Anti-egfr / her3 antibodies and uses thereof

Abstract

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

Description

[0001] This application claims the benefit of PCT application No. PCT / CN2023 / 143082, filed on December 29, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to multispecific anti-EGFR (epidermal growth factor receptor) / HER3 (human epidermal growth factor receptor 3) antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof), 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 dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivery of payloads to target sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and belintomab (anti-CD19 and anti-CD3) represents a significant milestone in the development of bispecific antibodies.

[0004] Bispecific antibodies have a wide range of applications; therefore, there is a need to continue developing various therapeutic agents based on bispecific antibodies. Summary of the Invention

[0005] This disclosure relates to anti-EGFR / HER3 antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to EGFR and HER3. In some embodiments, the antibodies or antigen-binding fragments thereof have the same light chain variable region. In some embodiments, the antibodies or antigen-binding fragments thereof have a common light chain. This disclosure also relates to antibody-pharmaceutical conjugates derived from these anti-EGFR / HER3 antibodies.

[0006] In one aspect, this disclosure relates to an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to EGFR; and a second antigen-binding domain that specifically binds to HER3.

[0007] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes 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 contains an amino acid sequence having at least 80% identity with a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region contains an amino acid sequence having at least 80% identity with a selected VH1 CDR3 amino acid sequence; and

[0009] The first light chain variable region (VL1) includes CDR1, 2, and 3, wherein the VL1 CDR1 region contains an amino acid sequence having at least 80% identity with a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region contains an amino acid sequence having at least 80% identity with a 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 are one of the following: (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22-24, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 25-27, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; and (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28-30, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3.

[0010] In some embodiments, the second heavy chain variable region (VH2) includes CDR1, 2, and 3, wherein the VH2CDR1 region contains an amino acid sequence having at least 80% identity with a selected VH2 CDR1 amino acid sequence, the VH2CDR2 region contains an amino acid sequence having at least 80% identity with a selected VH2 CDR2 amino acid sequence, and the VH2CDR3 region contains an amino acid sequence having at least 80% identity with a selected VH2 CDR3 amino acid sequence; and

[0011] The second light chain variable region (VL2) includes CDR1, 2, and 3, wherein the VL2 CDR1 region contains an amino acid sequence having at least 80% identity with a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region contains an amino acid sequence having at least 80% identity with a 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 are one of the following: (1) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (2) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (3) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31-33, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (5) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34-36, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; and (6) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37-39, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3.

[0012] In some embodiments, (1) the selected VH1 CDR1, 2, and 3 amino acid sequences are as shown in SEQ ID NO: 4-6, the selected VL1 CDR1, 2, and 3 amino acid sequences are as shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are as shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are as shown in SEQ ID NO: 1-3; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (7) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (8) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3; or (9) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3.

[0013] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 41, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 44, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0014] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 41, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 45, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0015] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 41, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 46, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0016] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 42, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 44, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0017] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 42, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 45, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0018] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 42, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 46, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0019] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 43, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 44, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0020] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 43, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 45, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0021] In some embodiments, the first heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 43, the first light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 46, and the second light chain variable region contains a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40.

[0022] In some embodiments, the VH1 comprises an amino acid sequence having at least 90% identity with a selected VH sequence, and the VL1 comprises an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 42, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 40.

[0023] In some embodiments, the VH1 comprises the same VH1 CDR1, VH1 CDR2, and VH1 CDR3 as the selected VH sequence's VH CDR1, VH CDR2, and VH CDR3; and the VL1 comprises the same VL1 CDR1, VL1 CDR2, and VL1 CDR3 as the selected VL sequence's VL CDR1, VL1 CDR2, and VL1 CDR3, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 42, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 40.

[0024] In some embodiments, the VH2 comprises an amino acid sequence having at least 90% identity with a selected VH sequence, and the VL2 comprises an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 44, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO: 40.

[0025] In some embodiments, the VH2 comprises the same VH2 CDR1, VH2 CDR2, and VH2 CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and the VL2 comprises the same VL2 CDR1, VL2 CDR2, and VL2 CDR3 as the VL CDR1, VLCDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 44, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO: 40.

[0026] In some embodiments, VH1 contains the sequence of SEQ ID NO: 41, and VL1 contains the sequence of SEQ ID NO: 40.

[0027] In some embodiments, VH1 contains the sequence of SEQ ID NO: 42, and VL1 contains the sequence of SEQ ID NO: 40.

[0028] In some embodiments, VH1 contains the sequence of SEQ ID NO: 43, and VL1 contains the sequence of SEQ ID NO: 40.

[0029] In some embodiments, VH2 comprises the sequence of SEQ ID NO: 44, and VL2 comprises the sequence of SEQ ID NO: 40.

[0030] In some embodiments, VH2 comprises the sequence of SEQ ID NO: 45, and VL2 comprises the sequence of SEQ ID NO: 40.

[0031] In some embodiments, VH2 comprises the sequence of SEQ ID NO: 46, and VL2 comprises the sequence of SEQ ID NO: 40.

[0032] In some embodiments, the first antigen-binding domain specifically binds to human or monkey EGFR; and / or the second antigen-binding domain specifically binds to human or monkey HER3.

[0033] In some implementations, the first antigen-binding domain is human or humanized; and / or the second antigen-binding domain is human or humanized.

[0034] In some implementations, the antibody is a multispecific antibody (e.g., a bispecific antibody).

[0035] In some implementations, the first antigen-binding domain is a single-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0036] In some implementations, the first light chain variable region and the second light chain variable region are the same.

[0037] In one aspect, this disclosure relates to an anti-EGFR / HER3 antibody or antigen-binding fragment thereof that cross-competes with the anti-EGFR / HER3 antibody or antigen-binding fragment thereof described herein.

[0038] In one respect, this disclosure relates to a nucleic acid comprising a polynucleotide encoding the anti-EGFR / HER3 antibody described herein or an antigen-binding fragment thereof.

[0039] In one respect, this disclosure relates to a carrier containing the nucleic acid described herein.

[0040] In one respect, this disclosure relates to a cell that comprises the vector described herein.

[0041] In some implementations, the cells are CHO cells.

[0042] In one respect, this disclosure relates to a cell that contains the nucleic acids described herein.

[0043] In one aspect, this disclosure relates to a method for generating an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof, the method comprising:

[0044] (a) The cells are cultured under conditions sufficient to induce the cells described herein to produce the anti-EGFR / HER3 antibody or its antigen-binding fragment; and

[0045] (b) Collect anti-EGFR / HER3 antibodies or antigen-binding fragments thereof produced by said cells.

[0046] In one aspect, this disclosure relates to an anti-EGFR / HER3 antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-EGFR / HER3 antibody or its antigen-binding fragment described herein.

[0047] In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

[0048] In some implementations, the therapeutic agent is MMAE or MMAF.

[0049] In some embodiments, the therapeutic agent is selected from... (CPT-1) (CPT-2) (CPT-3) or (CPT-4).

[0050] 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: .

[0051] In some embodiments, the antibody-drug conjugate has the following structure: or , In some implementations, n = 1-8; in some implementations, "Ab" represents the antibody or its antigen-binding fragment.

[0052] In some implementations, the drug-antibody ratio (DAR) is approximately 4 or 8.

[0053] In one aspect, this disclosure relates to a method of treating a subject suffering from cancer, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising an anti-EGFR / HER3 antibody as described herein or an antigen-binding fragment thereof or an anti-EGFR / HER3 antibody pharmaceutical conjugate as described herein.

[0054] In some implementations, the subject has cancer that expresses EGFR and / or HER3 (e.g., simultaneously expresses EGFR and HER3).

[0055] In some implementations, the cancer is esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial cancer, lung cancer, melanoma, ovarian cancer, bladder cancer, non-Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, lung adenocarcinoma, and cervical cancer.

[0056] In some implementations, the subject is a human being.

[0057] In some implementations, the method further includes administering an anti-PD1 antibody to the subject.

[0058] In some implementations, the method further includes administering chemotherapy to the subject.

[0059] In one aspect, this disclosure relates to a method for reducing tumor growth rate, the method comprising contacting the tumor cells with an effective amount of a composition comprising an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof as described herein, or an anti-EGFR / HER3 antibody-drug conjugate as described herein.

[0060] In one aspect, this disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof as described herein, or an anti-EGFR / HER3 antibody-drug conjugate as described herein.

[0061] In one aspect, this disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

[0062] (a) The anti-EGFR / HER3 antibody or its antigen-binding fragment described herein, and / or

[0063] (b) The anti-EGFR / HER3 antibody drug conjugate described herein.

[0064] In one aspect, this disclosure relates to an anti-EGFR / HER3 antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment comprising: a first antigen-binding domain specifically binding to EGFR; and a second antigen-binding domain specifically binding to HER3.

