the therapeutic agent is linked to an antibody or its antigen-binding fragment via a linker. In some embodiments, the linker has the following structure. [ka]

[0039] In some embodiments, the antibody-drug conjugate has the following structure: [ka] In some embodiments, n=1 to 8, and in some embodiments, "Ab" represents an antibody or its antigen-binding fragment.

[0040] In some embodiments, the drug-to-antibody ratio (DAR) is approximately 4 or 8.

[0041] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-HER3 / MUC1 antibody or its antigen-binding fragment as described herein, or an anti-HER3 / MUC1 antibody-drug conjugate as described herein.

[0042] In some embodiments, the subjects have cancer that expresses HER3 and / or MUC1 (e.g., both HER3 and MUC1).

[0043] In some embodiments, cancers include esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial carcinoma, lung cancer, melanoma, ovarian cancer, bladder cancer, gastric cancer, non-Hodgkin lymphoma, head and neck cancer, pancreatic cancer, lung adenocarcinoma, and cervical cancer.

[0044] In some embodiments, the subject is human.

[0045] In some embodiments, the method further includes administering an anti-PD1 antibody to the subject.

[0046] In some embodiments, the method further includes administering chemotherapy to the subject.

[0047] In one embodiment, the present disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of a composition comprising an anti-HER3 / MUC1 antibody or its antigen-binding fragment as described herein, or an anti-HER3 / MUC1 antibody-drug conjugate as described herein.

[0048] In one embodiment, the present disclosure relates to a method for killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising an anti-HER3 / MUC1 antibody or its antigen-binding fragment as described herein, or an anti-HER3 / MUC1 antibody-drug conjugate as described herein.

[0049] In one aspect, the disclosure relates to a pharmaceutically acceptable carrier, (a) an anti-HER3 / MUC1 antibody or its antigen-binding fragment as described herein, and / or (b) Anti-HER3 / MUC1 antibody drug conjugate as described herein and This relates to pharmaceutical compositions, including those containing the above.

[0050] In one embodiment, the present disclosure relates to an anti-HER3 / MUC1 antibody-drug conjugate (ADC) comprising a bispecific antibody or a therapeutic agent covalently bound to an antigen-binding fragment thereof, the ADC comprising a first antigen-binding domain that specifically binds to HER3 and a second antigen-binding domain that specifically binds to MUC1.

[0051] As used herein, the term “antigen-binding domain” refers to one or more protein domains (e.g., formed from amino acids derived from a single polypeptide, or from amino acids derived from two or more polypeptides (e.g., identical or different polypeptides)) that can specifically bind to one or more different antigens (e.g., an effector antigen or a control antigen). In some examples, the antigen-binding domain can bind to an antigen or epitope with similar specificity and affinity to naturally occurring antibodies. In some embodiments, the antigen-binding domain may be an antibody or a fragment thereof. An example of an antigen-binding domain is one 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 VHH. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are well known in the art. In some examples, the antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other examples, the antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen).

[0052] The term "antibody" is used herein 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. Antibodies include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a protein complex containing two heavy chains and two light chains. Further examples of antibodies are described herein.

[0053] As used herein, the term “multispecific antibody” refers to an antibody comprising two or more different antigen-binding domains that collectively and specifically bind to two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on a cell surface) or on different antigens (e.g., different proteins present on the surface of the same cell, or different proteins present on the surfaces of different cells). In some embodiments, a multispecific antibody binds to two different epitopes (i.e., a “bispecific antibody”). In some embodiments, a multispecific antibody binds to three different epitopes (i.e., a “triplespecific antibody”). In some embodiments, a multispecific antibody binds to four different epitopes (i.e., a “quadrispecific antibody”). In some embodiments, a multispecific antibody binds to five different epitopes (i.e., a “quintuplespecific antibody”). Each binding specificity may exist at any preferred titer. Non-limiting examples of multispecific antibodies are described herein.

[0054] As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. Epitopes may be present on the same antigen or on different antigens.

[0055] 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, which 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, which can specifically bind to different antigens or epitopes. In some embodiments, an antibody or its antigen-binding fragment may have a common light chain. In some embodiments, an anti-HER3 / MUC1 antibody or its antigen-binding fragment may have a common light chain variable region.

[0056] As used herein, the term "anti-HER3 / MUC1 antibody or its antigen-binding fragment" refers to an antibody or antigen-binding fragment that binds to both MUC1 and HER3.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. While methods and materials for use in the invention are described herein, other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail.

[0058] Other features and advantages of the present invention will become apparent from the following detailed description and figures, as well as from the claims. [Brief explanation of the drawing]

[0059] [Figure 1] This figure shows the mean tumor volume in different groups of B-NDG mice that were injected with NUGC-4 cells and treated with phosphate-buffered saline (PBS) or ADC. [Figure 2] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived gastric tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 3] The heavy chain variable region (CDR) sequences of the anti-HER3 antigen-binding domain (1B2, 3G6, 3E1) and anti-MUC1 antigen-binding domain (10D1) of anti-HER3 / MUC1 antibodies, according to Kabat's definition, are listed below. [Figure 4] The heavy chain variable region (CDR) sequences of the anti-HER3 antigen-binding domain (1B2, 3G6, 3E1) and anti-MUC1 antigen-binding domain (10D1) of anti-HER3 / MUC1 antibodies, according to Chothia's definition, are listed below. [Figure 5]The following lists the specific amino acid sequences discussed in this disclosure. [Figure 6] This figure shows the mean tumor volume in different groups of B-NDG mice injected with HCC70 cells and treated with PBS or ADC. [Figure 7] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived pancreatic tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 8] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived lung tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figures 9A-9C] This figure shows the endocytosis rates of anti-HER3 antibodies, anti-MUC1 antibodies, or anti-HER3 / MUC1 bispecific antibodies against ADCs in NUGC-4 cells. [Figure 10] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived lung tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 11] This figure shows the mean tumor volume in different groups of B-NDG mice transplanted with NUGC-4 cells and treated with PBS or ADC. [Figure 12A] This figure shows the mean tumor volume in different groups of B-NDG mice transplanted with HCC70 cells and treated with PBS or ADC. [Figure 12B] This figure shows the average body weight in different groups of B-NDG mice transplanted with HCC70 cells and treated with PBS or ADC. [Figure 13] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived colorectal tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 14] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived colorectal tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 15A]This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived pancreatic tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 15B] This figure shows the average body weight in each group of B-NDG mice that were transplanted with patient-derived pancreatic tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 16] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived mammary tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figures 17A-17B] This figure shows the binding activity of anti-HER3 antibodies, anti-MUC1 antibodies, or anti-HER3 / MUC1 bispecific antibodies against NUGC-4 cells and NCI-H226 cells. [Figure 18] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived gastric tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 19] This figure shows the average tumor volume in each group of B-NDG mice that were transplanted with patient-derived colorectal tumor fragments (2mm x 2mm x 2mm) and treated with PBS or ADC. [Figure 20] This figure shows the average tumor volume in each group of BALB / c nude mice that were transplanted with patient-derived lung tumor fragments (2mm x 2mm x 2mm) and administered 5% glucose or ADC. [Figure 21] This figure shows the average tumor volume in each group of BALB / c nude mice that were transplanted with patient-derived ovarian tumor fragments (2mm x 2mm x 2mm) and administered 5% glucose or ADC. [Modes for carrying out the invention]

[0060] A bispecific antibody or its antigen-binding fragment is an artificial protein capable of simultaneously binding to two different epitopes (e.g., on two different antigens). In some embodiments, the bispecific antibody or its antigen-binding fragment may have two arms. Each arm may have one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or antigen-binding region). In some embodiments, the bispecific antibody has a common light chain.

[0061] This disclosure relates to anti-HER3 / MUC1 antibodies (e.g., bispecific antibodies or their antigen-binding fragments) that specifically bind to HER3 and MUC1, and antibody-drug conjugates derived from these anti-HER3 / MUC1 antibodies.

[0062] Anti-HER3 / MUC1 antibody HER3 is a pseudokinase member of the EGFR family and is involved in both tumor progression and drug resistance. HER3 is an exceptional member of the EGFR family; while it is not oncogenic on its own, it can induce tumorigenesis, metastasis, and drug resistance in conjunction with other receptors. HER3 is an attractive cancer therapeutic target. In contrast to EGFR and HER2, which have been widely targeted with TKIs, HER3 has low kinase activity and has therefore been targeted primarily with monoclonal or bispecific antibodies by either inhibiting ligand binding or heterodimerization with other receptors.

[0063] Uccentric expression of HER3 has been detected in various cancers, including breast cancer, ovarian cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, and pancreatic cancer. High expression of HER3 is also associated with disease progression and poor prognosis in many cancer types. While HER3 alone does not cause tumorigenesis, the HER2:HER3 heterodimer has the highest transformative potential among all possible EGFR family dimers. Due to the high oncogenic potential of the dimer pair, HER3 is crucial for HER2-mediated tumorigenesis in various types of tumors. In breast cancer cell lines, HER3 has been shown to be important for maintaining cell viability, while EGFR is not required.

[0064] HER3 expression acts as a bypass mechanism for various targeted therapies, and elevated HER3 signaling confers resistance to multiple therapeutic agents. For example, because HER3 dimerizes with non-EGFR receptors, including HER2 and the MET receptor, HER3 may confer resistance to EGFR-targeted therapies through dimerization with non-EGFR partners. HER3 expression is also associated with resistance to hormone therapy. HER3 plays a crucial role in HER2 phosphorylation in breast cancer cells, and HER3 downregulation reversed resistance to anti-estrogen receptor (ER) tamoxifen in breast cancer cell lines.

[0065] Detailed reviews of HER3 and its function are published in Haikala, Heidi M., and Pasi A. Jaenne, “Thirty Years 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); and Liu, Xiaolong, et al., “Development of effective therapeutics targeting HER3 for cancer treatment,” Biological procedures online 21.1(2019):1-10. These are incorporated herein by reference in their entirety.

[0066] Mucin 1 (MUC1; also known as epicyalin, PEM, H23Ag, EMA, CA15-3, and MCA) is a single-pass type I transmembrane protein with a highly glycosylated extracellular domain that extends 200–500 nm from the cell surface. MUC1 is typically expressed in glandular or luminal epithelial cells of the mammary gland, esophagus, stomach, duodenum, pancreas, uterus, prostate, and lung, but is less expressed in hematopoietic cells. It is absent in skin epithelium and mesenchymal cells. In healthy tissue, MUC1 protects the underlying epithelium. The extended, negatively charged glycans of MUC1 form a physical barrier, conferring anti-adhesion to MUC1, thereby restricting access to MUC1 and preventing pathogenic colonization. The glycans oligomerize to form a mucous gel, which lubricates and protects the underlying epithelium from drying, pH changes, contaminants, and microorganisms. MUC1, which has abnormally attached sugar chains, is overexpressed in most human epithelial cancers and is attracting attention as a carcinogenic molecule.

[0067] MUC1 is overexpressed in cancer cells, and the loss of cell polarity leads to the redistribution of TA-MUC1 to the cell surface and cytoplasm. The lack of cell polarity also causes the redistribution of cell surface growth factors, which are normally limited to the basal surface of epithelial cells. Growth factors paralleling MUC1, as well as intracellular kinases such as ZAP-70, PKC-g, GSK-3b, and c-Src, phosphorylate serine, tyrosine, and threonine residues on MUC1 CT. Furthermore, it is thought that deglycosylation removes the mask from the peptide core of TA-MUC1, allowing for cleavage and release of MUC1-N by extracellular proteases. The release of MUC1-N causes conformational changes in MUC1-C, altering its ligand state and subsequently activating downstream cellular signaling pathways such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3 kinase (P13K / Akt), and the wingless (Wnt) pathway. As a result, MUC1-positive pancreatic, breast, lung, and colon cancer cells commonly exhibit hyperactivation of these important signaling pathways. MUC1-C also associates with various transcription factors (STAT3, NF-κB, p53, β-catenin) and binds to the promoter regions of target genes to promote their expression. Several studies have shown that MUC1 plays a crucial role in the transcriptional regulation of genes related to tumor invasion, metastasis, angiogenesis, proliferation, apoptosis, drug resistance, inflammation, and immune regulation.

[0068] A detailed review of MUC1 and its function is included in its entirety by reference 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.

[0069] In some embodiments, the bispecific anti-HER3 / MUC1 antibodies described herein can be designed to have an IgG1 subtype structure with a knob-in-hole (KIH) mutation that promotes heterodimerization and avoids mispairs between the two heavy chains. In some embodiments, the bispecific anti-HER3 / MUC1 antibodies have a higher endocytosis rate than the corresponding monoclonal antibody or control bispecific antibody.

[0070] In some embodiments, the bispecific anti-HER3 / MUC1 antibodies described herein can conjugate with a therapeutic agent to form an antibody-drug conjugate (ADC). In some embodiments, the drug-to-antibody ratio (DAR) of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4. 0.4, approximately 3.7-4.4, approximately 3.8-4.4, approximately 3.9-4.4, approximately 4.0-4.4, approximately 4.1-4.4, approximately 4.2-4.4, approximately 4.3-4.4, approximately 3.5-4.3, approximately 3.6-4.3, approximately 3.7-4.3, approximately 3.8-4.3, approximately 3.9-4.3, approximately 4.0-4.3, approximately 4.1-4.3, approximately 4. 2-4.3, 3.5-4.2, 3.6-4.2, 3.7-4.2, 3.8-4.2, 3.9-4.2, 4.0-4.2, 4.1-4.2, 3.5-4.1, 3.6-4.1, 3.7-4.1, 3.8-4.1, 3.9-4.1, 4.0-4.1, 3.5-4.0 The ranges are approximately 3.6-4.0, 3.7-4.0, 3.8-4.0, 3.9-4.0, 3.5-3.9, 3.6-3.9, 3.7-3.9, 3.8-3.9, 3.5-3.8, 3.6-3.8, 3.7-3.8, 3.5-3.7, 3.6-3.7, or 3.5-3.6.In some embodiments, the DAR of the ADC described herein is approximately 7.5 to 8.5, approximately 7.6 to 8.5, approximately 7.7 to 8.5, approximately 7.8 to 8.5, approximately 7.9 to 8.5, approximately 8.0 to 8.5, approximately 8.1 to 8.5, approximately 8.2 to 8.5, approximately 8.3 to 8.5, approximately 8.4 to 8.5, approximately 7.5 to 8.4, approximately 7.6 to 8.5 0.4, approximately 7.7~8.4, approximately 7.8~8.4, approximately 7.9~8.4, approximately 8.0~8.4, approximately 8.1~8.4, approximately 8.2~8.4, approximately 8.3~8.4, approximately 7.5~8.3, approximately 7.6~8.3, approximately 7.7~8.3, approximately 7.8~8.3, approximately 7.9~8.3, approximately 8.0~8.3, approximately 8.1~8.3, approximately 8. 2-approx. 8.3, approx. 7.5-approx. 8.2, approx. 7.6-approx. 8.2, approx. 7.7-approx. 8.2, approx. 7.8-approx. 8.2, approx. 7.9-approx. 8.2, approx. 8.0-approx. 8.2, approx. 8.1-approx. 8.2, approx. 7.5-approx. 8.1, approx. 7.6-approx. 8.1, approx. 7.7-approx. 8.1, approx. 7.8-approx. 8.1, approx. 7.9-approx. 8.1, approx. 8.0-approx. 8.1, approx. 7.5-approx. 8.0 The ranges are approximately 7.6-8.0, 7.7-8.0, 7.8-8.0, 7.9-8.0, 7.5-7.9, 7.6-7.9, 7.7-7.9, 7.8-7.9, 7.5-7.8, 7.6-7.8, 7.7-7.8, 7.5-7.7, 7.6-7.7, or 7.5-7.6.