[0065] As used herein, the term "antigen-binding domain" refers to one or more protein domains (e.g., formed from amino acids from a single polypeptide or from amino acids from two or more polypeptides, e.g., the same or different polypeptides) capable of specifically binding to one or more different antigens (e.g., effector antigens or control antigens). In some embodiments, the antigen-binding domain may 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 an antigen-binding domain formed by a VH-VL dimer. In some embodiments, the antigen-binding domain may include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Other examples of antigen-binding domains are known in the art. In some embodiments, the antigen-binding domain may bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other embodiments, the antigen-binding domain may bind to two different antigens (e.g., an effector antigen and a control antigen).

[0066] As used herein, the term "antibody" is used in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a protein complex comprising two heavy chains and two light chains. Other examples of antibodies are described herein.

[0067] As used herein, the term "multispecific antibody" is an antibody comprising two or more different antigen-binding domains that commonly and specifically bind to two or more different epitopes. The two or more different epitopes can be epitopes of the same antigen (e.g., a single polypeptide present on the cell surface) or epitopes of different antigens (e.g., different proteins present on the same or different cell surfaces). In some aspects, multispecific antibodies bind to two different epitopes (i.e., "bispecific antibodies"). In some aspects, multispecific antibodies bind to three different epitopes (i.e., "trispecific antibodies"). In some aspects, multispecific antibodies bind to four different epitopes (i.e., "tetraspecific antibodies"). In some aspects, multispecific antibodies bind to five different epitopes (i.e., "pentaspecific antibodies"). Each binding specificity can be present in any suitable valence state. Non-limiting examples of multispecific antibodies are described herein.

[0068] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. Epitopes can be on the same antigen or on different antigens.

[0069] As used herein, the term "common light chain" refers to a light chain that can interact with two or more different heavy chains to form different antigen-binding sites, wherein these different antigen-binding sites can specifically bind different antigens or epitopes. Similarly, the term "common light chain variable region" refers to a light chain variable region that can interact with two or more different heavy chain variable regions to form different antigen-binding sites, wherein these different antigen-binding sites can specifically bind different antigens or epitopes. In some embodiments, the antibody or its antigen-binding fragment may have a common light chain. In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment may have a common light chain variable region.

[0070] As used herein, the term "anti-EGFR / HER3 antibody or its antigen-binding fragment" refers to an antibody or antigen-binding fragment that binds to both EGFR and HER3.

[0071] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0072] Other features and advantages of the invention will become clear from the detailed description, accompanying drawings, and claims below. Attached Figure Description

[0073] Figure 1 The heavy chain variable region and light chain variable region CDR sequences of the anti-EGFR antigen-binding domain (9F3, 9D2, and 9A6) and anti-HER3 antigen-binding domain (1B2, 3E1, and 3G6) of anti-EGFR / HER3 antibodies, as defined by Kabat, are listed.

[0074] Figure 2 The heavy chain variable region and light chain variable region CDR sequences of the anti-EGFR antigen-binding domain (9F3, 9D2, and 9A6) and anti-HER3 antigen-binding domain (1B2, 3E1, and 3G6) of anti-EGFR / HER3 antibodies, as defined by Chothia, are listed.

[0075] Figure 3 The amino acid sequences discussed in this disclosure are listed.

[0076] Figure 4 The average tumor volume of different groups of B-NDG mice injected with NUGC-4 cells and treated with phosphate-buffered saline (PBS) or ADC is shown.

[0077] Figure 5 The average tumor volume of B-NDG mice in different groups injected with NCI-H292 cells and treated with PBS or ADC is shown.

[0078] Figure 6 The average tumor volume of B-NDG mice from different groups inoculated with gastric tumor fragments (2 mm × 2 mm × 2 mm) derived from patients and treated with PBS or ADC is shown.

[0079] Figure 7 The average tumor volume of B-NDG mice from different groups inoculated with gastric tumor fragments (2 mm × 2 mm × 2 mm) derived from patients and treated with PBS or ADC is shown.

[0080] Figure 8 The average tumor volume of B-NDG mice from different groups is shown. The mice were inoculated with fragments of colorectal tumors derived from patients (2 mm × 2 mm × 2 mm) and treated with PBS or ADC.

[0081] Figure 9 The average tumor volume of B-NDG mice from different groups is shown. The mice were inoculated with fragments of colorectal tumors derived from patients (2 mm × 2 mm × 2 mm) and treated with PBS or ADC.

[0082] Figure 10 The average tumor volume of B-NDG mice from different groups is shown. The mice were inoculated with fragments of colorectal tumors derived from patients (2 mm × 2 mm × 2 mm) and treated with PBS or ADC.

[0083] Figure 11 The average tumor volume of B-NDG mice from different groups inoculated with lung tumor fragments (2 mm × 2 mm × 2 mm) derived from patients and treated with PBS or ADC is shown.

[0084] Figure 12 The average tumor volume of B-NDG mice from different groups inoculated with lung tumor fragments (2 mm × 2 mm × 2 mm) derived from patients and treated with PBS or ADC is shown.

[0085] Figure 13 The average tumor volume of B-NDG mice from different groups is shown. The mice were inoculated with fragments of colorectal tumors derived from patients (2 mm × 2 mm × 2 mm) and treated with PBS or ADC.

[0086] Figure 14 The average tumor volume of B-NDG mice from different groups is shown. The mice were inoculated with fragments of colorectal tumors derived from patients (2 mm × 2 mm × 2 mm) and treated with PBS or ADC.

[0087] Figure 15 The blocking effect of anti-EGFR / HER3 antibody and its ADC on EGF-induced signal transduction was demonstrated.

[0088] Figure 16 The blocking effect of anti-EGFR / HER3 antibody and its ADC on NGR1-induced signal transduction was demonstrated.

[0089] Figure 17 The blocking effect of anti-EGFR / HER3 antibody and its ADC on NGR1-induced signal transduction was demonstrated. Detailed Implementation

[0090] 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 has a heavy chain variable region and a light chain variable region, forming an antigen-binding domain (or antigen-binding region). In some embodiments, the bispecific antibody has a common light chain.

[0091] This disclosure relates to anti-EGFR / HER3 antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to EGFR and HER3, and antibody-drug conjugates derived from these anti-EGFR / HER3 antibodies.

[0092] Anti-EGFR / HER3 antibody

[0093] Epidermal growth factor receptor (EGFR, ErbBI, or HER1) is a 170 kDa type 1 transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. EGFR is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases (EGFR (ErbB-1), HER2 / neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4)). Mutations affecting EGFR expression or activity can contribute to cancer in many cancer types. EGFR signaling is initiated by ligand binding, followed by conformational changes, homodimerization or heterodimerization of the receptor with other ErbB family members, and trans-autophosphorylation of the receptor, thereby initiating a signal transduction cascade that ultimately affects a variety of cellular functions, including cell proliferation and survival. Increased EGFR expression or kinase activity is associated with a range of human cancers, making EGFR an attractive target for therapeutic intervention. In non-small cell lung cancer, increased EGFR gene copy number and protein expression are associated with a good response to the EGFR tyrosine kinase inhibitor IRESSA™ (gefitinib).

[0094] The binding of ligands such as EGF (epidermal growth factor) to EGFR promotes receptor dimerization, autophosphorylation, activation of the receptor's intracellular cytoplasmic tyrosine kinase domain, and initiates various signal transduction and transactivation pathways, participating in the regulation of DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of EGFR signaling may lead to the inhibition of one or more EGFRs. In some embodiments, EGFR ligands include EGF, TGFα, heparin-binding EGF (HB-EGF), amphotericin B (AR), and epithelial protein (EPI).

[0095] For a detailed description of EGFR, see Sabbah, Dima A., Rima Hajjo, and Kamal Sweidan. "Review on epidermal growth factor receptor (EGFR) structure, signaling pathways, interactions, and recent updates of EGFR inhibitors." Currenttopics in medicinal chemistry (2020), the full text of which is incorporated herein by reference.

[0096] HER3 is a unique member of the EGFR family. While it is not carcinogenic on its own, it can cooperate with other receptors to induce tumorigenesis, metastasis, and drug resistance. HER3 is an attractive target for cancer therapy. Unlike EGFR and HER2, which are widely targeted with tyrosine kinase inhibitors (TKIs), HER3 has extremely low kinase activity and is primarily targeted by monoclonal or bispecific antibodies, either by blocking ligand binding or preventing heterodimerization with other receptors.

[0097] HER3 expression is ubiquitous in various cancers, including breast cancer, ovarian cancer, colon cancer, gastric cancer, lung cancer, skin cancer, and pancreatic cancer. High HER3 expression is also associated with disease progression and / or poor prognosis in many cancer types. Although HER3 itself does not cause tumorigenesis, the HER2:HER3 heterodimer has the highest transforming capacity among all possible EGFR family dimers. This stronger oncogenic capacity of the dimer pair makes HER3 essential for HER2-mediated tumorigenesis in multiple tumor types. In breast cancer cell lines, HER3 has been shown to be essential for maintaining cell viability, while EGFR is not required.

[0098] HER3 expression acts as a bypass mechanism for various targeted therapies, and elevated HER3 signaling leads to resistance to multiple therapeutic agents. For example, HER3 can dimerize with receptors other than EGFR (including HER2 and MET receptors), leading to resistance to EGFR-targeted therapies through dimerization with other non-EGFR chaperones. HER3 expression is also associated with resistance to hormone therapy. HER3 plays a crucial role in HER2 phosphorylation in breast cancer cells, and downregulation of HER3 reverses resistance to the anti-estrogenic receptor (ER) tamoxifen in breast cancer cell lines.

[0099] For a detailed review of HER3 and its functions, see Haikala, Heidi M. and Pasi A. Jänne, "Thirty Years of HER3: From Basic Biology to Therapeutic Interventions 30 Years of HER3." Clinical Cancer Research 27.13 (2021): 3528-3539; Mishra, Rosalin et al., "HER3 signaling and targeted therapy in cancer." Oncology reviews 12.1 (2018); Liu, Xiaolong et al., "Development of effective therapeutics targeting HER3 for cancer treatment." Biological procedures online 21.1 (2019): 1-10; all of which are incorporated herein by reference.

[0100] In some embodiments, the bispecific anti-EGFR / HER3 antibody described herein can be designed as an IgG1 subtype with a club-and-mortar (KIH) mutation, which promotes heterodimerization and avoids mismatch between the two heavy chains. In some embodiments, the bispecific anti-EGFR / HER3 antibody exhibits a higher endocytosis rate than the corresponding monoclonal antibody or control bispecific antibody.

[0101] In some embodiments, the bispecific anti-EGFR / HER3 antibody described herein may be conjugated with a therapeutic agent to form an antibody-drug conjugate (ADC). In some embodiments, the drug-to-antibody ratio (DAR) of the ADC described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADC described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4.4, about 3... .7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 to About 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.2, about 3.8 to about 4.2, about 3.9 to about 4.2, about 4.0 to about 4.2, about 4.1 to about 4.2, about 3.5 to about 4.1, about 3.6 to about 4.1, about 3.7 to about 4.1, about 3.8 to about 4.1, about 3.9 to about 4.1, about 4.0 to about 4.1, about 3.5 to about 4. 0, about 3.6 to about 4.0, about 3.7 to about 4.0, about 3.8 to about 4.0, about 3.9 to about 4.0, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7, or about 3.5 to about 3.6.In some implementations, the DAR of the ADC described herein is approximately 7.5 to approximately 8.5, approximately 7.6 to approximately 8.5, approximately 7.7 to approximately 8.5, approximately 7.8 to approximately 8.5, approximately 7.9 to approximately 8.5, approximately 8.0 to approximately 8.5, approximately 8.1 to approximately 8.5, approximately 8.2 to approximately 8.5, approximately 8.3 to approximately 8.5, approximately 8.4 to approximately 8.5, approximately 7.5 to approximately 8.4, approximately 7.6 to approximately 8.4, and approximately 7... .7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8.2 to About 8.3, about 7.5 to about 8.2, about 7.6 to about 8.2, about 7.7 to about 8.2, about 7.8 to about 8.2, about 7.9 to about 8.2, about 8.0 to about 8.2, about 8.1 to about 8.2, about 7.5 to about 8.1, about 7.6 to about 8.1, about 7.7 to about 8.1, about 7.8 to about 8.1, about 7.9 to about 8.1, about 8.0 to about 8.1, about 7.5 to about 8. 0, about 7.6 to about 8.0, about 7.7 to about 8.0, about 7.8 to about 8.0, about 7.9 to about 8.0, about 7.5 to about 7.9, about 7.6 to about 7.9, about 7.7 to about 7.9, about 7.8 to about 7.9, about 7.5 to about 7.8, about 7.6 to about 7.8, about 7.7 to about 7.8, about 7.5 to about 7.7, about 7.6 to about 7.7, or about 7.5 to about 7.6.

[0102] In some embodiments, the anti-EGFR / HER3 ADC described herein can effectively inhibit the growth of cancer cells in vitro at concentrations less than 10 μg / ml, less than 3.33 μg / ml, less than 1.11 μg / ml, less than 0.37 μg / ml, less than 0.12 μg / ml, less than 0.04 μg / ml, or less than 0.01 μg / ml. In some embodiments, the anti-EGFR / HER3 ADC described herein can inhibit the growth of cancer cells (e.g., lung cancer, gastric cancer, or skin cancer) in xenograft mouse models at dose levels less than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg.

[0103] In some embodiments, the anti-EGFR / HER3 antibodies described herein have a common light chain. In some embodiments, the anti-EGFR / HER3 antibody includes an anti-EGFR antigen-binding domain (e.g., 9F3, 9D2, 9A6) or an anti-HER3 antigen-binding domain (e.g., 1B2, 3E1, 3G6). In some embodiments, the anti-EGFR / HER3 antibody has a heavy chain variable region targeting EGFR (e.g., any of the EGFR-targeting VH described herein), a heavy chain variable region targeting HER3 (e.g., any of the HER3-targeting VH described herein), and two identical common light chain variable regions.

[0104] The CDR sequence of the 9F3 antigen-binding domain includes the CDRs of the heavy chain variable domain (SEQ ID NO: 4-6) and the light chain variable domain (SEQ ID NO: 1-3) as defined by Kabat. The CDRs can also be determined by Chothia. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 22-24, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 1-3. The fully human light chain variable region and the fully human heavy chain variable region of 9F3 are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

[0105] The CDR sequence of the 9D2 antigen-binding domain includes the CDRs of the heavy chain variable domain (SEQ ID NO: 7-9) and the light chain variable domain (SEQ ID NO: 1-3) as defined by Kabat. The CDRs can also be determined by Chothia. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 25-27, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 1-3. The fully human light chain variable region and the fully human heavy chain variable region of 9D2 are shown in SEQ ID NO: 40 and SEQ ID NO: 42, respectively.

[0106] The CDR sequence of the 9A6 antigen-binding domain includes the CDRs of the heavy chain variable domain (SEQ ID NO: 10-12) and the light chain variable domain (SEQ ID NO: 1-3) as defined by Kabat. The CDRs can also be determined by Chothia. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 28-30, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 1-3. The fully human light chain variable region and the fully human heavy chain variable region of 9A6 are shown in SEQ ID NO: 40 and SEQ ID NO: 43, respectively.

[0107] The CDR sequence of the 1B2 antigen-binding domain includes the CDRs of the heavy chain variable domain (SEQ ID NO: 13-15) and the light chain variable domain (SEQ ID NO: 1-3) as defined by Kabat. The CDRs can also be determined by Chothia. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 31-33, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 1-3. The fully human light chain variable region and the fully human heavy chain variable region of 1B2 are shown in SEQ ID NO: 40 and SEQ ID NO: 44, respectively.

[0108] The CDR sequence of the 3E1 antigen-binding domain includes the CDRs of the heavy chain variable domain (SEQ ID NO: 16-18) and the light chain variable domain (SEQ ID NO: 1-3) as defined by Kabat. The CDRs can also be determined by Chothia. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 34-36, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 1-3. The fully human light chain variable region and the fully human heavy chain variable region of 3E1 are shown in SEQ ID NO: 40 and SEQ ID NO: 45, respectively.

[0109] The CDR sequence of the 3G6 antigen-binding domain includes the CDRs of the heavy chain variable domain (SEQ ID NO: 19-21) and the light chain variable domain (SEQ ID NO: 1-3) as defined by Kabat. The CDRs can also be determined by Chothia. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 37-39, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 1-3. The fully human light chain variable region and the fully human heavy chain variable region of 3G6 are shown in SEQ ID NO: 40 and SEQ ID NO: 46, respectively.

[0110] In some embodiments, the anti-EGFR / HER3 antibody described herein may comprise one, two, or three heavy chain variable regions (CDRs) selected from SEQ ID NO: 4-6, SEQ ID NO: 7-9, SEQ ID NO: 10-12, SEQ ID NO: 13-15, SEQ ID NO: 16-18, SEQ ID NO: 19-21, SEQ ID NO: 22-24, SEQ ID NO: 25-27, SEQ ID NO: 28-30, SEQ ID NO: 31-33, SEQ ID NO: 34-36, and SEQ ID NO: 37-39; and / or one, two, or three light chain variable regions (CDRs) selected from SEQ ID NO: 1-3.