[0071] In some embodiments, the anti-HER3 / MUC1 ADCs described herein can effectively inhibit cancer cell proliferation in vitro at concentrations of less than 10 μg / ml, less than 3.33 μg / ml, less than 1.11 μg / ml, less than 0.37 μg / ml, less than 0.12 μg / ml, less than 0.04 μg / ml, or less than 0.01 μg / ml. In some embodiments, the anti-HER3 / MUC1 ADCs described herein can inhibit cancer cell proliferation in vivo (e.g., lung cancer, gastric cancer, or skin cancer) in xenograft mouse models at dose levels of less than 10 mg / kg, less than 9 mg / kg, less than 8 mg / kg, less than 7 mg / kg, less than 6 mg / kg, less than 5 mg / kg, less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg.

[0072] In some embodiments, the anti-HER3 / MUC1 antibodies described herein have a common light chain. In some embodiments, the anti-HER3 / MUC1 antibody comprises 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 antibody has a heavy chain variable region targeting HER3 (e.g., any one of the HER3-targeting VHs described herein), a heavy chain variable region targeting MUC1 (e.g., any one of the MUC1-targeting VHs described herein), and two identical common light chain variable regions.

[0073] The CDR sequence of the 1B2 antigen-binding domain includes the CDR of the heavy chain variable domain, sequence numbers 4-6, and the CDR of the light chain variable domain, sequence numbers 1-3, according to the Kabat definition. CDRs can also be defined by the Chothia definition. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are described in sequence numbers 16-18, and the CDR sequences of the light chain variable domain are described in sequence numbers 13-15. The human light chain variable region and human heavy chain variable region of 1B2 are shown in sequence numbers 25 and 26, respectively.

[0074] The CDR sequence of the 3E1 antigen-binding domain includes the CDR of the heavy chain variable domain, sequence numbers 7-9, and the CDR of the light chain variable domain, sequence numbers 1-3, according to Kabat's definition. According to Chothia's definition, the CDR sequence of the heavy chain variable domain is described in sequence numbers 19-21, and the CDR of the light chain variable domain is described in sequence numbers 13-15. The human light chain variable region and human heavy chain variable region of 3E1 are shown in sequence number 25 and sequence number 27, respectively.

[0075] The CDR sequence of the 3G6 antigen-binding domain includes the CDR of the heavy chain variable domain according to Kabat's definition, as well as sequence numbers 7, 42, and 43, and the CDR of the light chain variable domain, as well as sequence numbers 1-3. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are described in sequence numbers 19, 20, and 44, and the CDRs of the light chain variable domain are described in sequence numbers 13-15. The human light chain variable region and human heavy chain variable region of 3E1 are shown in sequence numbers 25 and 45, respectively.

[0076] The CDR sequence of the 10D1 antigen-binding domain includes the CDR of the heavy chain variable domain, sequence numbers 10-12, and the CDR of the light chain variable domain, sequence numbers 1-3, according to Kabat's definition. According to Chothia's definition, the CDR sequence of the heavy chain variable domain is described in sequence numbers 22-24, and the CDR of the light chain variable domain is described in sequence numbers 13-15. The human light chain variable region and human heavy chain variable region of 3E1 are shown in sequence number 25 and sequence number 28, respectively.

[0077] In some embodiments, the anti-HER3 / MUC1 antibodies described herein may include one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, 7-9, 10-12, 7, 42, 43, 16-18, 19-21, 19, 20, 44, and 22-24; and / or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3 and 13-15.

[0078] In some embodiments, an anti-HER3 / MUC1 antibody or antigen-binding fragment may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence, and the CDR1 region comprises or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, and the CDR2 region comprises a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, and the CDR1 region comprises or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, and the CDR2 region comprises a heavy chain variable region (VH) comprising a selected VL The CDR2 amino acid sequence contains or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to the CDR2 amino acid sequence, and the CDR3 region contains or is composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to the selected VL CDR3 amino acid sequence. The selected VH CDR, 1, 2, and 3 amino acid sequences and the selected VL CDR, 1, 2, and 3 amino acid sequences are shown in Figures 3 and 4.

[0079] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 4 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 6 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0080] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 16 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0081] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may include heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 7 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 9 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0082] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0083] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 7 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 43 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0084] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 44 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0085] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0086] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain heavy chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 22 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 24 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0087] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain light chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 1 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 3 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0088] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein may contain light chain variable domains comprising one, two, or three of the CDRs of SEQ ID NO: 13 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 15 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0089] Insertions, deletions, and substitutions can be performed within the CDR sequence or at one or both ends of the CDR sequence.

[0090] In some embodiments, the anti-HER3 / MUC1 antibody comprises a heavy chain variable region (VH) containing or composed of amino acid sequences that are at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) containing or composed of amino acid sequences that are 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.

[0091] In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment may have three VH CDRs identical to those of any VH sequence CDR described herein. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment may have three VL CDRs identical to those of any VL sequence CDR described herein.

[0092] This 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 a CDR as shown in Figure 3, Figure 4, or Figure 5. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to MUC1 and / or HER3.

[0093] The anti-HER3 / MUC1 antibody may also be an anti-HER3 / MUC1 antibody variant (including derivatives and conjugates) of the anti-HER3 / MUC1 antibody or antibody fragment. Additional anti-HER3 / MUC1 antibodies provided herein include polyclonal antibodies, monoclonal antibodies, multispecific antibodies (multimers, e.g., bispecific antibodies), human antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. The anti-HER3 / MUC1 antibody may be 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 its antigen-binding fragment.

[0094] The anti-HER3 / MUC1 antibody fragments are suitable for use in the provided method, provided they retain the desired affinity and specificity for both MUC1 and HER3. Therefore, the anti-HER3 / MUC1 antibody fragments maintain their ability to bind to both MUC1 and HER3.

[0095] Antibodies and their antigen-binding fragments In some embodiments, multispecific anti-HER3 / MUC1 antibodies (e.g., bispecific antibodies) include 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 their antigen-binding fragments can take on a variety of forms.

[0096] Generally, antibodies (also called immunoglobulins) consist of two types of polypeptide chains: a light chain and a heavy chain. The non-limiting anti-HER3 / MUC1 antibodies of this disclosure may be intact four-chain immunoglobulin antibodies containing two heavy chains and two light chains. The heavy chains of the anti-HER3 / MUC1 antibody may be isotypes containing IgM, IgG, IgE, IgA, or IgD, or subisotypes containing IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chains may be kappa light chains or lambda light chains.

[0097] The hypervariable regions, known as complementary determining regions (CDRs), form loops that constitute the primary antigen-binding surface of the antibody. The four framework regions mostly adopt a β-sheet structure, and the CDRs form loops connecting these β-sheet structures, and in some cases, form parts of them. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDR of the other chain, contribute to the formation of the antigen-binding domain.

[0098] Methods for identifying CDR regions by analyzing the amino acid sequence of antibodies are well-known, and many definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al., Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et This is described in al., Nature 342(6252):877-83 (Dec. 1989); and Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007). Each of these is incorporated herein by reference in its entirety.

[0099] CDRs are important for recognizing the epitopes of antigens. As used herein, “epitope” refers to the smallest portion of a target molecule that can specifically bind to the antigen-binding domain of an antibody. The minimum size of an epitope is about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be a continuous linear sequence of the primary structure of the antigen, as epitopes may depend on the stereochemistry of the antigen based on its secondary and tertiary structures.

[0100] In some embodiments, the anti-HER3 / MUC1 antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, IgG4) are highly conserved, but differ in their constant regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are known in the art, for example, as described in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5(2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2(2015):171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016. Each of these is incorporated herein by reference in whole.

[0101] Anti-HER3 / MUC1 antibodies may also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camelids). The antigen-binding domain or antigen-binding fragment is a portion of the antibody that retains the specific binding activity of the intact antibody, i.e., any portion of the antibody that can specifically bind to an epitope on the target molecule of the intact antibody. These include, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, anti-HER3 / MUC1 antibodies or their antigen-binding fragments may consist of, 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 that is an antibody-binding domain or a binding domain homologous to an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs from either the heavy or light chain of an intact antibody.

[0102] In some embodiments, the scFv of 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 the two antigen-binding regions can bind to their respective target antigens with different affinities.

[0103] In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment may include one, two, or three heavy chain variable region CDRs selected from Figures 3 and 4.

[0104] In some embodiments, the anti-HER3 / MUC1 antibodies described herein can be conjugated to a therapeutic agent. The anti-HER3 / MUC1 antibody-drug conjugate, comprising the antibody or its antigen-binding fragment, can be covalently or non-covalently bonded to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or cell activator (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, cortisine, doxorubicin, daunorubicin, dihydroxyanthracine, meitansinoids (DM-1, DM-4, ​​etc.), zione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, cyclophosphamide and its analogues). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, such as a VC linker. Details of the linkers used in ADCs are described, for example, in Su, Z. et al. “Antibody-drug conjugates: Recent advances in linker chemistry.” Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0105] In some embodiments, anti-HER3 / MUC1 antibodies are bispecific antibodies. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers obtained from recombinant cell cultures. For example, the interface may include at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with small amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is formed at the interface of the second antibody molecule. This provides a mechanism that increases the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in International Publication No. 96 / 27011, which is incorporated in its entirety by reference.

[0106] Any anti-HER3 / MUC1 antibody or its antigen-binding fragment described herein may be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in the body 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). By conjugating with a stabilizing molecule, the half-life or biological activity of the anti-HER3 / MUC1 antibody or antigen-binding fragment can be extended in vitro (e.g., when stored in tissue culture or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0107] Anti-HER3 / MUC1 antibodies or their antigen-binding fragments can also take various forms. Many different formats of bispecific antibodies or their antigen-binding fragments are known in the art, for example, described 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.

[0108] In some embodiments, the anti-HER3 / MUC1 antibody is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. In some embodiments, the anti-HER3 / MUC1 antibody is VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, diabody, crossMab, DAF(2-in-one), DAF(4-in-one), DutaMab, DT-IgG, knobs-in-holes common light chain, knob-in-hole assembly, charge pair, Fab-arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH These include IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Miniantibody, Minibody, TriBiMinibody, 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 & Lock, lmmTAC, IgG-IgG Conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0109] In some embodiments, the anti-HER3 / MUC1 antibody may be a TrioMab. In a TrioMab, the two heavy chains are derived from different species, and different sequences restrict heavy-light chain pairing.

[0110] In some embodiments, the anti-HER3 / MUC1 antibody has two distinct heavy chains and one common light chain. Heterodimerization of the heavy chains can be based on knob-in-hole or other heavy chain pairing techniques.

[0111] In some embodiments, bispecific anti-HER3 / MUC1 antibodies can be produced using CrossMAb technology. CrossMAb technology can be used to modify the light chain binding of bispecific heterodimeric IgG antibodies, enabling the creation of various bispecific antibody formats, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) based antibodies. These formats can be derived from existing antibody pairs using domain crossovers without requiring identification of a common light chain, post-translational processing / in vitro chemical assembly, or introduction of a series of mutations to force correct light chain binding. This method is described in Klein et al., “The use of CrossMAb technology for the generation of bi- and multispecific antibodies.” MAbs. Vol.8. No.6. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. In some embodiments, the CH1 domain of the heavy chain and the CL domain of the light chain are swapped.

[0112] Anti-HER3 / MUC1 antibodies can be duobody antibodies. The Fab exchange mechanism that occurs spontaneously with IgG4 antibodies is mimicked in a controlled manner with IgG1 antibodies, a mechanism called controlled Fab exchange. This format ensures specific pairing between the heavy and light chains.

[0113] In bivariable domain antibodies (DVD-Ig), a VH domain and a variable light chain (VL) domain are added to the N-terminus of each antibody for dual targeting. This format is similar to IgG-scFv, but instead of binding scFv to the N-terminus of each heavy chain, the added binding domains are bound to the N-terminus of each antibody.

[0114] In scFv-IgG, two scFv molecules are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format is also called tetravalent because it has two types of divalent binding sites. scFv-IgG does not have the problem of heavy-light chain pairing.

[0115] In some embodiments, the anti-HER3 / MUC1 antibody may have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminuses of their heavy chains.

[0116] Anti-HER3 / MUC1 antibodies can also be produced in Fab-scFv-Fc format. In the Fab-scFv-Fc format, the light chain, heavy chain, and third chain, containing the Fc region and scFv, are combined. This allows for efficient production and purification.

[0117] In some embodiments, the anti-HER3 / MUC1 antibody may be a TF (transfer facilli). Three Fab fragments are linked by disulfide crosslinks. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF format lacks an Fc region.

[0118] ADAPTIR has two scFvs bound to both sides of the Fc region. This construct is based on intact IgG but preserves the Fc region to extend its half-life and facilitate purification.

[0119] Dual affinity retargeting (DART) involves linking opposing fragments with two peptide chains, such as VLA and VHB, or VLB and VHA, and fusing them with a sulfur bond at the C-terminus. In DART, the sulfur bond can provide greater stability than in BiTE.

[0120] In DART-Fc, the Fc region is bound to the DART structure. This can be produced by assembling three chains (two via disulfide bonds), similar to DART. One chain contains half of the Fc region and dimerizes with a third chain expressing only the Fc region. The addition of the Fc region extends the half-life and increases the effective concentration, thus avoiding the need for continuous infusion.