[0111] In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment may have heavy chain variable regions (VH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR1 amino acid sequence; CDR2 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR2 amino acid sequence; and CDR3 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR3 amino acid sequence; and light chain variable regions (VL) including CDRs 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL CDR1 amino acid sequence; and CDR2 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL CDR1 amino acid sequence; and CDR2 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL CDR1 amino acid sequence; and CDR3 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL CDR1 amino acid sequence; and CDR2 ... The CDR2 amino acid sequence has or is composed of an amino acid sequence with at least 80%, 85%, 90%, or 95% identity with the selected VL CDR3 amino acid sequence, and the CDR3 region contains or is composed of an amino acid sequence with at least 80%, 85%, 90%, or 95% identity with the selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: Figure 1 and Figure 2 As shown.

[0112] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 4 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 6 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0113] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 7 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 9 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0114] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 10 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 12 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0115] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 13 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 15 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0116] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 16 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 18 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0117] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 19 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 21 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0118] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 22 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 24 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0119] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 25 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 27 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0120] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 28 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 30 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0121] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 31 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 33 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0122] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 34 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 36 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0123] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 37 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 39 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0124] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment described herein may comprise a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 1 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 3 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0125] Insertion, deletion, and substitution can occur within the CDR sequence or at one or both ends of the CDR sequence.

[0126] In some embodiments, the anti-EGFR / HER3 antibody comprises: a heavy chain variable region (VH), the VH comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH sequence; and a light chain variable region (VL), the VL comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 41, 42, 43, 44, 45, or 46, and the selected VL sequence is SEQ ID NO: 40.

[0127] In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment may have the same three VH CDRs as any VH sequence described herein. In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment may have the same three VL CDRs as any VL sequence described herein.

[0128] This disclosure also provides a nucleic acid comprising a polynucleotide encoding an anti-EGFR / HER3 antibody. The anti-EGFR / HER3 antibody immunoglobulin heavy chain or immunoglobulin light chain comprises, for example... Figure 1 , Figure 2 or Figure 3 The CDR is shown. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to EGFR and / or HER3.

[0129] Anti-EGFR / HER3 antibodies can also be anti-EGFR / HER3 antibody variants (including derivatives and conjugates) or antibody fragments thereof. Other anti-EGFR / HER3 antibodies described herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (multimeric antibodies, such as bispecific antibodies), fully human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly generated antibodies (i.e., intrabody antibodies), and their antigen-binding fragments. Anti-EGFR / HER3 antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the anti-EGFR / HER3 antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or its antigen-binding fragment.

[0130] Anti-EGFR / HER3 antibody fragments are suitable for use in the methods presented herein, provided they retain the desired affinity and specificity for EGFR and HER3. Therefore, fragments of anti-EGFR / HER3 antibodies will retain the ability to bind to both EGFR and HER3.

[0131] Antibodies and their antigen-binding fragments

[0132] In some embodiments, the multispecific anti-EGFR / HER3 antibody (e.g., a bispecific antibody) comprises an antigen-binding domain derived from an anti-EGFR antibody and an antigen-binding domain derived from an anti-HER3 antibody. These anti-EGFR / HER3 antibodies and their antigen-binding fragments can take many forms.

[0133] Typically, antibodies (also known as immunoglobulins) are composed of two types of polypeptide chains: light chains and heavy chains. The non-restrictive anti-EGFR / HER3 antibody disclosed herein can be a complete tetraimmunoglobulin chain antibody containing two heavy chains and two light chains. The heavy chain of the anti-EGFR / HER3 antibody can be any isotype, including IgM, IgG, IgE, IgA, or IgD; or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain.

[0134] The hypervariable region, called the complementarity-determining region (CDR), forms a loop containing the main antigen-binding surface of the antibody. The four framework regions primarily employ a β-sheet conformation, and the CDRs form a loop structure that is connected and, in some cases, forms part of a β-sheet structure. The CDRs in each chain remain closely adjacent through the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding domain.

[0135] Identifying the CDR region of an antibody by analyzing its amino acid sequence is a well-known method, and many definitions of CDR are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in the following: for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecularimmunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp.Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., BiophysChem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan .1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each is incorporated into this paper in its entirety through citation.

[0136] CDR is important for recognizing antigenic epitopes. As used in this article, an "epitaph" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not necessarily have to be located in a continuous linear sequence of the primary antigen structure, as epitopes can depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0137] In some implementations, anti-EGFR / HER3 antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing only in their constant regions, particularly their hinges and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described, for example, in the following: Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0138] Anti-EGFR / HER3 antibodies can also be immunoglobulin molecules derived from any species (e.g., human, rodent, mouse, rat, camelid). An antigen-binding domain or antigen-binding fragment is an antibody portion that retains the specific binding activity of the intact antibody; that is, any portion of the antibody capable of specifically binding 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, an anti-EGFR / HER3 antibody or its antigen-binding fragment may comprise, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, biantibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies, and any polypeptide containing 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 chain and / or light chain CDR of the intact antibody, the variable region of the heavy chain and / or light chain of the intact antibody, the full-length heavy chain or light chain of the intact antibody, or a single CDR derived from the heavy chain or light chain of the intact antibody.

[0139] In some embodiments, the scFv in the anti-EGFR / HER3 antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the anti-EGFR / HER3 scFv has two antigen-binding regions, and these two antigen-binding regions can bind to their respective target antigens with different affinities.

[0140] In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment may contain one, two, or three components selected from... Figure 1 and Figure 2 The heavy chain variable region CDR.

[0141] In some embodiments, the anti-EGFR / HER3 antibodies described herein may be conjugated to a therapeutic agent. Anti-EGFR / HER3 antibody-pharmaceutical conjugates comprising the antibody or its antigen-binding fragment may be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., monomethylaurestatin E, monomethylaurestatin F, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxine, maytansine alkaloids (such as DM-1 and DM-4), diketones, mitoxantrone, styromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues). In some embodiments, the therapeutic agent is MMAE or MMAF. In some implementations, the therapeutic agent is coupled via a linker (e.g., a VC linker). For more information on the linkers used in ADCs, see Su, Z et al., "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), the full text of which is incorporated herein by reference.

[0142] In some embodiments, the anti-EGFR / HER3 antibody is a bispecific antibody. Bispecific antibodies can be prepared by modifying the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size targeting the large side chains are generated at the interface of the second antibody molecule by replacing the larger amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products (e.g., homodimers). This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0143] Any anti-EGFR / HER3 antibody or its antigen-binding fragment described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation to a stabilizing molecule may prolong the half-life or enhance the biological activity of the anti-EGFR / HER3 antibody or antigen-binding fragment in vitro (e.g., in tissue cultures or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0144] Anti-EGFR / HER3 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 and described, for example, in Suurs et al., "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges," Pharmacology & therapeutics (2019), which is incorporated herein by reference in its entirety.

[0145] In some embodiments, the anti-EGFR / HER3 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-EGFR / HER3 antibody is VHH-scAb, VHH-Fab, dual scFab, F(ab')2, biantibody, crossMab, DAF (dual-component), DAF (quadrivalent-component), DutaMab, DT-IgG, knots-in-holes co-light chain, knots-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triple antibody, Micro antibody, Micro antibody, TriBi micro antibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Quadrivalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intracellular antibody, Dock and lock antibody, lmm TAC, IgG-IgG conjugate, Cov-X-Body or scFv1-PEG-scFv2.

[0146] In some implementations, the anti-EGFR / HER3 antibody may be TrioMab. In TrioMab, the two heavy chains are from different species, and the different sequences restrict heavy chain-light chain pairing.

[0147] In some implementations, the anti-EGFR / HER3 antibody has two distinct heavy chains and a common light chain. The heterodimerization of the heavy chains can be performed according to a mortise and tenon joint or some other heavy chain pairing technique.

[0148] In some implementations, CrossMAb technology can be used to generate bispecific anti-EGFR / HER3 antibodies. CrossMAb technology can be used to enforce correct light chain association in bispecific heterodimeric IgG antibodies, allowing for the generation of various bispecific antibody formats, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as antibodies based on non-Fc tandem antigen-binding fragments (Fab). These formats can be derived from any existing antibody pair using domain cross-referencing without identifying a common light chain, post-translational processing / in vitro chemical combinations, or introducing a set of mutations to enforce correct light chain association. This approach 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 implementations, the CH1 domain in the heavy chain and the CL domain in the light chain are interchanged.

[0149] Anti-EGFR / HER3 antibodies can be duobody. The naturally occurring Fab exchange mechanism in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies; this mechanism is called controlled Fab exchange. This format ensures specific pairing between the heavy and light chains.

[0150] In dual variable domain antibodies (DVD-Ig), an additional VH and variable light chain (VL) domains are added to each N-terminus to achieve bispecific targeting. This format is similar to IgG-scFv, but the added binding domains bind individually to their respective N-termini, rather than scFv binding to the N-terminus of each heavy chain.

[0151] In scFv-IgG, two scFvs are attached to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two distinct bivalent binding sites and is therefore also called tetravalent. There is no heavy chain and light chain pairing issue in scFv-IgG.

[0152] In some implementations, the anti-EGFR / HER3 antibody may be in IgG-IgG format. Two complete IgG antibodies are conjugated by chemically linking the C-terminus of the heavy chain.

[0153] Anti-EGFR / HER3 antibodies can also be produced in the Fab-scFv-Fc format. In the Fab-scFv-Fc format, the light chain, heavy chain, and a third chain containing the Fc region and scFv are assembled together. This ensures efficient manufacturing and purification.

[0154] In some implementations, the anti-EGFR / HER3 antibody may be a TF (fiber molecule). Three Fab fragments are linked by disulfide bonds. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF format does not have an Fc region.

[0155] ADAPTIR has two scFvs that bind to each side of the Fc region. It abandons the complete IgG as the basis of its construct but retains the Fc region to prolong the half-life and facilitate purification.

[0156] Amphiphilic targeted peptides (DART) have two peptide chains linked with opposite segments, so VLA is linked to VHB and VLB to VHA, and their C-termini are fused together via a sulfur bond. In DART, the sulfur bond provides better stability compared to BiTE.

[0157] In DART-Fc, the Fc region is attached to the DART structure. It can be generated by combining three chains, two of which are connected by disulfide bonds, just like DART. One chain contains half of the Fc region, which dimers with the third chain that expresses only the Fc region. The addition of the Fc region increases the half-life, allowing the effective concentration to be maintained for a longer period of time, avoiding continuous intravenous injection (IV).

[0158] In tetravalent DART, four peptide chains are combined. Essentially, this results in two DART molecules, each containing half of the Fc region, which dimerize. This format has the ability to bind divalently to two targets, hence it is a tetravalent molecule.

[0159] Tandem biantibodies (TandAbs) consist of two biantibodies. Each biantibody comprises one VHA and VLB fragment and another VHA and VLB fragment, covalently associated. The two biantibodies are linked by peptide chains. This provides improved stability compared to biantibodies composed of two scFvs. It has two bivalent binding sites.

[0160] ScFv-scFv-toxin comprises a toxin and two scFvs with stable linkers. It can be used for specific delivery of payloads.

[0161] In some embodiments, the anti-EGFR / HER3 antibody is a bispecific antibody. In some embodiments, the bispecific antibody of this disclosure is designed as 1+1 (each target is monovalent) and has an IgG1 subtype structure. This can reduce affinity for cells that express low levels of EGFR and HER3 and increase affinity for cells that co-express EGFR and HER3 to achieve enhanced targeting function.

[0162] In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment has a light chain constant region having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 47 and a heavy chain constant region having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with either SEQ ID NO: 48 or 49.

[0163] In some embodiments, the anti-EGFR / HER3 antibody includes a KIH mutation. In some embodiments, the anti-EGFR / HER3 antibody includes a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to HER3. In some embodiments, the first antigen-binding domain includes a heavy chain containing one or more acetabular mutations (acetabular heavy chain), and the second antigen-binding domain includes a heavy chain containing one or more acetabular mutations (acetabular heavy chain). In some embodiments, the first antigen-binding domain includes a heavy chain containing one or more acetabular mutations (acetabular heavy chain), and the second antigen-binding domain includes a heavy chain containing one or more acetabular mutations (acetabular heavy chain). In some embodiments, the anti-EGFR / HER3 antibody includes a acetabular heavy chain containing a constant region having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 48. In some embodiments, the anti-EGFR / HER3 antibody includes a mortar heavy chain containing a constant region having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 49.

[0164] Antibody characteristics

[0165] Anti-EGFR / HER3 antibodies may include an anti-EGFR antigen-binding domain and any of the anti-HER3 antigen-binding domains described herein.

[0166] This disclosure provides anti-EGFR / HER3 antibodies that specifically bind to EGFR and their antigen-binding fragments. These anti-EGFR / HER3 antibodies can be agonists or antagonists. The anti-EGFR / HER3 antibodies or their antigen-binding fragments described herein can bind to EGFR and block the binding between EGFR and its ligand. By blocking the binding between EGFR and its ligand, anti-EGFR / HER3 antibodies can inhibit EGFR-related signaling pathways and thus treat cancer. In some embodiments, the anti-EGFR / HER3 antibodies or their antigen-binding fragments can induce CDC or ADCC.

[0167] The affinity of an antibody for an antigen can be measured using common techniques, including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and surface plasmon resonance (SPR). Affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some embodiments, anti-EGFR / HER3 antibodies or their antigen-binding fragments can bind EGFR (e.g., human EGFR, monkey EGFR, mouse EGFR, and / or chimeric EGFR) at the following dissociation rates (koff): less than 0.1 s. -1 Less than 0.01 s -1 Less than 0.001 s -1 Less than 0.0001 s -1 or less than 0.00001 s -1 In some implementations, the dissociation rate (koff) is greater than 0.01 s⁻¹. -1 Greater than 0.001 s -1 Greater than 0.0001 s -1 Greater than 0.00001 s -1 or greater than 0.000001 s -1 .

[0168] In some implementations, the kinetic association rate (kon) is greater than 1 x 10⁻⁶. 2 / Ms, greater than 1 x 10 3 / Ms, greater than 1 x 10 4 / Ms, greater than 1 x 10 5 / Ms or greater than 1 x 10 6 / Ms. In some implementations, the kinetic association rate (kon) is less than 1 x 10 5 / Ms, less than 1 x 10 6 / Ms or less than 1 x 10 7 / Ms.

[0169] In some implementations, the anti-EGFR / HER3 antibody or its antigen-binding fragment can bind EGFR (e.g., human EGFR, monkey EGFR, mouse EGFR, and / or chimeric EGFR) using the following KD: less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M or less than 1 x 10 -10 M. In some embodiments, KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10 -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M or greater than 1 x 10 -10 M.

[0170] Anti-EGFR / HER3 antibodies or their antigen-binding fragments may also include an antigen-binding domain that specifically binds to HER3. The anti-EGFR / HER3 antibodies or their antigen-binding fragments described herein can block the binding between HER3 and its ligand. In some embodiments, by binding to HER3, anti-EGFR / HER3 antibodies can also inhibit HER3-related signaling pathways and suppress cell proliferation, differentiation, and / or metastasis. Therefore, in some embodiments, the anti-EGFR / HER3 antibodies described herein are HER3 agonists. In some embodiments, anti-EGFR / HER3 antibodies are HER3 antagonists.

[0171] In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment can bind HER3 (e.g., human HER3, monkey HER3, mouse HER3, and / or chimeric HER3) at the following dissociation rate (koff): less than 0.1 s. -1 Less than 0.01s -1 Less than 0.001 s -1 Less than 0.0001 s -1 or less than 0.00001 s -1 In some implementations, the dissociation rate (koff) is greater than 0.01 s⁻¹. -1 Greater than 0.001 s -1 Greater than 0.0001 s -1 Greater than 0.00001 s -1 or greater than 0.000001 s -1 .

[0172] In some implementations, the kinetic association rate (kon) is greater than 1 x 10⁻⁶. 2 / Ms, greater than 1 x 10 3 / Ms, greater than 1 x 10 4 / Ms, greater than 1 x 10 5 / Ms or greater than 1 x 10 6 / Ms. In some implementations, the kinetic association rate (kon) is less than 1 x 10 5 / Ms, less than 1 x 10 6 / Ms or less than 1 x 10 7 / Ms.

[0173] The affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some implementations, KD is less than 1 x 10⁻⁶. -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M or less than 1 x 10 -10 M. In some embodiments, KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10⁻⁶. -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M or greater than 1 x 10 -10 M.

[0174] Because anti-EGFR / HER3 antibodies (such as bispecific antibodies) can bind to both EGFR and HER3 simultaneously, they exhibit higher binding affinity for cells that co-express EGFR and HER3. Affinity can be used to measure the binding affinity of an antibody to these cells. Affinity refers to the cumulative binding strength of multiple independent nonvalently binding interactions.

[0175] In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment is capable of binding to human HER3 or monkey HER3. In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment is not capable of binding to human HER3 or monkey HER3. In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment is not capable of binding to human EGFR or monkey EGFR.