[0121] In tetravalent DART, four peptide chains are combined. Essentially, two DART molecules are created, each possessing half of the Fc region, and then dimerize. This format allows for divalent binding to both targets, resulting in a tetravalent molecule.

[0122] A tandem diabody (TandAb) consists of two diabodies. Each diabody is composed of a VHA and a VLB fragment, and a covalently bonded VHA and VLB fragment. The two diabodies are linked by a peptide chain. This improves stability compared to a diabody consisting of two scFvs. It has two divalent binding sites.

[0123] The ScFv-scFv-toxin contains two scFvs, one with a toxin and the other with a stabilizing linker. This can also be used for specific payload deliveries.

[0124] In some embodiments, the anti-HER3 / MUC1 antibody is a bispecific antibody. In some embodiments, the bispecific antibody in this disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This reduces binding affinity to cells with low levels of HER3 and MUC1 expression, increases avidity to cells co-expressing HER3 and MUC1, and enhances targeting function.

[0125] In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment has a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 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 either SEQ ID NO: 30 or 31.

[0126] In some embodiments, the anti-HER3 / MUC1 antibody contains a KIH mutation. In some embodiments, the anti-HER3 / MUC1 antibody contains 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 contains a heavy chain (knob heavy chain) containing one or more knob mutations, and the second antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations. In some embodiments, the first antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations, and the second antigen-binding domain contains a heavy chain (knob heavy chain) containing one or more knob mutations. In some embodiments, the anti-HER3 / MUC1 antibody includes a knob heavy chain containing a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the anti-HER3 / MUC1 antibody includes a hole heavy chain containing a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31.

[0127] Characteristics of antibodies The anti-HER3 / MUC1 antibody may also contain the anti-HER3 antigen-binding domain and any anti-MUC1 antigen-binding domain described herein.

[0128] The present disclosure provides anti-HER3 / MUC1 antibodies and antigen-binding fragments thereof that specifically bind to HER3. These anti-HER3 / MUC1 antibodies can function as either agonists or antagonists. The anti-HER3 / MUC1 antibodies or antigen-binding fragments thereof described herein can bind to HER3 and inhibit the binding of HER3 to its ligand. By inhibiting the binding of HER3 to its ligand, the anti-HER3 / MUC1 antibodies can inhibit the HER3-related signaling pathway and treat cancer. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof can initiate CMC or ADCC.

[0129] Common techniques that can be used to measure the affinity of an antibody for an antigen include, for example, ELISA, RIA, surface plasmon resonance (SPR), etc. Affinity can be inferred from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment thereof has a dissociation rate (koff) of less than 0.1 s -1 less than, less than 0.01 s -1 less than, less than 0.001 s -1 less than, less than 0.0001 s -1 less than, or less than 0.00001 s -1 and can bind to HER3 (e.g., human HER3, monkey HER3, mouse HER3, and / or chimeric HER3). In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 greater than, greater than 0.001 s -1 greater than, greater than 0.0001 s -1 greater than, greater than 0.00001 s -1 greater than, or greater than 0.000001 s -1 greater than.

[0130] In some embodiments, the reaction association 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 6The reaction association rate (kon) is greater than / Ms. In some embodiments, the reaction association rate (kon) is 1 × 10⁻⁶. 5 / Ms less than 1 × 10 6 Less than / Ms, or 1 × 10⁻⁶ 7 It is less than / Ms.

[0131] In some embodiments, an anti-HER3 / MUC1 antibody or its antigen-binding fragment is used to target HER3 (e.g., human HER3, monkey HER3, mouse HER3, and / or chimeric HER3) at a rate of 1 × 10⁻¹⁴ -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 KD can be bonded at less than M. In some embodiments, KD is less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, KD is 1 × 10⁻⁶ -7 Super M, 1×10 -8 Super M, 1×10 -9 M or 1 × 10 -10 It is greater than M.

[0132] The anti-HER3 / MUC1 antibody or its antigen-binding fragment may also include an antigen-binding domain that specifically binds to MUC1. The anti-HER3 / MUC1 antibody or its antigen-binding fragment described herein can inhibit the binding of MUC1 to its ligand. In some embodiments, by binding to MUC1, the anti-HER3 / MUC1 antibody can also inhibit MUC1-related signaling pathways, thereby inhibiting cell proliferation, differentiation, and / or translocation. Therefore, in some embodiments, the anti-HER3 / MUC1 antibody described herein is a MUC1 agonist. In some embodiments, the anti-HER3 / MUC1 antibody is a MUC1 antagonist.

[0133] In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment is 0.1s -1 Less than 0.01s-1 Less than 0.001s -1 Less than 0.0001s -1 Less than, or 0.00001s -1 It can bind to MUC1 (e.g., human MUC1, monkey MUC1, mouse MUC1, and / or chimeric MUC1) with a dissociation rate (koff) of less than 0.01s. In some embodiments, the dissociation rate (koff) is 0.01s. -1 Larger than 0.001s -1 Larger than 0.0001s -1 Larger than 0.00001s -1 Greater than, or 0.000001s -1 Larger.

[0134] In some embodiments, the reaction association rate (kon) is 1 × 10⁻⁶ 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms greater than, or 1 × 10⁻⁶ 6 The reaction association rate (kon) is greater than / Ms. In some embodiments, the reaction association rate (kon) is 1 × 10⁻⁶. 5 / Ms less than 1 × 10 6 Less than / Ms, or 1 × 10⁻⁶ 7 It is less than / Ms.

[0135] The affinity can be estimated from the quotient of the velocity constant (KD = koff / kon). In some embodiments, KD is 1 × 10⁻⁶ -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, KD is less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, KD is 1 × 10⁻¹⁶ -7 Super M, 1×10 -8 Super M, 1×10 -9 M or 1 × 10 -10 It is greater than M.

[0136] Anti-HER3 / MUC1 antibodies (e.g., bispecific antibodies) bind to both MUC1 and HER3, and therefore have a higher binding affinity to cells that express both MUC1 and HER3. Avidity can be used to measure the binding affinity of an antibody to these cells. Avidity is the cumulative strength of multiple affinities of individual non-covalent interactions.

[0137] Thermal stability can also be measured. The anti-HER3 / MUC1 antibodies or their antigen-binding fragments described herein may have a Tm of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. Because IgG is a multi-domain protein, the melting curve may show two transitions between a first denaturation temperature Tm D1 and a second denaturation temperature Tm D2. The presence of these two peaks often indicates denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When there are two peaks, Tm usually refers to Tm D2. Therefore, in some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment described herein has a Tm D1 higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the anti-HER3 / MUC1 antibody or antigen-binding fragment described herein has a Tm D2 higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, Tm, Tm D1, and Tm D2 are 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 less than 95°C.

[0138] In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment can bind to human HER3 or monkey HER3. In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment cannot bind to human HER3 or monkey HER3. In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment can bind to human MUC1 or monkey MUC1. In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment cannot bind to human MUC1 or monkey MUC1.

[0139] In some embodiments, the anti-HER3 / MUC1 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.

[0140] In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has a purity of 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 residence time of greater than 2 minutes, greater than 3 minutes, greater than 4 minutes, or greater than 5 minutes in hydrophobic interaction chromatography (HIC). In some embodiments, the HIC residence time is less than 2 minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes, or less than 6 minutes.

[0141] In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has a purity of over 85%, over 86%, over 87%, over 88%, over 89%, over 90%, over 91%, over 92%, over 93%, over 94%, over 95%, over 96%, over 97%, over 98%, or over 99%, as determined by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).

[0142] In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has a main peak that accounts for more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of the total sample, as determined by capillary isoelectric focusing (cIEF). In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has an acidic peak that accounts for 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).

[0143] In some embodiments, the anti-HER3 / MUC1 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) 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 / MUC1 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate of less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, less than 110%, less than 120%, less than 130%, less than 140%, or less than 150%. TGI (%) can be measured, for example, on days 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 after the start of treatment. When used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated by the following formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100% 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, and V0 is the mean tumor volume of the control group on day 0.

[0144] 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 functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0145] 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 its antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the anti-HER3 / MUC1 antibody or its antigen-binding fragment described herein has an Fc region that does not have effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (EU numbers L234A and L235A mutations) or an LALA-PG mutation (EU numbers L234A, L235A, and P329G mutations).

[0146] The Fc region can be modified in several other ways. For example, introducing a cysteine ​​residue into the Fc region can enable the formation of interchain disulfide bonds in this region. The homodimer fusion protein thus produced may have an increased half-life in vitro and / or in vivo.

[0147] In some embodiments, IgG4 has the S228P mutation (EU numbered). The S228P mutation inhibits IgG4 Fab-arm exchange in vivo and in vitro.

[0148] In some embodiments, an Fc region is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an Fc region composition can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the total of all sugar structures (e.g., complex structures, hybrid structures, high-mannose structures) bound to Asn297 as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located approximately 297th in the Fc region (Eu numbering of Fc region residues; or 314th in Kabat numbering). However, due to slight sequence variations in the Fc region sequence, Asn297 may also be located ±3 amino acids upstream or downstream of 297, i.e., between 294th and 300th. Such fucosylated variants may enhance ADCC function. In some embodiments, to reduce heterogeneity of the sugar chain, the Fc region can be further manipulated to replace the asparagine at position 297 with alanine (N297A).

[0149] In some embodiments, the main peaks of HPLC-SEC account for at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 100% of the protein complex described herein after purification by protein A-based affinity chromatography and / or size-exclusive chromatography.

[0150] 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.

[0151] In some embodiments, the bispecific anti-HER3 / MUC1 antibodies described herein have a higher endocytosis rate than the corresponding monoclonal antibodies and / or control bispecific antibodies described herein. In some embodiments, the anti-HER3 / MUC1 antibodies described herein have a higher endocytosis rate than the patritumab analogs and / or gatipotuzumab analogs.

[0152] Antibody-drug conjugate (ADC) The anti-HER3 / MUC1 antibodies or their antigen-binding fragments described herein can be conjugated to therapeutic agents (drugs). The therapeutic agent can be covalently or noncovalently 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.

[0153] In some embodiments, the therapeutic agent is a cytotoxic agent or a cell activator (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, cortisine, doxorubicin, daunorubicin, dihydroxyanthracine, meitansinoids (DM-1, DM-4, ​​etc.), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, cyclophosphamide and its analogs). Useful classes of cytotoxic agents, cell quiescent agents, and immunomodulators include, for example, antitubulins, DNA sulcus binding agents, DNA replication inhibitors, and alkylating agents.

[0154] In some embodiments, the therapeutic agent may include, but is not limited to, cytotoxic reagents such as chemotherapeutic agents and immunotherapeutic agents, antiviral agents, or antibacterial agents. In some embodiments, the conjugated therapeutic agent may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0155] Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this invention, chemical elements are defined using the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75). th Identified according to the edition (inside cover), specific functional groups are generally defined as described therein. Furthermore, for general principles of organic chemistry, specific functional groups and reactivity, see Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 thEdition, John Wiley&Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3 rd This information is found in Edition, Cambridge University Press, Cambridge, 1987.

[0156] All scopes referenced herein are inclusive unless expressly stated otherwise. Where a range of values ​​is given, it is intended to include each value and subrange within that range. For example, "C 1~6 " refers to 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 , C 5~6 It is intended to encompass.

[0157] The compounds of this disclosure or any formula describing and explaining the compounds may have one or more chiral centers. The present invention encompasses all stereoisomers or any formula of the compounds describing and explaining the compounds of the present invention. All asymmetric centers present in the compounds of the present invention or formulas describing the compounds may independently have (R) or (S) configurations. When a bond to a chiral carbon is depicted as a straight line in a structural formula, or when a compound name is listed without a (R) or (S) chiral designation for the chiral carbon, it is understood that both the (R) and (S) stereoconfigurations of each such chiral carbon, and therefore each enantiomer or diastereomer and mixtures thereof, are encompassed in the formula or by name.

[0158] This disclosure includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, for example, mixtures of enantiomers and / or diastereomers in all proportions. Thus, enantiomers are the subject of this disclosure in both levorotatory and dextrorotatory pairs, in enantiomerically pure forms, in racemic forms, and in mixtures of two enantiomers in all proportions. In the case of cis / trans isomers, this disclosure includes both cis and trans forms, and mixtures of these in all proportions. Preparation of individual stereoisomers can be carried out, if necessary, by separation of mixtures by conventional methods, e.g., chromatography or crystallization, by the use of stereochemically homogeneous starting materials for synthesis, or by stereoselective synthesis. Optionally, derivatization may be carried out before separation of stereoisomers. Separation of mixtures of stereoisomers can be carried out as an intermediate step in the synthesis of the compound, or against the final racemic product. Absolute stereochemistry can be determined, if necessary, by X-ray crystallography of crystalline products or intermediates derivatized with reagents containing chiral centers of known configurations. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0159] Unless otherwise specified, the structures shown in this specification include compounds that differ only in the presence of one or more isotope-enriched atoms, i.e., compounds in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number from the naturally predominant atomic mass or mass number. Such compounds are referred to as "isotope variants." This disclosure is intended to include compounds that depict and describe the compounds of the invention or all pharmaceutically acceptable isotope variants of any formula. Examples of isotopes suitable for inclusion in the compounds of the invention include 2 H (i.e., D) and 3 isotopes of hydrogen such as 11 C, 13 C, and 14 C, etc., of carbon; 36 Cl, etc., of chlorine; 18 F, etc., of fluorine; 123 I and 125 I, etc., of iodine; 13 N, 15 N, etc., of nitrogen; 15 O, 17 O, 18 O, etc., of oxygen; 32 P, etc., of phosphorus; 35 S, etc., of sulfur, but are not limited thereto. Compounds that depict and describe the compounds of this disclosure or specific isotope variants of any formula, such as those incorporating radioactive isotopes, may be useful in the study of drug and / or substrate tissue distribution. In particular, compounds having depicted structures that differ only in substitution with heavier isotopes, such as substitution of hydrogen by deuterium ( 2 H, or D), can provide certain therapeutic advantages resulting from, for example, higher metabolic stability, increased in vivo half-life, or a reduced need for dosage, and can thus be utilized in some specific situations. The compounds of this disclosure or isotope variants of any formula that depict and describe the compounds can generally be prepared by techniques known to those of ordinary skill in the art or by synthesis using appropriate isotope-labeled reagents in processes similar to those described in the accompanying examples and using the appropriate isotope-labeled reagents in place of the previously used unlabeled reagents.