[0176] In some embodiments, the anti-EGFR / HER3 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 by, for example, 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 by, for example, HPLC.

[0177] In some embodiments, the purity of the anti-EGFR / HER3 antibody, antigen-binding fragment, or ADC is 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 hydrophobic interaction chromatography (HIC) retention time of the anti-EGFR / HER3 antibody, antigen-binding fragment, or ADC is greater than 2 minutes, greater than 3 minutes, greater than 4 minutes, or greater than 5 minutes. In some embodiments, the HIC retention time is less than 2 minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes, or less than 6 minutes.

[0178] In some implementations, the purity of the anti-EGFR / HER3 antibody, antigen-binding fragment, or ADC is greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, as determined by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).

[0179] In some implementations, the main peak of the anti-EGFR / HER3 antibody, antigen-binding fragment, or ADC accounts for more than 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the total sample volume, as determined by capillary isoelectric focusing (cIEF). In some implementations, the acidic peak of the anti-EGFR / HER3 antibody, antigen-binding fragment, or ADC accounts for less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the total sample volume, as determined by capillary isoelectric focusing (cIEF).

[0180] In some embodiments, the anti-EGFR / HER3 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-EGFR / HER3 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. TGI% can be determined, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the start of treatment. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula: TGI (%)=[1-(Ti-T0) / (Vi-V0)]×100 Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0181] In some embodiments, the anti-EGFR / HER3 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 both ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0182] In some embodiments, the anti-EGFR / HER3 antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-EGFR / HER3 antibody or its antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the anti-EGFR / HER3 antibody or its antigen-binding fragment described herein has an effector-free Fc region. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbers) or LALA-PG mutations (L234A, L235A, P329G mutations according to EU numbers).

[0183] The Fc region can undergo several other modifications. For example, cysteine ​​residues can be introduced into the Fc region, allowing interchain disulfide bonds to form there. The resulting homodimeric fusion protein may have an increased half-life in vitro and / or in vivo.

[0184] In some implementations, IgG4 has an S228P mutation (EU number). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0185] In some embodiments, an Fc region with a sugar structure is provided that lacks fucose attached (directly or indirectly) 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, as described, for example, in WO 2008 / 077546, by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, heterozygous, and high-mannose structures) attached to Asn297, by MALDI-TOF mass spectrometry. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu number of the Fc region residue; or position 314 in the Kabat number); however, due to minor sequence variations in the Fc region sequence, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylation variants can possess improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further modified to replace the asparagine at position 297 with alanine (N297A).

[0186] In some embodiments, after purification by protein A-based affinity chromatography and / or size exclusion chromatography, the main peak of HPLC-SEC accounts for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the protein complex described herein.

[0187] In some implementations, the bispecific anti-EGFR / HER3 antibody described herein has a higher endocytosis rate than the corresponding monoclonal antibody and / or positive control antibody described herein.

[0188] Antibody-drug conjugates (ADCs)

[0189] The anti-EGFR / HER3 antibodies or their antigen-binding fragments described herein can be conjugated to therapeutic agents (medications). The therapeutic agents can be covalently or non-covalently bound to the anti-EGFR / HER3 antibody. In some embodiments, the anti-EGFR / MUC1 antibody is an anti-EGFR / HER3 bispecific antibody. In some embodiments, the bispecific antibody has a common light chain.

[0190] In some implementations, the therapeutic agent is a cytotoxic agent or cell inhibitor (e.g., monomethylolpropamine E, monomethylolpropamine F, camptothecin, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxine, maytansine alkaloids (such as DM-1 and DM-4), diketones, mitoxantrone, styracin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues). Useful classes of cytotoxic, cell growth inhibitors, or immunomodulators include, for example, antituberculosis drugs, DNA small groove binders, DNA replication inhibitors, and alkylating agents.

[0191] In some embodiments, the therapeutic agent may include, but is not limited to, cytotoxic agents such as chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antibacterial agents. In some embodiments, the therapeutic agent to be conjugated may be selected from, but is not limited to, MMAE (monomethylauristan E), MMAD (monomethylauristan D), or MMAF (monomethylauristan F).

[0192] Specific definitions of functional groups and chemical terms are detailed below. For the purposes of this invention, the identification of chemical elements is based on the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75. th Edition, inside cover, and the definition of specific functional groups is generally as described therein. Furthermore, the general principles of organic chemistry, as well as specific functional groups and reactivity, are described in the following references: Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smithand March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCHPublishers, Inc., New York, 1989; Carruthers, Some Modem Methods of OrganicSynthesis, 3 rdEdition, Cambridge University Press, Cambridge, 1987.

[0193] Unless otherwise expressly stated, all ranges referenced in this document are included. When listing a range of values, the range should include every value within the range and its subranges. For example, "C 1-6 "Includes C1, C2, C3, C4, C5, C6, C 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 .

[0194] The compounds disclosed herein, or any formula describing or depicting compounds, may have one or more chiral (asymmetric) centers. This invention includes all stereoisomers of the compounds described herein, or any formula describing or depicting compounds. Asymmetric centers present in any formula of the compounds of this invention may have (R) or (S) configurations independently of each other. When the bonds of the chiral carbons in the structural formula are depicted as straight lines, or when the name of the compound does not contain a (R) or (S) chiral name for the chiral carbons, it should be understood that each chiral carbon has both (R) and (S) configurations, and therefore each enantiomer or diastereomer and mixtures thereof are included in the formula or name.

[0195] This disclosure includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, such as mixtures of enantiomers and / or diastereomers present in various proportions. Therefore, the enantiomers of this disclosure include enantiomeric purees, levorotatory and dextrorotatory enantiomers, racemates, and mixtures of two enantiomers in various proportions. If cis / trans isomers are present, mixtures of cis and trans isomers in various proportions are included. If desired, the individual stereoisomers can be prepared by separating the mixture using conventional methods, such as chromatography or crystallization, by using stereochemically homogeneous starting materials, or by stereoselective synthesis. Optionally, derivatization can be performed prior to stereoisomer separation. Separation of stereoisomer mixtures can be carried out as an intermediate step in compound synthesis or on the final racemic product. Absolute stereochemistry can be determined by X-ray crystallography analysis of the crystalline product or crystalline intermediate, where necessary, the crystalline intermediate can be derivatized using a reagent containing a stereocenter of known configuration. Alternatively, the absolute stereochemical structure can also be determined by vibrational circular dichroism (VCD) spectroscopy.

[0196] Unless otherwise stated, the structures described herein also include compounds that differ only in the presence of one or more isotopically enriched atoms; in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Such compounds are referred to as “isotope variants.” This disclosure is intended to include all pharmaceutically acceptable isotope variants of any formula of the compounds of the invention or of which compounds are depicted and described. Examples of isotopes suitable for inclusion in the compounds of the invention include, but are not limited to, isotopes of hydrogen, such as… 2 H (i.e., D) and 3 H; carbon, such as 11 C 13 C, and 14 C; chlorine, such as 36 Cl; fluorine, such as 18 F; Iodine, such as 123 I and 125 I; Nitrogen, such as 13 N and 15 N; oxygen, such as 15 O、 17 O and 18 O; phosphorus, such as 32 P; and sulfur, such as 35 S. Certain isotopic variants of the compounds disclosed herein, or any formulas of compounds depicted and described herein, such as those containing radioactive isotopes, may be helpful in studies of drug and / or substrate tissue distribution. In particular, compounds having the described structure, differing only in the substitution of a heavier isotope, such as with deuterium (… 2Replacing hydrogen with H or D can provide certain therapeutic advantages, such as greater metabolic stability, prolonged in vivo half-life, or reduced dose requirements, and therefore may be used in certain specific situations. The compounds disclosed herein, or isotopic variants of any formula of the compounds depicted and described, are generally prepared by techniques known to those skilled in the art, or by processes similar to those described in the appended examples, and are synthesized using appropriate isotopic labeling reagents instead of previously used unlabeled reagents.

[0197] The compounds described herein are illustrated with reference to general formulas and specific compounds. Furthermore, the compounds disclosed herein may exist in a variety of 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, regioisomers, prodrugs, solvated forms, different crystal forms or polymorphs, and active metabolites.