[0160] The compounds provided herein are described by reference to both their general formulas and specific compounds. Furthermore, the compounds of this disclosure may exist in many different forms or derivatives, all of which are within the scope of this disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvates, different crystalline forms or polymorphs, and active metabolites.

[0161] As used herein, the term “pharmaceutically acceptable salt” includes, unless otherwise specified, salts that retain the biological efficacy of the free acid / base form of a particular compound and are biologically or otherwise undesirable. A pharmaceutically acceptable salt may include salts formed with an inorganic base or acid and an organic base or acid. Where a compound of the Disclosure contains one or more acidic or basic groups, the Disclosure also includes the corresponding pharmaceutically acceptable salts thereof. Thus, compounds of the Invention containing acidic groups such as carboxyl groups can exist in the form of salts and, according to the Invention, can be used, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts, or ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines, such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts can be readily obtained, for example, by reacting a compound having an acidic group with a suitable base, such as lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of the compounds of this disclosure include, but are not limited to, copper(I), copper(II), iron(II), iron(III), manganese(II), and zinc salts. Compounds of this disclosure containing one or more basic groups, such as protonable groups, can exist in the form of salts and can be used in accordance with this disclosure in the form of addition salts with inorganic or organic acids.Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, diethylacetic acid pivalate, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, as well as other acids well known to those skilled in the art. The salts formed are, in particular, hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), tosylate, carbonate, bicarbonate, formate, acetate, sulfoacetate, triflate, oxalate, malonate, maleate, succinate, tartrate, malate, embonate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate, glutamate, and the like. The stoichiometry of the salts formed from the compounds of this disclosure may further be an integer multiple of 1 or a non-integer multiple.

[0162] The compounds of this disclosure containing a basic nitrogen-containing group are C 1~4 Alkyl halides, such as methyl, ethyl, isopropyl, and tert-butyl chloride, bromide, and iodide; diC 1~4 Alkyl sulfates, such as dimethyl, diethyl, and diamyl sulfates; C 10~18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aryl C 1~4 Quaternization can be performed using alkyl halides, such as benzyl chloride and phenethyl bromide.

[0163] Where the compounds of this disclosure contain both acidic and basic groups in their molecules, this disclosure also includes intramolecular salts or betaines (zwitterions) in addition to the salt forms mentioned. Each salt can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. This disclosure also includes all salts of the compounds of this disclosure that are not directly suitable for pharmaceutical use due to their poor physiological compatibility, but can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. For a general overview of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0164] The compounds or any formulas describing and illustrating the compounds of this disclosure and their pharmaceutically acceptable salts may exist in both non-solvated and solvated forms. As used herein, the term “solvate” refers to a molecular complex comprising the compound of formula (I) or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable solvent molecules. For example, the term “hydrate” is used when the solvent is water.

[0165] Examples of pharmaceutically acceptable solvates in accordance with this disclosure include those in which the crystallization solvent may be isotope-substituted, such as D2O, d6-acetone, and d6-DMSO.

[0166] Linker (binding compound) In some embodiments, the therapeutic agent is conjugated via a linker (or coupling compound). As used herein, the terms “linker” or “coupling compound” refer to a compound that can link a ligand (e.g., an antibody or its antigen-binding fragment as described herein) and a therapeutic agent (e.g., any of the therapeutic agents as described herein) together to form a ligand-drug conjugate by reacting with the groups of the ligand compound and the therapeutic agent compound, respectively, for example, by a coupling reaction.

[0167] In some embodiments, the linker described herein is a compound having the following formula: QL Equation (I) A compound that is a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein Q represents a junction portion capable of binding to a ligand via a bond selected from the group consisting of carbonyl bonds, thioether bonds, amide bonds, disulfide bonds, and hydrazone bonds; and L represents a linker portion capable of linking Q to a therapeutic agent.

[0168] In some embodiments, the joint portion (Q in formula (I)) has the following structure. [ka]

[0169] In some embodiments, the linker portion (L in formula (I)) has the following formula: [ka] In the formula, L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, and includes an amino acid residue having at least one side-chain carboxyl group, such as a glutamic acid residue or an aspartic acid residue, and "-COOH" indicates the carboxyl group of the amino acid residue at the C-terminus of the polypeptide residue; L2 is either absent or is a mono-, di-, or tri-hydrophilic group bonded to the side-chain carboxyl group on the amino acid residue of the polypeptide residue L1, and L2 has the structure of -NHC(R L2a )(R L2b )(R L2c ), where R L2a , R L2b and R L2c are each independently selected from the group consisting of H, -(CH2O)(CH2CH2O) m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d , and R L2d is H or C 1~6 alkyl optionally substituted with 1 to 6 hydroxy groups, each m is independently an integer from 0 to 10, preferably 0 to 4, such as 0, 1, 2, 3, or 4, and particularly preferably m is 0, and each p is independently an integer from 1 to 4, such as 1, 2, 3, or 4;

Chemical formula

[0170] In some embodiments, the polypeptide residue L1 is NH -Glu-Val-Ala-[[ID=�0]] COOH . In some embodiments, the hydrophilic group L2 has the following structure,

Chemical formula

[0171] In some embodiments, the linker described herein is a compound having the following structure.

Chemical formula

[0172] In some embodiments, the linker is a VC linker. Details of the linker used in ADCs are, for example, described 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.

[0173] Therapeutic drugs In some embodiments, the therapeutic agent conjugated to the antibody or antigen-binding fragment described herein is described as follows.

[0174] In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, its analogue or derivative. In some preferred embodiments, the camptothecin compound is a compound having the following structure: [ka] In the formula, X is selected from the group consisting of -CH2-, O, and S; Y is selected from the group consisting of H, D, and F.

[0175] 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]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione) (CPT-1). The structure of CPT-1 is shown below. [ka]

[0176] 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]indolidino[1,2-b]quinoline-10,13(2H)-dione (CPT-2). The structure of CPT-2 is shown below. [ka]

[0177] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below. [ka]

[0178] 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]indolidino[1,2-b]quinoline-10,13(2H)-dione (CPT-4). The structure of CPT-4 is shown below. [ka]

[0179] In some embodiments, the therapeutic agent is auristatin, for example, auristatin E (also known in the art as a derivative of drastatin-10) or a derivative thereof. The auristatin may be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other representative auristatins include AFP, MMAF, and MMAE. Exemplary auristatin synthesis and structure are described in U.S. Patent Application Publication No. 2003-0083263, International Patent Publication No. 04 / 010957, International Patent Publication No. 02 / 088172, and U.S. Patents No. 7,498,298, No. 6,884,869, No. 6,323,315, No. 6,239,104, No. 6,034,065, No. 5,780,588, No. 5,665,860, and No. 5,663,149. These documents are described in the following specifications: Specification No. 5,635,483, No. 5,599,902, No. 5,554,725, No. 5,530,097, No. 5,521,284, No. 5,504,191, No. 5,410,024, No. 5,138,036, No. 5,076,973, No. 4,986,988, No. 4,978,744, No. 4,879,278, No. 4,816,444, and No. 4,486,414. These documents are incorporated herein by reference in whole and for all purposes.

[0180] Auristatin has been shown to inhibit microtubule dynamics, nuclear mitosis, and cell division, and to have anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell division-inhibiting effects on cancer cells. There are several different assays known in the art that can be used to determine whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation-inhibiting or cytotoxic effects on desired cells.

[0181] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, methuludopa, and urdopa; ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethiophosphaoramide, and trimethylolmelamine; chlorambucil, chlornafadin, colophosphamide, and estram Nitrogen mustards such as stine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kakutinomycin, calitiamicin, carabicin, carminomycin, cardinophilin, and chromoma Antibiotics such as ycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, merceromycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; methotrexate, 5-fluorouracil (5-F Antimetabolites such as U; folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmopher, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone;Anti-adrenergic agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofsphamide glycosides; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edatraxate; defofamin; demecolsin; diazicon; elfornitine; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet Pirarubicin; Podophyllic acid; 2-Ethyl hydrazide; Procarbazine; PSK7; Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2',2',2'-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxanes, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ and doxetaxel (TAXOTERE®, 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; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition also includes antiestrogens such as tamoxifen, raloxifen, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston);This also includes anti-hormone agents that act to modulate or inhibit the hormonal effects on tumors, such as flutamide, nilutamide, bicalutamide, leuprolide, and anti-androgens such as goserelin; as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. A detailed description of the chemotherapeutic agents can be found, for example, in U.S. Patent Application Publication No. 20180193477A1, which is incorporated in its entirety by reference.

[0182] Linker - therapeutic compound In some embodiments, a linker (e.g., any of the linkers described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) can be linked to form a "linker-therapeutic agent" compound.

[0183] In some embodiments, the linker-therapeutic compound has the following structure. [ka]

[0184] In some embodiments, the linker-therapeutic compound has the following structure. [ka]

[0185] In some embodiments, an antibody ("Ab"), for example, either an antibody described herein or an antigen-binding fragment thereof, can be linked to a linker-therapeutic compound (for example, any of the linker-therapeutic compounds described herein) to generate an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate has the following structure: [ka] In the formula, n = 1 to 8. In some embodiments, n = 1 to 8. In some embodiments, n is approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8. In some embodiments, n is approximately 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 multiple or a non-integer multiple of 1.

[0186] 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 sulfoSMCC. Selecting an appropriate linker for a given ADC can be easily done by a person skilled in the art, taking into account relevant factors such as the binding site to the anti-HER3 / MUC1 antibody, the structural constraints of the drug, and the hydrophobicity of the drug (see, for example, the overview in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). Numerous specific linker-toxin combinations have been described and can be used in certain embodiments with the anti-HER3 / MUC1 antibody or its antigen-binding fragment described herein to prepare an ADC. Examples of cleavable peptide-based linkers include, but are not limited to, auristatins such as MMAE and MMAF, camptothecin such as SN-38, duocalmycin and PBD dimer; insoluble MC-based linkers with auristatin MMAF and MMAE; acid-soluble hydrazone-based linkers with calichiamicin and doxorubicin; disulfide-based linkers with mayansinoids such as DM1 and DM4; and bis-maleimide-trioxyethylene glycol (BMPEO)-based linkers with mayansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Application Publication No. 2015 / 0374847; and U.S. Patent Application Publication No. US20180193477A1, which are incorporated herein by reference as a whole.

[0187] Depending on the desired drug and the selected linker, those skilled in the art can select an appropriate method for coupling them. For example, several conventional coupling methods, such as amine coupling, can be used to form a desired drug-linker complex containing a reactive group for covalently binding to an anti-HER3 / MUC1 antibody or its antigen-binding fragment. In some embodiments, a drug-maleimide complex (i.e., a maleimide-linked drug) can be used in the payload having the reactive group in this disclosure. In ADC preparation, maleimide is the most common reactive group that can bind to a thiol group. In addition, organobromids and iodides are also frequently used.

[0188] Anti-HER3 / MUC1 ADCs can be prepared by one of several routes known in the art using organic chemical reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G. Thermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) reacting the nucleophile or electrophile of the antibody with a divalent linker reagent to form an antibody-linker intermediate Ab-L via covalent bonding, and then reacting it with an activated drug moiety D; or (2) reacting the nucleophile or electrophile of the drug moiety with a linker reagent to form a drug-linker intermediate DL via covalent bonding, and then reacting it with the nucleophile or electrophile of the antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug sites, and linkers to prepare the anti-HER3 / MUC1 ADCs described herein. A variety of linkers, linker components, and toxins are commercially available, but can also be prepared using standard organic synthesis techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G. Thermanson, 2013, Academic Press); U.S. Patent Application Publication No. 20210379193A1 and U.S. Patent Application Publication No. 20180193477A1, which are incorporated herein by reference in their entirety. In addition, a number of pre-formed drug linkers suitable for reaction with selected anti-HER3 / MUC1 antibodies or antigen-binding fragments are commercially available. For example, linker toxins containing DM1, DM4, MMAE, MMAF, or Duocarmycin SA are available from Creative BioLabs (Shirley, NY).

[0189] Several specific examples of methods for preparing anti-HER3 / MUC1 ADCs are known in the art and are described in U.S. Patent No. 8,624,003 (POT method), U.S. Patent No. 8,163,888 (one-step method), and U.S. Patent No. 5,208,020 (two-step method) and U.S. Patent No. US20180193477A1, which are incorporated herein by reference in their entirety. Other methods are known in the art and are described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0190] Drug load is expressed as the number of drug moieties per antibody in the ADC molecule. Depending on the antibody-drug conjugate, the drug load may be limited by the number of binding sites on the antibody. For example, if the binding is cysteinethiol, as in certain exemplary embodiments described herein, the drug load may range from 0 to 8 drug moieties per antibody. In certain embodiments, a high drug load, e.g., p≧5, may cause aggregation, insolubilization, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug load of an anti-HER3 / MUC1 antibody-drug conjugate ranges from 1 to about 8; about 2 to about 6; or about 3 to about 5. In fact, for certain antibody-drug conjugates, the optimal ratio of drug moieties per antibody has been shown to be around 4. In some embodiments, the DAR of an 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 / MUC1 ADC composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0191] In some embodiments, anti-HER3 / MUC1 antibody variants are provided that have carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such antibodies can be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all sugar structures (e.g., complex structures, hybrid structures, high-mannose structures) bound to Asn297 as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located approximately 297th in the Fc region (Eu numbering of Fc region residues; 314th in Kabat numbering); however, due to slight sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of 297, i.e., between 294th and 300th. Such fucosylated variants may enhance ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-HER3 / MUC1 antibody can be further manipulated to replace asparagine at position 297 with alanine (N297A).

[0192] In some embodiments, to avoid Fab-arm exchange and promote production efficiency, the Fc region of the anti-HER3 / MUC1 antibody or its antigen-binding fragment was further engineered to replace serine at position 228 (EU number) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al. “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.” Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated herein by reference in its entirety.

[0193] In some embodiments, the methods described herein are designed to produce bispecific anti-HER3 / MUC1 antibodies. Bispecific anti-HER3 / MUC1 antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers obtained from recombinant cell culture. For example, the interface can include at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with small amino acid side chains (e.g., alanine or threonine), a compensatory “hole” of the same or similar size as the large side chain is formed at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011 pamphlet, which is incorporated herein by reference in its entirety.

[0194] In some embodiments, the KIH (knobs-into-holes) technique can be used. This involves engineering the CH3 domain to form either a "knob" or a "hole" in each heavy chain to promote heterodimerization. The KIH technique 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 hereby 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 can have one or more substitutions of E356C, T366S, L368A, Y407V (EU number). Additionally, a substitution (-ppcpScp-->-ppcpPcp-) can also be introduced into the hinge region of both substituted IgGs.

[0195] Recombinant vector The present disclosure also includes recombinant vectors (e.g., expression vectors) containing the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotide and / or the vector containing the polynucleotide), and the production of anti-HER3 / MUC1 antibody polypeptides or fragments thereof by recombinant techniques.

[0196] As used herein, “vector” is a 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” can deliver and express one or more polynucleotides of interest as encoded polypeptides in the host cell into which the expression vector has been introduced. In an expression vector, the polynucleotides of interest are positioned for expression within the vector by being operably ligated with regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or near the integration site of the polynucleotides of interest or in the host cell genome near thereto, so that the polynucleotides of interest are translated in the host cell into which the expression vector has been introduced.

[0197] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0198] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia virus or other smallpox virus, retrovirus, or adenovirus), which may include the use of non-pathogenic (deficient), replication-competent viruses, or replication-deficient viruses. In the latter case, viral replication generally occurs only in complementary viral packaging cells. Suitable systems include, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NYA Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Patents No. 4,603,112, No. 4,769,330, and No. 5,017,487, International Publication No. 89 / 01973; U.S. Patent No. 4,777,127; British Patent No. 2,200,651; European Patent No. 0,345,242; International Publication No. 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. This is disclosed in al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692.By coating DNA onto biodegradable beads and efficiently transporting it into cells, the uptake of naked DNA can be increased.

[0199] For expression, a DNA insert containing a polypeptide-coding polynucleotide disclosed herein can be operably ligated to a suitable promoter (e.g., a heterologous promoter), such as the phage-lambda PL promoter, the Escherichia coli (E. coli) lac, trp and tac promoters, the SV40 early and late promoters, and the promoter of retroviral LTRs. Other suitable promoters are well known to those skilled in the art. The expression construct may further include a transcription start site, a termination site, and a transcription region containing a ribosome-binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation start codon and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide being translated.

[0200] As shown, the expression vector may contain at least one selection marker. Such markers include dihydrofolate reductase and neomycin resistance for eukaryotic cell cultures, and tetracycline resistance genes and ampicillin resistance genes for Escherichia coli (E. coli) and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli (E. coli), Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 cells and Spodoptera Sf9 cells; animal cells such as CHO cells, COS cells, Bowes melanoma cells, and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0201] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0202] Suitable non-restrictive bacterial promoters include the Escherichia coli (E. coli) lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic promoters include the CMV immediate-type early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters for retroviral LTRs such as Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0203] In budding yeast (Saccharomyces cerevisiae), many vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH can be used. For an overview, 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).

[0204] The introduction of constructs into host cells can be carried out 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 experimental manuals, e.g., Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference in their entirety.

[0205] The transcription of the DNA encoding the anti-HER3 / MUC1 antibody of this disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, typically 10-300 bp in length, and function to increase the transcriptional activity of the promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located 100-270 base pairs late at the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located late at the origin of replication, and the adenovirus enhancer.

[0206] Appropriate secretory signals can be incorporated into the expressed polypeptide to secrete the translated protein into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. The signal may be endogenous to the polypeptide or it may be a heterologous signal.

[0207] Polypeptides (e.g., anti-HER3 / MUC1 antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST fusions) or histidine tags, and may contain not only secretory signals but also heterologous functional regions. For example, amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and persistence within host cells, during purification, or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to promote secretion or efflux, improve stability, or facilitate purification is a well-known and common technique in the art.

[0208] This disclosure also relates to 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%, and 99% identical to any nucleotide sequence described herein. We also provide 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%, and 99% identical to the amino acid sequences described herein.

[0209] This disclosure also relates to 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%, and 99% homology to any nucleotide sequence described herein. We also provide amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology to any amino acid sequence described herein.

[0210] In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence 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.

[0211] In some embodiments, the amino acid sequence consists of (i) an amino acid sequence; or (ii) an amino acid sequence, which is one of the sequences described herein.

[0212] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) comprises a nucleic acid sequence, which is any one of the sequences described herein.

[0213] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Next, the amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then 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 percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For example, the comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using a Blossum62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0214] The percentage of sequence identity (e.g., amino acid sequence identity or nucleic acid identity) can also be determined. Methods for determining the percentage of sequence identity are well known in the art. In some embodiments, amino acid residues conserved with similar physicochemical properties (percent identity), such as leucine and isoleucine, can be used in the measurement of sequence similarity. Families of amino acid residues with similar physicochemical properties are defined in the art. These families include, for example, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (threonine, valine, isoleucine, etc.), aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine, etc.). The percentage of homology is often higher than the percentage of identity.

[0215] The present disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) includes a polynucleotide encoding a heavy chain polypeptide as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a light chain polypeptide as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding an scFv polypeptide as described herein.

[0216] In some embodiments, the vector can have two of the nucleic acids described herein, where the vector encodes a VL region and a VH region that together bind to HER3. In some embodiments, a pair of vectors is provided, each vector including one of the nucleic acids described herein, and the pair of vectors together encode a VL region and a VH region that bind to HER3.

[0217] In some embodiments, the vector comprises two nucleic acids described herein, where the vector encodes a VL region and a VH region that bind together to MUC1. In some embodiments, a pair of vectors is provided, each vector comprising one nucleic acid described herein, where the pair of vectors encodes a VL region and a VH region that bind together to MUC1.

[0218] Treatment method The methods described herein include the treatment of cancer-related disorders. Generally, these methods involve administering a therapeutically effective dose of the anti-HER3 / MUC1 antibody or anti-HER3 / MUC1 antibody-drug conjugate described herein to a subject who is in need of such treatment or who has been determined to be in need of such treatment.

[0219] In this context, "treatment" means improving at least one symptom of a cancer-related disorder. Since cancer is often fatal, treatment can extend life expectancy (for example, by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). Administration of a therapeutically effective dose of the drugs described herein for the treatment of a cancer-related condition will result in a reduction in the number of cancer cells and / or a reduction in symptoms.

[0220] As used herein, the term “cancer” refers to a state or condition characterized by autonomous cell proliferation, i.e., rapid cell proliferation. This term is intended to include all types of cancerous proliferation or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. As used herein, the term “tumor” refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems affecting the lungs, breasts, thyroid, lymphatic system, gastrointestinal tract, and genitourinary system, as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung carcinomas, small intestine cancers, and esophageal cancers. In some embodiments, the agents described herein are designed to treat or diagnose carcinomas in a subject. The term "carcinoma" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissue, including respiratory carcinomas, gastrointestinal carcinomas, genitourinary carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine carcinomas, and melanomas. In some embodiments, carcinoma is renal carcinoma or melanoma. Exemplary carcinomas include those formed from tissues of the cervix, lungs, prostate, breast, head and neck, colon, and ovaries. The term also includes carcinomas, for example, malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to carcinomas originating from glandular tissue, or carcinomas in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to malignant tumors of mesenchymal origin.

[0221] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0222] In one embodiment, the disclosure also provides a method for treating cancer in a subject, a method for reducing the rate of increase in tumor volume over time in a subject, a method for reducing the risk of developing metastasis, or a method for reducing the risk of developing further metastasis in a subject. In some embodiments, the treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0223] In one embodiment, the Disclosure is characterized by a method comprising administering a therapeutically effective amount of the anti-HER3 / MUC1 antibody or anti-HER3 / MUC1 antibody-drug conjugate disclosed herein to a subject in need thereof, e.g., cancer, e.g., solid tumors, 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, central nervous system cancer, liver cancer, nasopharyngeal cancer, brain tumor, colon cancer, bladder cancer, oral squamous cell carcinoma, cervical cancer, or esophageal cancer. In some embodiments, cancers include esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial carcinoma, lung cancer, melanoma, ovarian cancer, bladder cancer, gastric cancer, non-Hodgkin lymphoma, head and neck cancer, pancreatic cancer, and cervical cancer.

[0224] Where used herein, the terms “subject” and “patient” are interchangeable throughout this specification and refer to a human or non-human animal to which treatment by the method of the present invention is provided. The present invention is intended for veterinary and non-veterinary applications. A human patient may be an adult human or a young human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (such as monkeys, chimpanzees, gorillas), rodents (such as rats, mice, gerbils, hamsters, ferrets, rabbits), ragmorphs, pigs (such as pigs, miniature pigs), horses, dogs, cats, cattle, and other livestock, farm animals, and zoo animals.

[0225] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Cancer patients can be identified in various ways known in the art.

[0226] As used herein, “effective dose” means an amount or dose sufficient to produce a beneficial or desired outcome, such as stopping, slowing, delaying, or inhibiting the progression of a disease, for example, cancer. The effective dose will vary depending on the age and weight of the person to whom the anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, anti-HER3 / MUC1 antibody-drug conjugate, anti-HER3 / MUC1 antibody-coding polynucleotide, polynucleotide-containing vector, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the administration may be determined on an individual basis.

[0227] The effective dose may be administered in one or more divided doses. For example, the effective dose of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 antibody-drug conjugate is sufficient to improve, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of autoimmune disease or cancer in a patient, or 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 is understood in the art, the effective dose of an anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 antibody-drug conjugate may vary depending on other factors, particularly the patient's medical history, as well as the type (and / or dosage) of the drug used.

[0228] The effective doses and schedules for administering the anti-HER3 / MUC1 antibodies, their anti-HER3 / MUC1 antigen-binding fragments, the polynucleotides encoding the anti-HER3 / MUC1 antibodies, the anti-HER3 / MUC1 antibody-drug conjugates, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the scope of the art. A person skilled in the art will understand that the dose to be administered will vary depending, for example, on the mammal to which the anti-HER3 / MUC1 antibody, its anti-HER3 / MUC1 antigen-binding fragments, the anti-HER3 / MUC1 antibody-encoding polynucleotides, the anti-HER3 / MUC1 antibody-drug conjugates, and / or compositions disclosed herein are administered, on the route of administration, on the specific type of drug or composition disclosed herein used, and on other drugs administered to the mammal.

[0229] The typical effective daily dose of anti-HER3 / MUC1 antibody or anti-HER3 / MUC1 ADC is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose is less than 100 mg / kg, less than 30 mg / kg, less than 20 mg / kg, less than 10 mg / kg, less than 9 mg / kg, less than 8 mg / kg, less than 7 mg / kg, less than 6 mg / kg, less than 5 mg / kg, less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, less than 1 mg / kg, less than 0.5 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dose can be 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or greater than 0.01 mg / kg. In some embodiments, the dosage is approximately 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.

[0230] In any of the methods described herein, at least one anti-HER3 / MUC1 antibody, its anti-HER3 / MUC1 antigen-binding fragment, an anti-HER3 / MUC1 antibody-drug conjugate, or a pharmaceutical composition (e.g., comprising an anti-HER3 / MUC1 antibody, an anti-HER3 / MUC1 antigen-binding antibody fragment, or an anti-HER3 / MUC1 ADC) may be administered to the target patient, and optionally, at least one additional therapeutic agent (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).

[0231] In some embodiments, one or more additional therapeutic agents can 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., one of the 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 and at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 antibody-drug conjugate are administered to the subject such that the biological activity periods of the one or more additional therapeutic agents and at least one anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 ADC overlap.

[0232] In some embodiments, the subject is administered at least one anti-HER3 / MUC1 antibody, an anti-HER3 / MUC1 antigen-binding antibody fragment, an anti-HER3 / MUC1 antibody-drug conjugate, or a pharmaceutical composition (e.g., comprising any of the anti-HER3 / MUC1 antibody, anti-HER3 / MUC1 antigen-binding antibody fragment, or anti-HER3 / MUC1 ADC) over a long period of time (e.g., 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 may determine the length of the treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., observation of at least one symptom of cancer). As described herein, experienced healthcare professionals may change (e.g., increase or decrease) the identity and number of anti-HER3 / MUC1 antibodies or anti-HER3 / MUC1 antigen-binding antibody fragments, anti-HER3 / MUC1 antibody-drug conjugates (and / or one or more additional therapeutic agents) administered to a subject, and may also adjust (e.g., increase or decrease) the dose or frequency of administration 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 additional therapeutic agents) to a subject based on an assessment of the efficacy of the treatment (e.g., using any of the methods described herein or known in the art).

[0233] In some embodiments, one or more additional therapeutic agents may be administered. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of B-Raf inhibitors, HER3 inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, MUC1 inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3 kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors, etc. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0234] In some embodiments, additional therapeutic agents may include one or more inhibitors selected from the group consisting of HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated Hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0235] In some embodiments, additional therapeutic agents include trabectedin, nab-paclitaxel, trevananib, pazopanib, sediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolisin, alimta, zykadia, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, and AGS. -003 may include one or more therapeutic agents selected from the group consisting of cabozantinib, vinflunin, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, salomib, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0236] In some embodiments, additional 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, therapeutic agents targeting CX3CL1, therapeutic agents targeting CXCL9, therapeutic agents targeting CXCL10, therapeutic agents targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0237] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the target patient.

[0238] In some embodiments, further therapeutic agents include anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA4 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-4-1BB antibody, anti-TIM3 antibody, or anti-GITR antibody.

[0239] Pharmaceutical composition and route of administration Furthermore, this specification provides pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the anti-HER3 / MUC1 antibodies described herein (e.g., bispecific antibodies), anti-HER3 / MUC1 antigen-binding fragments, or anti-HER3 / MUC1 antibody-drug conjugates. The pharmaceutical compositions may be formulated in any form known in the art.

[0240] Pharmaceutical compositions are formulated to be compatible with the intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), fixatives, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, phosphates), isotonic agents (e.g., sugars, glucose), polyhydric alcohols (e.g., mannitol, sorbitol), or their salts (e.g., sodium chloride), or combinations thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). The formulations of the composition can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. If necessary (for example, as in injectable formulations), adequate fluidity can be maintained by the use of coatings such as lecithin or surfactants. The absorption of anti-HER3 / MUC1 antibodies, their anti-HER3 / MUC1 antigen-binding fragments, or anti-HER3 / MUC1 ADCs can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, release control can be achieved by microencapsulation delivery systems, which may include implants or biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0241] Compositions comprising one or more of the anti-HER3 / MUC1 antibodies, anti-HER3 / MUC1 antigen-binding fragments, or anti-HER3 / MUC1 antibody-drug conjugates described herein can be formulated in dose unit form (i.e., physically discontinuous units containing a predetermined amount of the active compound for ease of administration and uniformity of dosage) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0242] The toxicity and therapeutic efficacy of a composition can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population): i.e., the therapeutic index (ratio of LD50 to ED50) can be determined. Drugs exhibiting a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., mitigate the undesirable side effects). Toxicity and therapeutic efficacy can be evaluated by other standard pharmaceutical procedures.