[0198] As used herein, unless otherwise stated, the term "pharmaceutically acceptable salt" includes salts that retain the bioavailability of a particular compound in its free acid / base form and have no adverse effects on the biological or other respects. Pharmaceutically acceptable salts can include salts formed with inorganic bases or acids, as well as organic bases or acids. Where the compounds of this disclosure contain one or more acidic or basic groups, this disclosure also includes their corresponding pharmaceutically acceptable salts. Thus, compounds of the invention containing acidic groups (such as carboxyl groups) can be present in the form of salts and can be used according to the invention, such as alkali metal salts, alkaline earth metal salts, aluminum salts, or ammonium salts. Further non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts having ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts are readily available, for example, by reacting compounds having acidic groups with a suitable base (e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide). Other basic salts of the compounds disclosed herein include, but are not limited to, copper (I), copper (II), iron (II), iron (III), manganese (II), and zinc salts. The compounds of this disclosure contain one or more basic groups, such as protonable groups, may exist in salt form, and may be used according to this disclosure as addition salts of 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, pentylamino acid, diethylacetic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, dihydroxynaphthalic acid, mandelic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids known to those skilled in the art. The salts formed include hydrochlorides, chlorides, hydrobromates, bromides, iodides, sulfates, phosphates, methanesulfonates, toluenesulfonates, carbonates, bicarbonates, formates, acetates, sulfoacetates, trifluoromethanesulfonates, oxalates, malonates, maleates, succinates, tartrates, malates, bis(hydroxynaphthyl)ate, mandelates, fumarates, lactates, citrates, glutarate, stearates, aspartate salts, and glutamates. Furthermore, the stoichiometric ratio of the salts formed from the compounds of this invention can be an integer multiple or a non-integer multiple of 1.

[0199] The compounds containing basic nitrogen-containing groups in this disclosure can be quaternized using the following agents: C 1-4 Alkyl halides, such as methyl, ethyl, isopropyl, and tert-butyl chlorides, bromides, and iodides; dicarbonyl halides. 1-4 Alkyl sulfates, such as dimethyl sulfate, diethyl sulfate, and dipentyl sulfate; C10-18 Alkyl halides, such as decyl, dodecyl, lauryl, tetradecyl and octadecyl chlorides, bromides and iodides; and aryl C 1-4 Alkyl halides, such as benzyl chloride and phenylethyl bromide.

[0200] If the compounds of this disclosure contain both acidic and basic groups in their molecules, this disclosure also includes, in addition to the salt forms mentioned, internal salts or betaines (zwitterions). The corresponding salts can be obtained by conventional methods known to those skilled in the art, such as by contacting these salts with organic or inorganic acids or bases in a solvent or dispersant, or by anion or cation exchange with other salts. This disclosure also includes all salts of the compounds of this disclosure that are unsuitable for direct use in pharmaceuticals due to low physiological compatibility, but can be used, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0201] The compounds disclosed herein, or any formula of compounds depicting and describing them, and their pharmaceutically acceptable salts, may exist in both insoluble and soluble forms. As used herein, the term "solvent" refers to a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules. For example, the term "hydrate" is used when the solvent is water.

[0202] Pharmaceutically acceptable solvates according to this disclosure may include those in which the crystallization solvent can be replaced by an isotope, such as D2O, d6-acetone, d6-DMSO.

[0203] Linker (linker compound)

[0204] In some embodiments, the therapeutic agent is coupled via a linker (or linker compound). As used herein, the terms "linker" or "linker compound" refer to a compound that can, for example, react with groups of a ligand compound and a therapeutic agent compound, respectively, to link a ligand (antibody, its antigen-binding fragment, or antigen-binding protein construct such as a bispecific antibody) and a therapeutic agent (any therapeutic agent described herein) together to form a ligand-drug conjugate.

[0205] In some embodiments, the connector described herein is a compound having the following formula:

[0206] Formula (I), Or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein Q represents a linker portion capable of coupling with a ligand via a bond selected from carbonyl, thioether, amide, disulfide, and hydrazone; and L represents a linker portion capable of connecting Q to a therapeutic agent.

[0207] In some implementations, the connecting portion (Q in formula (I)) has the following structure: .

[0208] In some implementations, the connecting portion (L in formula (I)) has the following structure: , L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue with a side chain carboxyl group, such as glutamic acid residue or aspartic acid residue, where "-COOH" represents the carboxyl group of the C-terminal amino acid residue of the polypeptide residue; L2 is a monodentate, bidentate, or tripentate hydrophilic group on the side chain carboxyl group of an amino acid residue that is absent or attached to the peptide residue L1, and L2 has NHC(R L2a (R) L2b (R) L2c The structure of ) where R L2a R L2b and R L2c Each can independently choose H, -(CH2O)(CH2CH2O) m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d The group consisting of R L2d It is H or C optionally substituted with 1 to 6 hydroxyl groups. 1-6 Alkyl groups, each m being an independent integer from 0 to 10, preferably from 0 to 4, for example 0, 1, 2, 3 or 4, particularly preferably m is 0, and each p being an independent integer from 1 to 4, for example 1, 2, 3 or 4; and This indicates that the polypeptide residues are covalently linked to the N-terminal side of the linker Q.

[0209] In some implementations, peptide residue L1 is NH -Glutamic acid-valine-alanine- COOH In some embodiments, the hydrophilic group L2 has the following structure: , in" "" indicates a site covalently linked to peptide residue L1, for example NH -Glutamic acid-valine-alanine- COOH The side chain of the glutamic acid residue.

[0210] In some embodiments, the linker described herein is a compound having the following structure:

[0211] CPT-L

[0212] In some implementations, the linker is the VC linker. For more information on the linkers used in ADCs, see Su, Z. et al., "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), the full text of which is incorporated herein by reference.

[0213] Therapeutic agents

[0214] In some implementations, therapeutic agents conjugated with the antibodies or their antigen-binding fragments described herein are discussed below.

[0215] In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, its analogues, or derivatives. In some preferred embodiments, the camptothecin compound is a compound having the following structure: , X is selected from the group consisting of -CH2-, O, and S; Y is selected from the group consisting of H, D, and F.

[0216] 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]inzazo[1,2-b]thiarano[4,3,2-de]quinoline-10,13(2H)-dione (CPT-1). The structure of CPT-1 is shown below: .

[0217] CPT-1

[0218] 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]inzazo[1,2-b]quinoline-10,13(2H)-dione (CPT-2). The structure of CPT-2 is shown below: .

[0219] CPT-2

[0220] In some implementations, the therapeutic agent is CPT3. The structure of CPT-3 is shown below: .

[0221] CPT-3

[0222] 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]inzazido[1,2-b]quinoline-10,13(2H)-dione (CPT-4). The structure of CPT-4 is shown below: .

[0223] CPT-4

[0224] In some embodiments, the therapeutic agent is auristatin, such as auristatin E (also known in the art as a derivative of dolasstatin-10) or a derivative thereof. For example, auristatin can be an ester formed between auristatin E and a keto acid. For example, auristatin E can react with acetylbenzoic acid or benzoylvaleric acid to generate AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. Exemplary synthesis and structures of auristatin are described in U.S. Patent Application Publication No. 2003-0083263, International Patent Publication No. WO 04 / 010957, and International Patent Publication No. WO 02 / 088172 and U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,09 Descriptions are found in 7; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, the full text of each of which is incorporated herein by reference and used for the purposes described herein.

[0225] Studies have shown that olistatin can interfere with microtubule dynamics, cell nucleus, and cell division, and possesses anticancer activity. Olistatin binds to tubulin, which can exert cytotoxic or inhibitory effects on cancer cells. Many different assay methods are known in the art for determining whether olistatin or its antibody-drug conjugates produce inhibitory or cytotoxic effects on desired cells.