[0243] Data obtained from cell culture assays and animal studies can be used when formulating any given drug in an appropriate dosage for use in subjects (e.g., humans). A therapeutically effective amount of anti-HER3 / MUC1 antibody, its anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 ADC reduces the severity, frequency, and / or duration of one or more symptoms of the disease in subjects (e.g., humans) in subjects (e.g., human subjects identified as having cancer) or subjects identified as being at risk of developing the disease (e.g., subjects who have previously had cancer but are now cured). The efficacy and dosage of any of the anti-HER3 / MUC1 antibodies, their anti-HER3 / MUC1 antigen-binding fragments, or anti-HER3 / MUC1 ADCs described herein can be determined by medical or veterinary experts by methods well known in the art and by observation of one or more symptoms of the disease in subjects (e.g., humans). Certain factors may influence the dosage and timing necessary to effectively treat the subject (e.g., severity of the disease or disability, previous treatments, the subject's general health status and / or age, presence of other diseases).

[0244] Exemplary doses include milligrams or micrograms of any of the anti-HER3 / MUC1 antibody, its anti-HER3 / MUC1 antigen-binding fragment, or anti-HER3 / MUC1 ADC described herein, per kg of body weight of the subject (e.g., approximately 1 μg / kg to 500 mg / kg; approximately 100 μg / kg to 500 mg / kg; approximately 100 μg / kg to 50 mg / kg; approximately 10 μg / kg to 5 mg / kg; approximately 10 μg / kg to 0.5 mg / kg; or approximately 0.1 mg / kg to 0.5 mg / kg). While these doses cover a wide range, those skilled in the art will understand that therapeutic agents vary in potency and that effective doses can be determined by methods well known in the art. Typically, a relatively low dose is administered initially, and the medical or veterinary expert (in therapeutic applications) or researcher (in developmental stages) may then gradually increase the dose until an appropriate response is obtained. Furthermore, it is understood that specific dose levels for a particular subject depend on various factors, including the activity of the specific compound employed, the subject's age, weight, general health status, sex, and diet, administration time, route of administration, excretion rate, and the half-life of the therapeutic agent in vivo.

[0245] The pharmaceutical composition may be placed in a container, pack, or dispenser along with instructions for administration. This disclosure also provides methods for producing anti-HER3 / MUC1 antibodies, their anti-HER3 / MUC1 antigen-binding fragments, or anti-HER3 / MUC1 ADCs for various applications as described herein. [Examples]

[0246] The present invention will be further illustrated by the following embodiments, but these will not limit the scope of the invention as described in the claims.

[0247] Example 1. Preparation of anti-HER3 / MUC1 bispecific antibody This specification provides bispecific antigen-binding molecules that target HER3 and MUC1. These antigen-binding molecules will hereafter be referred to as anti-HER3 / MUC1 bispecific antibodies.

[0248] Bispecific antibodies can be formed by pairing 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). Vectors encoding the light and heavy chains of the antibodies were constructed. CHO-S cells were co-transfected with three vectors: 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.

[0249] To confirm the binding affinity of the bispecific antibodies, anti-HER3 control bispecific antibodies and anti-MUC1 control bispecific antibodies were also prepared. One arm of these control bispecific antibodies recognizes HER3 or MUC1, and the other arm recognizes CD28. These control bispecific antibodies were prepared using a similar method, for example, by immunizing RenLite® mice to obtain the VH sequence. Exemplary control bispecific antibodies are named 3E1-CD28, 3G6-CD28, 1B2-CD28, and 10D1-CD28.

[0250] Various methods can be used to reduce the possibility of the two heavy chains mispairing. For example, knob-in-hole mutations have been introduced into the Fc regions of the anti-HER3 arm heavy chain, anti-MUC1 arm heavy chain, and anti-CD28 arm heavy chain. For example, in 3E1-10D1, the heavy chain constant region of 3E1 contains a knob mutation, and the heavy chain constant region of 10D1 contains a hole mutation. In 3E1-CD28, the heavy chain constant region of 3E1 contains a knob mutation, and the heavy chain constant region of CD28 contains a hole mutation.

[0251] The sequences of the light chain constant region, the heavy chain constant region with a knob mutation, and the heavy chain constant region with a hole mutation are shown in SEQ ID NOs. 29, 30, and 31, respectively.

[0252] Example 2. Heterospecific binding activity of anti-HER3 / MUC1 bispecific antibody 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 each measured in 5 × 10⁻¹⁴ cells. 4 The cells were transferred to a 96-well plate at a cell / well density. An anti-HER3 / MUC1 bispecific antibody was added to the 96-well plate and incubated at 4°C for 30 minutes. Subsequently, the cells were incubated with the 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, followed by flow cytometry analysis.

[0253] CHO-hHER3 cells, CHO-fasHER3 cells, and CHO-fasMUC1 cells were obtained by transfecting CHO-S cells with vectors expressing human HER3 (hHER3, SEQ ID NO: 32), cynomolgus monkey (Macaca fascicularis) HER3 (fasHER3, SEQ ID NO: 33), and cynomolgus monkey (Macaca fascicularis) MUC1 (fasMUC1, SEQ ID NO: 35), respectively. H293F-hMUC1 cells were obtained by transfecting H293F cells with a vector expressing human MUC1 (hMUC1, positions 961-1152 of SEQ ID NO: 34). RNA sequence analysis revealed that the expression levels of HER3 and MUC1 in NUGC-4 cells were 155 and 79, respectively, while the expression levels in HCC827 cells were 27 and 41, respectively.

[0254] Gatipotuzumab is a glycosylated humanized monoclonal antibody that recognizes tumor-associated epitopes of MUC1, and is currently in Phase II clinical development by Glycotope Inc. as a treatment for recurrent epithelial ovarian cancer, fallopian tube cancer, and primary peritoneal cancer. The heavy chain variable region and light chain variable region of gatipotuzumab are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively.

[0255] 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.

[0256] [Table 1]

[0257] In another experiment, the binding activity of anti-HER3 / MUC1 bispecific antibodies against tumor cells NUGC-4 or NCI-H226 was measured by flow cytometry. The secondary antibody used in the experiment was as follows: Alexa Fluor® 647 anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc. catalog number: 109-606-170). Human IgG1 was used as the isotype control (ISO). MFI was measured using serially diluted sample antibodies (maximum concentration: 30 μg / mL, 3-fold dilution). A fitted curve was obtained with concentration (μg / mL) on the X axis and MFI on the Y axis. The results are shown in Figures 17A-17B.

[0258] Example 3. Internalization of anti-HER3 / MUC1 bispecific antibody Anti-HER3 antibody, anti-MUC1 antibody, anti-HER3 / MUC1 bispecific antibody, anti-HER3 / CD28 bispecific antibody, or anti-MUC1 / CD28 bispecific antibody, along with pHAb-AffiniPure Fab goat anti-human IgG secondary antibody, were added to HCC70 cells (ATCC, catalog number: CRL-2315) and incubated for 6 hours. The cells were centrifuged and washed with FACS buffer. MFI was measured using a flow cytometer. The endocytosis rate of the antibodies was calculated. Human IgG1 protein was used as the isotype control (ISO). The results are shown in the table below.

[0259] RNA sequence analysis revealed that the expression levels of HER3 and MUC1 in HCC70 cells were 19 and 110, respectively.

[0260] Patritumab is an anti-HER3 fully human monoclonal antibody developed by Daiichi Sankyo, and its heavy chain variable region and light chain variable region are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0261] [Table 2]

[0262] As a result, in HCC70 cells, the endocytosis rates of the bispecific antibodies 3E1-10D1, 3G6-10D1, and 1B2-10D1 were equivalent to or higher than those of the corresponding monoclonal antibodies 3E1, 3G6, 1B2, and 10D1.

[0263] Example 4. Binding activity of anti-HER3 / MUC1 bispecific antibody The binding activity of anti-HER3 / MUC1 bispecific antibodies against human HER3, human MUC1, monkey HER3, and monkey MUC1 was validated by surface plasmon resonance (SPR) using a Biacore® 8K biosensor (Biacore, Inc., Piscataway NJ) equipped with a pre-immobilized protein A sensor chip.

[0264] Specifically, hHER3-His (ACROBiosystems Inc., catalog number: ER3-H5223), hMUC1(24-1158)-His (positions 14-1158 of SEQ ID NO: 34), hMUC1(961-1152)-His (positions 961-1152 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 with 1× HBS-EP + buffer (pH 7.4) and injected into a Biacore® 8K biosensor at 10 μL / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 200 response units (RU)). Next, purified antibody at a concentration of 2 μg / ml was injected into 1×HBS-EP + buffer (pH 7.4) at a rate of 10 μL / min for 50 seconds. Dissociation was monitored for 400 seconds. After the final injection of each titration, the tip was regenerated with glycine solution (pH 1.5) at a rate of 30 μL / min for 30 seconds.

[0265] The reaction association rate (kon) and dissociation rate (koff) were simultaneously determined by globally fitting the data to a 1:1 Langmuir coupling model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore® 8K Evaluation Software 3.0. Affinity was estimated from the quotient of the velocity constant (KD = koff / kon).

[0266] As those skilled in the art would understand, the same method, with appropriately adjusted parameters (such as antibody concentration), was performed for each antibody tested.

[0267] The results of the tested antibodies are summarized in the table below. The anti-HER3 / MUC1 bispecific antibodies 3E1-10D1, 3G6-10D1, and 1B2-10D1 showed good binding affinity to human HER3, monkey HER3, human MUC1, and monkey MUC1.

[0268] [Table 3]

[0269] Example 5. Stability of anti-HER3 / MUC1 bispecific antibody The anti-HER3 / MUC1 bispecific antibodies 3E1-10D1 and 1B2-10D1 were buffered to pH 6.0 (3 mg / ml histidine, 80 mg / ml sucrose, 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 to evaluate thermal stability. Alternatively, the bispecific antibodies were loaded onto a Protein A column and eluted with pH 3.5 buffer (0.1 mol / L HAc). Half of the antibody solution was pH 7.5 with 2 M Tris buffer. The remaining half was maintained at pH 3.5 for 6 hours and then adjusted to pH 7.5. The diluted antibody was placed in an Eppendorf tube and stored at pH 3.5 ± 0.1 and 25 ± 2°C (hereinafter referred to as pH 3.5) for 6 or 24 hours to examine its stability at low pH.

[0270] After the above processing, 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 relative to the total peak area (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 alkaline component); (4) Capillary electrophoresis under non-reducing (CE-SDS(NR)) conditions - change in antibody purity with sodium dodecyl sulfate (CE-SDS) (purity, %); (5) Percentage of the main peak area relative to the total presence of soluble and insoluble particles.

[0271] In the SEC-HPLC experiment, the antibody sample was diluted to 1 mg / mL with purified water and an Agilent 1290 chromatograph system (connected to an XBridge Protein BEH SEC column (200 Å, Waters Corporation)) was used. 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 tray temperature: approximately 8°C; and analysis time: 7 minutes.

[0272] In the HIC-HPLC experiment, an Agilent 1260 chromatograph system (connected to a ProPac HIC-10 column (4.6 × 250 mm, Thermo Scientific)) was used, and the sample was diluted 10-fold 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, 45 min 100% A; column temperature: 30°C; detection wavelengths: 280 nm, 220 nm; injection volume: 10 μg; sample tray temperature: approximately 10°C; and analysis time: 50 minutes.

[0273] For the cIEF experiment, the Maurice cIEF Method Development Kit (Protein Simple, catalog number: PS-MDK01-C) was used for sample preparation. Specifically, 8 μL of a 30 ug protein sample was mixed with the following reagents from the kit: 1 μL Maurice cIEF pI Marker-7.05, 1 μL Maurice cIEF pI Marker-10.10, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500 mM arginine, 1.33 μL amphoteric solvent (Pharmalyte pH range 3-10), 6.66 μL amphoteric solvent (Pharmalyte pH range 8-10.5), and water (added to a final volume of 100 μL). Imaging capillary isoelectric focusing spectra were prepared using a Maurice analyzer (Protein Simple, Santa Clara, CA) and a Maurice cIEF cartridge (PS-MC02-C). The samples were focused for a total of 10 minutes. The absorbance of the protein focused at 280 nm was integrated using the analysis software installed on the instrument.

[0274] For the CE-SDS(NR) experiment, Maurice (Protein simple, Maurice®) and the Maurice CE-SDS Size Application Kit (Protein simple, catalog number: PS-MAK02-S) were used. For CE-SDS(NR), 30 μL of sample buffer, 30 μL of 30 ug of antibody sample, 1.5 μL of 25 × internal standard, and 3 μL of 250 nM iodacetamide (SIGMA, catalog number: 16125) were added to a microcentrifuge tube, centrifuged at 3000 rpm for 1 minute, and then heated in a 70°C water bath for 10 minutes. The sample was then cooled to room temperature and centrifuged at 10000 rpm for 3 minutes. Next, the supernatant sample preparation was transferred to a 96-well plate and tested with Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, separation time 40 minutes.

[0275] Detailed results for the anti-HER3 / MUC1 bispecific antibodies are shown in the table below. The results showed that 3E1-10D1 and 1B2-10D1 exhibited good physical and chemical properties, as well as good stability.

[0276] [Table 4]

[0277] Example 6. Preparation of anti-HER3 / MUC1 antibody-drug conjugate (ADC) Each purified antibody (3E1, 3G6, 1B2, 10D1, 3E1-10D1, 3G6-10D1, or 1B2-10D1) was conjugated to MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0278] The antibody-drug conjugate is named by directly adding "ADC" after the antibody name. For example, if 3E1-10D1, which has an IgG1 constant region, is bound to MMAE, it is named 3E1-10D1-ADC. Other antibody-drug conjugates prepared using a similar method include patritumab-ADC, gatipotuzumab-ADC, and 1H7-ADC. As an isotype control, an ISO-ADC was created by binding human IgG1 to MMAE.

[0279] 1H7 is a mouse monoclonal IgG antibody that targets 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.

[0280] 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 a ProPac® 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: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, 45 min 100% A; column temperature: 30°C; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: approximately 6°C; and analysis time: 45 minutes.

[0281] HIC-HPLC detection results indicate that the drug-to-antibody ratio (DAR) for each ADC is approximately 4.

[0282] Example 7. Antitumor activity in a NUGC-4 xenograft model The effect of ADCs on tumor growth in vivo was investigated using a gastric cancer NUGC-4 xenograft model. The expression levels of HER3 and MUC1 in NUGC-4 cells were measured by RNA sequencing at 154.99 and 79.44, respectively. Specifically, approximately 5 × 10⁻⁶ 6 Nine NUGC-4 cells were injected subcutaneously into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., catalog number: B-CM-002). The tumor volume of the mice was approximately 200 mm². 3 Upon reaching a certain tumor volume, the mice were randomly divided into different groups based on tumor volume. Subsequently, the mice were administered intravenously (iv) with phosphate-buffered saline (PBS), patritumab-ADC, 1H7-ADC, 3E1-10D1-ADC, 1B2-10D1-ADC, 3E1-ADC, 1B2-ADC, or 10D1-ADC. The administration frequency was once per week (a total of two doses). Details are shown in the table below.

[0283] [Table 5]

[0284] Tumor volume was measured twice a week, and the mouse's body weight was also recorded. The mouse's tumor volume was 3000 mm³. 3 They were euthanized when they reached that point.

[0285] Measure the length of the long axis and short axis of the tumor, and calculate the tumor volume as 0.5 × (long axis) × (short axis). 2 The following calculations were performed. The tumor growth inhibition rate (TGI) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100. 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, and V0 is the mean tumor volume of the control group on day 0. A t-test was used for statistical analysis. The threshold for showing a significant difference was set at P < 0.05.

[0286] During the experiment, no significant differences in body weight were observed between the groups.

[0287] The table below summarizes the results of this experiment, including tumor volume, TGI (%), and statistical differences (P-values) between the treatment and control groups in body weight and tumor volume on day 21, day 11, day 21, and day 32.

[0288] [Table 6]

[0289] Figure 1 shows the tumor volume in the group treated with ADCs. Each treatment group showed different tumor suppression effects. The anti-HER3 / MUC1 bispecific antibody ADCs 3E1-10D1-ADC (G4) and 1B2-10D1-ADC (G5) showed superior antitumor activity compared to monoclonal ADCs (G6, G7, G8) and positive controls (G2, G3) in a gastric cancer model.

[0290] Example 8. Antitumor activity in a xenograft model derived from gastric cancer patients. The effect of ADCs on tumor growth in vivo was investigated using a xenograft model derived from gastric cancer patients. Immunofluorescence staining was performed on patient-derived gastric tumor fragments, and images were analyzed using HALO 3.2. The results showed that in human gastric tumor tissue, HER3-positive cells accounted for 60.23% of the total cells, and MUC1-positive cells accounted for 91.30% of the total cells. Specifically, patient-derived gastric tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The tumor volume of the mice was approximately 250-300 mm². 3 When tumor volume was reached, the mice were randomly divided into different groups based on tumor volume. The mice were then injected with PBS, patritumab-ADC, 1H7-ADC, gatipotuzumab-ADC, 3E1-10D1-ADC, or 1B2-10D1-ADC. Details are shown in the table below.

[0291] [Table 7]

[0292] Body weight was measured twice a week. Since no significant differences in body weight were observed between the groups during the experiment, it was shown that the ADC tested was well tolerable to mice and did not have any apparent toxicity.

[0293] The tumor volumes of mice from different groups are shown in Figure 2. The treatment groups (G2-G6) showed a higher tumor suppression effect compared to the PBS group (G1). In addition, both 1B2-10D1-ADC (G6) and 3E1-10D1-ADC (G5) had higher TGI% at day 20 (50.1% and 69.1%) than the positive controls patritumab-ADC (G2, 18.9%), 1H7-ADC (G3, 24.8%), and gatipotuzumab-ADC (G4, 19.2%). Tumor volume and survival rate of mice were also continuously monitored from day 20 onward. When the tumor volume of a mouse reached 3000 mm³... 3Euthanasia was administered when the target was reached. 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 antitumor efficacy compared to the positive controls: patritumab-ADC (G2, TGI%=10.4%), 1H7-ADC (G3, TGI%=28.4%), and gatipotuzumab-ADC (G4, TGI%=24.9%).

[0294] Example 9. Antitumor activity in an HCC70 xenograft model The effect of ADCs on tumor growth in vivo was investigated using a xenograft model of ductal carcinoma HCC70 cells. Specifically, approximately 1 × 10⁻⁶ cells were used. 7 Individual HCC70 cells were injected subcutaneously into B-NDG mice. The tumor volume of the mice was approximately 200 mm². 3 At this point, the mice were randomly divided into different groups based on tumor volume. Subsequently, the mice were administered intravenously (iv) with PBS, patritumab-ADC, 1H7-ADC, 3E1-10D1-ADC, 3G6-10D1-ADC, or 1B2-10D1-ADC. The administration frequency was once per week (a total of two doses). Details are shown in the table below.

[0295] [Table 8]

[0296] Tumor volume was measured twice a week, and the mouse's body weight was also recorded. The mouse's tumor volume was 3000 mm³. 3 They were euthanized when they reached that point.

[0297] All mice in the different groups showed an increase in body weight. On day 0 of the experiment, the average body weight of each group ranged from 21.6g to 22.0g. At the end of the experiment (day 39), the average body weight of each group ranged from 24.3g to 26.6g. The average body weight of each group ranged from 112.5% ​​to 121.2%. As a result, the tested ADC was well-tolerated, and no apparent toxicity was observed in the mice.

[0298] The table below summarizes the results of this experiment, including tumor volume, mouse survival rate, TGI (%), and statistical differences (P-values) in body weight and tumor volume between the treatment group and the control group on the day of group assignment (Day 0), 18 days after group assignment (Day 18), 28 days after group assignment (Day 28), and 39 days after group assignment (Day 39).

[0299] [Table 9]

[0300] The tumor volume in the group treated with ADCs is shown in Figure 6. 3E1-10D1-ADC(G5), 3G6-10D1-ADC(G6), and 1B2-10D1-ADC(G7) showed superior antitumor activity compared to the positive controls, patritumab-ADC(G2), 1H7-ADC(G3), and gatipotuzumab-ADC(G4), in a ductal carcinoma model.

[0301] Example 10. Antitumor activity in a xenograft model derived from pancreatic cancer patients. The effect of ADCs on tumor growth in vivo was investigated using a xenograft model derived from pancreatic cancer patients. Immunofluorescence staining was performed on patient-derived pancreatic tumor fragments, and images were analyzed using HALO 3.2. The results showed that in human pancreatic tumor tissue, HER3-positive cells accounted for 26.78% of the total cells, and MUC1-positive cells accounted for 64.77% of the total cells. Specifically, patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The tumor volume in the mice was approximately 250-300 mm³. 3 At this point, the mice were randomly divided into different groups based on tumor volume (5 mice per group). The mice were then administered intravenously (iv) at a dose of 3 mg / kg either PBS (G1), patritumab-ADC (G2), 1H7-ADC (G3), gatipotuzumab-ADC (G4), 3E1-10D1-ADC (G5), or 1B2-10D1-ADC (G6). Administration was once per week (a total of two doses).

[0302] The tumor volumes of mice from different groups are shown in Table 10 and Figure 7. The treatment groups (G2-G6) showed a significant tumor-suppressing effect compared to the PBS group (G1). In addition, 3E1-10D1-ADC (G5) and 1B2-10D1-ADC (G6) showed a higher TGI% at day 56 compared to the positive controls patritumab-ADC (G2), 1H7-ADC (G3), and gatipotuzumab-ADC (G4). Anti-HER3 / MUC1 bispecific ADCs demonstrated robust and sustained antitumor effects in the pancreatic cancer model.

[0303] [Table 10]

[0304] Example 11. Antitumor activity in a xenograft model derived from lung cancer patients The effect of ADCs on tumor growth in vivo was investigated using a xenograft model derived from lung cancer patients. Immunofluorescence staining was performed on patient-derived lung tumor fragments, and images were analyzed using HALO 3.2. The results showed that in human lung tumor tissue, HER3-positive cells accounted for 22.84% of the total cells, and MUC1-positive cells accounted for 82.88% of the total cells. Specifically, patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The tumor volume in the mice was approximately 250-300 mm³. 3 At this point, the mice were randomly divided into different groups based on tumor volume (5 mice per group). The mice were then administered intravenously (iv) with PBS, patritumab-ADC, 1H7-ADC, gatipotuzumab-ADC, 3E1-10D1-ADC, 1B2-10D1-ADC, 3E1-ADC, 1B2-ADC, or 10D1-ADC. Details are shown in the table below.

[0305] [Table 11]

[0306] The tumor volumes of mice from different groups are shown in Figure 8. All mice in groups G1, G2, G3, and G4 reached the euthanasia criteria by day 39 after group assignment, therefore tumor size at day 39 could not be obtained.

[0307] 3E1-10D1-ADC and 1B2-10D1-ADC showed the best tumor growth inhibition at both 3 mg / kg and 6 mg / kg (G5-G8). In addition, 3E1-ADC, 1B2-ADC, and 10D1-ADC (G9-G11) showed superior efficacy compared to the positive controls, patritumab-ADC, 1H7-ADC, and gatipotuzumab-ADC (G2-G4).

[0308] Example 12. Internalization of anti-HER3 / MUC1 bispecific ADCs NUGC-4 cells cultured in cell culture plates were treated with an anti-HER3 antibody, an anti-MUC1 antibody, or an anti-HER3 / MUC1 bispecific antibody and an ADC. After culturing for 15-24 hours using IncuCyte (Sartorius AG, IncuCyte® S3), internalization activity was detected. The results are shown in Figures 9A-9B.

[0309] The data showed that the endocytosis activity of 3E1-10D1 and 1B2-10D1 remained unchanged even after MMAE binding. In addition, 3E1-10D1 and 1B2-10D1 showed superior internalization activity compared to their corresponding parental monoclonal antibodies (3E1, 1B2, 10D1) and positive controls (patritumab analog, 1H7 analog, gatipotuzumab analog).

[0310] Example 13. Antibody-drug conjugate Purified antibodies (1B2, 3E1, 10D1, 1B2-10D1, 3E1-10D1, 1H7 analog, gatipotuzumab analog) were conjugated to CPT-1, CPT-2, CPT-3, or CPT-4 via a CPT-L linker. The antibody-drug conjugate is named by adding CPTx (x=1, 2, 3, 4) immediately after the antibody name. For example, when 1B2 is conjugated to CPT-1, it is named 1B2-CPT1. As another example, when 1B2-10D1 is conjugated to CPT-2, it is named 1B2-10D1-CPT2. Exemplary ADCs obtained by this method include: 1B2-10D1-CPT2, 3E1-10D1-CPT2, 1H7-CPT2, and gatipotuzumab-CPT2.

[0311] Patritumab analogs and gatipotuzumab analogs were also conjugated to Dxd via GGFG linkers for comparison, and the resulting ADCs were named patritumab-Dxd and gatipotuzumab-Dxd, respectively.

[0312] IgG1 monoclonal antibodies targeting unrelated targets were conjugated to CPT-2 via the CPT-L linker and to Dxd via the GGFG linker. This resulted in the formation of isotypes CPT2 (ISO-CPT2) and Dxd (ISO-Dxd), which were used as isotype controls.

[0313] Mass spectrometry (MS) was used to detect the binding of antibodies to drug molecules. MS detection revealed that the drug-to-antibody ratio (DAR) of the ADCs was approximately 4 or 8. All controls (including 1H7-CPT2, gatipotuzumab-CPT2, ISO-CPT2, gatipotuzumab-Dxd, patrizumab-Dxd, and ISO-Dxd) had a DAR of 8. Regarding the naming of 1B2-10D1-CPT2 and 3E1-10D1-CPT2, when the DAR is approximately 4, the ADCs are named 1B2-10D1-CPT2(DAR4) and 3E1-10D1-CPT2(DAR4). When the DAR is approximately 8, the ADCs are named 1B2-10D1-CPT2(DAR8) and 3E1-10D1-CPT2(DAR8).

[0314] Example 14. Antitumor activity in a lung cancer PDX model The antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested in vivo using a lung cancer PDX model with low HER3 expression and high MUC1 expression. Specifically, patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The mouse tumors were approximately 200-300 mm in size. 3 Upon reaching a certain tumor volume, mice were randomly assigned to different groups (5 mice per group) based on tumor volume and administered PBS, 1B2-10D1-CPT2, or 3E1-10D1-CPT2 via intravenous injection. Tumor volume was measured twice weekly.

[0315] As shown in Figure 10, tumor volume was smaller in all treatment groups (G2-G13) than in the control group (G1). As a result, 1B2-10D1-CPT2 and 3E1-10D1-CPT2, which possess DAR4 and DAR8, showed dose-dependently different tumor suppressive effects. When administered at doses of 3 mg / kg or higher, 1B2-10D1-CPT2 and 3E1-10D1-CPT2 could significantly inhibit tumor growth, with TGI% (e.g., day 24) exceeding 100% (G7, G9, G10, G11, G12, and G13).

[0316] The tumor volume and survival rate of the mice were continued to be monitored from day 24 onward. On day 28, all mice in group G1 died, but groups G2-G13 had high survival rates. At the end of the experiment on day 49, all mice in groups G1-G5 had died, and only one mouse each in groups G6, G8, G9, and G11 had died. Since all mice in groups G7, G10, G12, and G13 survived, it was demonstrated that 1B2-10D1-CPT2 and 3E1-10D1-CPT2 have excellent tumor-suppressing effects and good safety.

[0317] Example 15. Antitumor activity in a NUGC-4 xenograft model The effect of ADC on tumor growth in vivo was investigated using a gastric cancer NUGC-4 xenograft model. Specifically, approximately 5 × 10⁻⁶ 5Nucleo-4 cells were injected subcutaneously into B-NDG mice. The tumors in the mice were approximately 200 mm. 3 Upon reaching a certain tumor volume, mice were randomly assigned to different groups based on tumor volume and administered PBS, patritumab-Dxd, gatipotuzumab-Dxd, 1B2-10D1-CPT2, or 3E1-10D1-CPT2 intravenously. Administration was once weekly (a total of two doses). Tumor volume was measured twice weekly, and the results are shown in Figure 11.

[0318] As a result, 1B2-10D1-CPT2 and 3E1-10D1-CPT2, which possess DAR4 and DAR8, inhibited tumor growth at a higher TGI% (e.g., day 35) than positive controls (G10 and G11). For example, at a dose of 6 mg / kg, the TGI% for G6-G9 was over 90%, while it was 50.7% for G10 and 40.4% for G11, respectively. 1B2-10D1-CPT2 and 3E1-10D1-CPT2 showed good tumor suppression effects in a gastric cancer model.

[0319] Example 16. Antitumor activity in an HCC70 xenograft model The effect of ADCs on tumor growth in vivo was investigated using a ductal carcinoma HCC70 xenograft model. Specifically, approximately 1 × 10⁻⁶ tumors grew. 7 Individual HCC70 cells were injected subcutaneously into B-NDG mice. The tumors in the mice were approximately 200 mm. 3 Upon reaching a certain tumor volume, mice were randomly assigned to different groups based on tumor volume and administered PBS, patritumab-Dxd, gatipotuzumab-Dxd, ISO-Dxd, 1B2-10D1-CPT2, or 3E1-10D1-CPT2 intravenously. Tumor volume was measured twice weekly, and mouse body weight was also recorded. The results are shown in Figures 12A and 12B.

[0320] Figure 12A shows the results of tumor volume measurements, where both 1B2-10D1-CPT2 and 3E1-10D1-CPT2 showed dose-dependent and different tumor suppressor effects using DAR4 and DAR8. At a dose level of 3 mg / kg, the TGI% (e.g., day 38) of the G6-G9 groups was higher than that of the control groups G10-G12. Specifically, the TGI% on day 38 was 89.1%, 106.1%, 100.0%, and 106.3% for the G6-G9 groups, respectively, but was lower in the G10 group (68.5%), the G11 group (52.1%), and the G12 group (19.7%), indicating that 1B2-10D1-CPT2 and 3E1-10D1-CPT2 have good tumor suppressor effects in the breast cancer model.

[0321] All mice in the different groups gained body weight (as shown in Figure 12B), indicating that all anti-HER3 / MUC1 ADCs were well tolerated and non-toxic in mice.

[0322] Example 17. Antitumor activity in a colorectal cancer PDX model The antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested in vivo using a colorectal cancer PDX model with high HER3 expression and low MUC1 expression. Specifically, patient-derived colorectal tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The mouse tumors were approximately 200-300 mm in size. 3 Upon reaching a certain tumor volume, mice were randomly assigned to different groups (5 mice per group) based on tumor volume and administered intravenously with PBS, 1B2-10D1-CPT2, 3E1-10D1-CPT2, 1H7-CPT2, or patritumab-Dxd. Tumor volume was measured twice weekly.

[0323] As shown in Figure 13, 1B2-10D1-CPT2 and 3E1-10D1-CPT2, which possess DAR4 and DAR8, inhibited tumor growth at a higher TGI% (e.g., day 31) than the positive controls 1H7-CPT2 and patrizumab-Dxd.

[0324] In a similar experiment, colorectal cancer PDX model mice were administered intravenously with PBS, ISO-CPT2, 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR8), 1H7-CPT2, or patritumab-Dxd. Tumor volume was measured twice weekly. As shown in Figure 14, 1B2-10D1-CPT2(DAR8) and 3E1-10D1-CPT2(DAR8) showed superior antitumor activity compared to the positive controls 1H7-CPT2 and patritumab-Dxd.

[0325] In another similar experiment, colorectal cancer PDX model mice were injected with PBS (G1), ISO-CPT2 (G2), 3E1-10D1-CPT2 (DAR8) (G3 and G4), or administered intravenously with a combination of 3E1-CPT2 (DAR8) and 10D1-CPT2 (DAR8) (G5), ISO-Dxd (G6), patrizumab-Dxd (G7), gatipotuzumab-Dxd (G8), or 3E1-10D1-Dxd (DAR8) (G9). The administration frequency was once a week (a total of two doses). Tumor volume was measured twice a week, and the results are shown in Figure 19. 3E1-10D1-CPT2 (DAR8) (G3 and G4) showed the most dose-dependent tumor suppressive effect compared to the positive control (G7 and G8) and combination therapy (G5). In addition, 3E1-10D1-Dxd(G9) also suppressed tumor growth with superior efficacy compared to the positive controls (G7 and G8).

[0326] Example 18. Antitumor activity in a pancreatic cancer PDX model. The antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested in vivo using a HER3 / MUC1 co-expressing pancreatic cancer PDX model. Patient-derived pancreatic tumor fragments (2mm x 2mm x 2mm) were transplanted into the right flank of B-NDG mice. The mouse tumors were approximately 200-300mm in size. 3Upon reaching a certain tumor volume, mice were randomly assigned to different groups (5 mice per group) based on tumor volume and administered intravenously with PBS, 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR8), 1H7-CPT2, gatipotuzumab-CPT2, or patritumab-Dxd. Tumor volume was measured twice weekly, and the body weight of the mice was also recorded.

[0327] As shown in Figure 15A, 1B2-10D1-CPT2(DAR8) and 3E1-10D1-CPT2(DAR8) at 3 mg / kg induced significant inhibition of tumor growth at a higher TGI% (e.g., day 31) than the positive controls 1H7-CPT2, gatipotuzumab-CPT2, or patrizumab-Dxd.

[0328] As shown in Figure 15B, the body weight of mice treated with 1B2-10D1-CPT2(DAR8) and 3E1-10D1-CPT2(DAR8) (G3 and G4) increased. On the day of group assignment, the average body weight was 21.8g for the G3 group and 21.6g for the G4 group. 31 days after group assignment, the average body weight of the G3 group was 24.8g and the average body weight of the G4 group was 24.4g, representing a body weight change of 114.4% and 113.1%, respectively. However, the body weight of the 1H7-CPT2, gatipotuzumab-CPT2, and patritumab-Dxd groups remained unchanged or slightly decreased. As a result, both 1B2-10D1-CPT2 and 3E1-10D1-CPT2 showed good tolerability without causing toxicity in mice.

[0329] Example 19. Antitumor activity in a breast cancer PDX model The antitumor activity of 1B2-10D1-CPT2 and 3E1-10D1-CPT2 was tested in vivo using a HER3-low / MUC1-high breast cancer PDX model. Patient-derived breast tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. Tumor volume was measured twice a week, and the results are shown in Figure 16.

[0330] 1B2-10D1-CPT2 and 3E1-10D1-CPT2, possessing DAR4 and DAR8 respectively, significantly inhibited tumor growth at a higher TGI% (e.g., day 16) than the positive controls, gatipotuzumab-CPT2 and patrizumab-Dxd. This suggests that anti-HER3 / MUC1 ADCs are promising for breast cancer treatment.

[0331] Example 20. Antitumor activity in a gastric cancer PDX model The effect of ADC on tumor growth in vivo was investigated using a xenograft model derived from gastric cancer patients. Specifically, a gastric tumor fragment (2 mm × 2 mm × 2 mm) derived from the patient was transplanted into the right flank of B-NDG mice. The tumor volume was approximately 250 mm². 3 When tumor volume was reached, mice were randomly divided into different groups based on tumor volume. Subsequently, 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), or administered intravenously (iv) with patritumab-Dxd (G6), gatipotuzumab-Dxd (G7), ISO-ADC (G8), or 3E1-10D1-ADC (G9). The administration frequency was once a week (a total of two doses).

[0332] Tumor volume was measured twice a week, and the results are shown in Figure 18. The treatment groups (G2-G9) showed different tumor-suppressing effects compared to the PBS group (G1). 3E1-10D1-CPT2(DAR8) (G3 and G4) showed dose-dependent superior tumor-suppressing effects compared to the positive controls (G6 and G7) and combination therapy (G5). In addition, 3E1-10D1-ADC (G9) also suppressed tumor growth with superior efficacy compared to the positive controls (G6 and G7).

[0333] Example 21. Antitumor activity in a lung cancer PDX model The effect of 3E1-10D1-CPT2(DAR8) on tumor growth in vivo was tested using a xenograft model derived from lung cancer patients. Immunohistochemical (IHC) staining of patient-derived tumor tissue revealed histochemical scores (H-scores) of 104.68 and 89.9 for HER3 and MUC1 expression levels, respectively. A patient-derived lung tumor fragment (2mm × 2mm × 2mm) was transplanted into the right flank of BALB / c nude mice. The tumor volume was approximately 200mm². 3 When tumor volume was reached, the mice were randomly divided into different groups based on tumor volume. Subsequently, the mice were administered intravenously (iv) either 5% glucose (G1) or 3 mg / kg of 3E1-10D1-CPT2(DAR8)(G2).

[0334] Figure 20 shows the results of measuring tumor volume twice a week, demonstrating that 3E1-10D1-ADC(DAR8) showed good tumor growth inhibition in a lung cancer model.

[0335] Example 22. Antitumor activity in an ovarian cancer PDX model 3E1-10D1-CPT2(DAR8) is being tested for its effect on tumor growth in a xenograft model derived from ovarian cancer patients. IHC staining of patient-derived tumor tissue revealed H-scores of 245.87 and 112.00 for HER3 and MUC1 expression levels, respectively. The tumor volume was approximately 200 mm². 3 When tumor volume was reached, the mice were randomly divided into different groups based on tumor volume. Subsequently, the mice were administered intravenously either 5% glucose (G1) or 6 mg / kg of 3E1-10D1-CPT2(DAR8)(G2).

[0336] Tumor volume was measured twice a week, and the regular results are shown in Figure 21. 3E1-10D1-ADC(DAR8) showed good tumor growth inhibition even in the ovarian cancer model.

[0337] Example 23. Toxicity Evaluation In preliminary experiments, to investigate safety and toxicological (TK) profiles, cynomolgus monkeys were administered IV injections of 1B2-10D1-CPT2(DAR4), 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR4), or 3E1-10D1-CPT2(DAR8) three times at 3-week intervals (days 1, 22, and 43). The dose formulations are shown in the table below. Subsequently, the animals were sacrificed on day 50 and examined macroscopically and histopathologically. Mortality / mortality rate, general observation, body weight, food intake, clinicopathological examinations (hematology, coagulation, serological chemistry, urinalysis), and macroscopic lesions were evaluated. Blood samples were also collected for TK analysis, and the payload, total antibodies, and key TK parameters of ADCs, e.g., Tmax, Cmax, AUC, were measured. (0-t) The following measurements were taken. As a result, it was revealed that T1B2-10D1-CPT2(DAR4), 1B2-10D1-CPT2(DAR8), 3E1-10D1-CPT2(DAR4), and 3E1-10D1-CPT2(DAR8) have good safety profiles.

[0338] [Table 12]

[0339] Other embodiments The present invention has been described in conjunction with its detailed description, but it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention as defined by the appended claims. Other embodiments, advantages, and modifications are included in the following claims.

Claims

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

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

5. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 4 to 6, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 10 to 12, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1 to 3, respectively. (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 16 to 18, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 13 to 15, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 22 to 24, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 13 to 15, respectively. (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 7 to 9, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 10 to 12, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1 to 3, respectively. (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 19 to 21, the selected VL1 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 13 to 15, the selected VH2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 22 to 24, and the selected VL2 CDR1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 13 to 15. An anti-HER3 / MUC1 antibody or its antigen-binding fragment according to any one of claims 2 to 4.

6. An anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5, wherein 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: 26, 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.

7. An anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 5, wherein 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: 27, 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.

8. The VH1 comprises an amino acid sequence that is at least 90% identical to the selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 90% identical to the selected VL sequence, and the selected VH sequence and the selected VL sequence are (1) The selected VH sequence is sequence number 26, and the selected VL sequence is sequence number 25, and (2) The selected VH sequence is sequence number 27, and the selected VL sequence is sequence number 25. An anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, which is one of the above.

9. The VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; the VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are (1) The selected VH sequence is sequence number 26, and the selected VL sequence is sequence number 25, (2) The selected VH sequence is sequence number 27, and the selected VL sequence is sequence number 25. An anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 8, which is one of the above.

10. The anti-HER3 / MUC1 antibody or antigen-binding fragment thereof according to any one of claims 2 to 9, wherein the VH2 comprises an amino acid sequence that is at least 90% identical to the selected VH sequence, the VL2 comprises an amino acid sequence that is at least 90% identical to the selected VL sequence, 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 according to any one of claims 2 to 10, wherein the VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and the VL2 comprises VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of the 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 according to any one of claims 2 to 11, wherein VH1 comprises the sequence of SEQ ID NO: 26 and VL1 comprises the sequence of SEQ ID NO:

25.

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

25.

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

18. The anti-HER3 / MUC1 antibody or antigen-binding fragment according to 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 an scFv.

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

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

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

22. A vector comprising the nucleic acid described in claim 21.

23. A cell comprising the vector according to claim 22.

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

25. A cell comprising the nucleic acid described in claim 21.

26. A method for producing an anti-HER3 / MUC1 antibody or its antigen-binding fragment, (a) Culturing the cells according to any one of claims 23 to 25 under conditions sufficient to cause the cells to produce the anti-HER3 / MUC1 antibody or the antigen-binding fragment thereof; (b) Recovering the anti-HER3 / MUC1 antibody or the antigen-binding fragment thereof produced by the cells. Methods that include...

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

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

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

30. The aforementioned therapeutic agent, 【Chemistry 1】 An antibody-drug conjugate according to claim 27, selected from the above.

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

32. The antibody-drug conjugate according to claim 31, wherein the linker has the following structure: 【Chemistry 2】

33. The antibody-drug conjugate 【Transformation 3】 The antibody-drug conjugate according to any one of claims 27 or 30-32, having the structure, wherein n = 1 to 8 in the formula; and "Ab" represents the antibody or its antigen-binding fragment, or the antigen-binding protein construct.

34. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-HER3 / MUC1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 20, or an anti-HER3 / MUC1 antibody-drug conjugate as described in any one of claims 27 to 33.

35. The method according to claim 34, wherein the subject has cancer expressing HER3 and / or MUC1 (for example, both HER3 and MUC1).

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

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

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

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

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

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

42. A pharmaceutically acceptable carrier, (a) an anti-HER3 / MUC1 antibody or an 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 and A pharmaceutical composition containing the following:

43. An anti-HER3 / MUC1 antibody-drug conjugate (ADC) comprising a bispecific antibody or an antigen-binding fragment thereof covalently bound to a bispecific 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.

44. An anti-HER3 / MUC1 ADC according to any one of claims 27 to 33 or 43, wherein the drug-to-antibody ratio (DAR) is approximately 4 or 8.

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