[0226] In some implementations, 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, indomethacin, and piperazofan); aziridines (such as benzodopa, carboquinone, meturedopa, and uredopa); ethyleneimines and methylmelamines (including hexamethylmelamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine); nitrogen mustards (such as chlorambucil, naphthiamethoxam, cholophosphamide, estradiol, ifosfamide, phenylalanine mustard, dichloromethyldiethylamine, and mechlorethamine oxide). Hydrochlorides, neonitrogen mustard, benzyl mustard cholesterol, prednimustine, trafosamide, uracil mustard; nitrosoureas (such as carmustine, chlorpromazine, formustine, lomustine, nimustine, ramustine); antibiotics (such as aclarubicin, actinomycin, atrazosin, azoserine, actinomycin C, chalcogenine, kalabicin, erythromycin, carcinomacin, chromomycin, actinomycin D, daunorubicin, detoxin, 6-hexamethasone Nitrogen-5-oxo-L-leucine, doxorubicin, epirubicin, isorubicin, idarubicin, methacycline, mitomycin, mycophenolic acid, nogamycin, oligomycin, pepromycin, potfiromycin, puromycin, triamcinolone acetonide, rodorubicin, streptomycin, streptozotocin, tuberculin, ubenmex, fenestrated lincomycin, zolrubicin; antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); Folic acid analogs (such as folate, methotrexate, pteroxetine, trimethoprim); purine analogs (such as fludarabine, 6-mercaptopurine, thioimidazoline, thioguanine); pyrimidine analogs (such as ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine, 5-FU); androgens (such as calotestosterone, drotaldone propionate, cyclothionol, meandrolone, testrolide); antiadrenergic drugs. (such as aminoglutethimide, mitotane, trilostertan); folic acid supplements (such as folinic acid); glucuronolactone; aldophosphamide glycoside; aminolevulinic acid; acridine; bestrabucil; bisacodyl; edatraxate; defofamine; colchicine; diacodyl; efornithine; erythritol;Etoglobulin; Gallium nitrate; Hydroxyurea; Lentinan; Clonidamine; Mitoguanidine hydrazone; Mitoantrone; Mopiperidine; Diamine nitroacetate; Pentostatin; Methamidoamine (phenamet); Pirarubicin; Podophyllotoxin; 2-ethylhydrazide; Procarbazine; PSK7; Razosen; Cizonan; Germanium spiroamine; Alternaria ketoacid; Triaminoquinone; 2',2',2'-Trichlorotriethylamine; Ethyl carbamate; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeugenol; Piperobromide; Cytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Taxanes (e.g., paclitaxel (TAXOL); ® (Bristol-Myers Squibb Oncology, Princeton, New Jersey) and TAXOTERE ® Rhone-Poulenc Rorer (Antoine de France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs (such as cisplatin and carboplatin); vincristine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; mitoxantrone (Novantrone); teniposide; donomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperapramycin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances. This definition also includes anti-hormonal agents that regulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, raloxifene, LY117018, onanasone, and toremifene (Faldon); and anti-androgens, such as flutamide, nilumid, bicalutamide, leuprorelin, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing substances. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, the entire text of which is incorporated herein by reference.

[0227] Linker-therapeutic compound

[0228] In some implementations, a linker (any linker described herein) and a therapeutic agent (any therapeutic agent described herein) may be linked to form a "linker-therapeutic agent" compound.

[0229] In some embodiments, the linker-therapeutic compound has the following structure: .

[0230] In some embodiments, the linker-therapeutic compound has the following structure: .

[0231] In some embodiments, an antibody (“Ab”), any antibody described herein or its antigen-binding fragment, may be linked to a linker-therapeutic agent compound (any linker-therapeutic agent compound described herein) to form an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate has the following structure: , Where n = 1-8. In some implementations, n = 1-8. In some implementations, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8. In some implementations, n is about 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8. In some implementations, n is an integer multiple or a non-integer multiple of 1.

[0232] In some embodiments, the anti-EGFR / HER3 antibody is conjugated to a drug via a cleavable linker (e.g., an SPBD linker or a maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker). In some embodiments, the anti-EGFR / HER3 antibody is conjugated to a drug via a non-cleavable linker (e.g., an MCC linker formed using SMCC or sulfonyl-SMCC). Those skilled in the art can readily select a suitable linker for a given ADC by considering relevant factors such as the linker site of the anti-EGFR / HER3 antibody, any structural limitations of the drug, and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). Many specific linker-toxin combinations have been described, and in some embodiments, they can be used in conjunction with the anti-EGFR / HER3 antibody described herein to prepare an ADC. Examples include, but are not limited to, cleavable peptide-based linkers containing aureatine (such as MMAE and MMAF) and camptothecin (such as SN-38, betamethasone, and PBD dimer); non-cleavable MC-based linkers containing aureatine MMAF and MMAE; acid-labile hydrazone-based linkers containing chachomycin and doxorubicin; disulfide-based linkers containing maytansine alkaloids (such as DM1 and DM4); and bis-maleimide-triethylene glycol (BMPEO)-based linkers containing maytansine alkaloid DM1. Some such therapeutic agents and connectors 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 Publications US 2015 / 0374847 and US20180193477A1, the entire contents of which are incorporated herein by reference.

[0233] Depending on the desired drug and the selected connector, those skilled in the art can choose a suitable method to couple them together. For example, some conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-connector complex, which still contains a reactive group covalently linked to the anti-EGFR / HER3 antibody. In some embodiments, a drug-maleimide complex (i.e., a maleimide-linked drug) can be used as a payload with a reactive group in this disclosure. In ADC preparation, the most common reactive group capable of bonding with thiol groups is maleimide. Additionally, organic bromides and iodides are also frequently used.

[0234] Anti-EGFR / HER3 ADCs can be prepared using one of several methods known in the art, employing organic chemical reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (GTHermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) reacting a nucleophilic or electrophilic group of the antibody with a divalent linker reagent to covalently form an antibody-linker intermediate Ab-L, which is then reacted with an activated drug moiety D; or (2) reacting a nucleophilic or electrophilic group of the drug moiety with a linker reagent to covalently form a drug-linker intermediate DL, which is then reacted with a nucleophilic or electrophilic group of the anti-EGFR / HER3 antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the ADCs described herein. Various prepared linkers, linker components, and toxins are commercially available or can be prepared using standard synthetic organic chemical techniques. These methods are described in, for example, 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 (GT Hermanson, 2013, Academic Press); US20210379193A1 and US20180193477A1, all of which are incorporated herein by reference in their entirety. Furthermore, many pre-formed drug-linkers suitable for responses to anti-EGFR / HER3 antibodies are commercially available, including linker-toxins such as DM1, DM4, MMAE, MMAF, or pyruvic acid SA, obtained from Creative BioLabs (Shirley, NY).

[0235] Several specific examples of methods for preparing anti-EGFR / HER3 ADCs are known in the art and described in U.S. Patent Nos. 8,624,003 (one-pot method), 8,163,888 (one-step method), 5,208,020 (two-step method), and US20180193477A1, all of which are incorporated herein by reference in their entirety. Other methods are known in the art and include those described in *Antibody-Drug Conjugates: Methods in Molecular Biology*, 2013, *Ducry* (Ed.), Springer.

[0236] Drug loading is expressed as the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, drug loading may be limited by the number of attachment sites on the antibody. For example, when cysteine ​​thiol is attached, as in some exemplary embodiments described herein, drug loading can range from 0 to 8 drug moieties per antibody. In some embodiments, higher drug loading, such as p ≥ 5, can cause aggregation, insolubility, toxicity, or loss of cell permeability in some antibody-drug conjugates. In some embodiments, the average drug loading of anti-EGFR / HER3 antibody-drug conjugates ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In fact, for some antibody-drug conjugates, the optimal ratio of drug moieties per antibody has been shown to be about 4. In some embodiments, the DAR of the anti-EGFR / HER3 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-EGFR / HER3 ADC composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0237] In some implementations, anti-EGFR / HER3 antibody variants are provided that have a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region. For example, the amount of fucose in such antibodies 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 within the glycan chain at Asn297 relative to the sum of all glycan structures linked to Asn297 (e.g., complex, heterozygous, and high-mannose structures), as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu number of the Fc region residue; or position 314 in the Kabat number); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants can possess improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-EGFR / HER3 antibody can be further engineered to replace the asparagine at position 297 (N297A) with alanine.

[0238] In some implementations, to improve production efficiency by avoiding Fab arm swapping, the Fc region of the anti-EGFR / HER3 antibody or its antigen-binding fragment is further modified by replacing the serine at position 228 (EU number) of IgG4 with proline (S228P). A detailed description of the S228 mutation can be found in Silva et al., "The S228P mutation prevents..." in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. Journal of Biological Chemistry 290.9 (2015): 5462-5469, the full text of which is incorporated herein by reference.

[0239] In some embodiments, the methods described herein are intended to prepare bispecific anti-EGFR / HER3 antibodies. Anti-EGFR / HER3 bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural 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 smaller amino acid side chains (e.g., alanine or threonine), a compensating "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism that allows for a higher yield of heterodimers than other unwanted end products (such as homodimers). This method is described, for example, in WO 96 / 27011, the entire contents of which are incorporated herein by reference.

[0240] In some implementations, the kilometre-hole (KIH) technique can be used, which involves engineering CH3 domains to create "kidneys" or "mortars" in each heavy chain to promote heterodimerization. The KIH technique is described, for example, by 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, the entire text of which is incorporated herein by reference. In some embodiments, one heavy chain has T366W and / or S354C (mortar) substitutions (EU number), while the other heavy chain has Y349C, T366S, L368A, and / or Y407V (mortar) substitutions (EU number). In some embodiments, one heavy chain has one or more of the following substitutions: Y349C and T366W (EU number). The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A, and Y407V (EU number). Furthermore, substitutions (-ppcpScp->-ppcpPcp-) may be introduced into the hinge regions of both substituted IgGs.

[0241] Recombinant vector

[0242] This disclosure also provides recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which these recombinant vectors are introduced (i.e., vectors containing polynucleotides and / or including polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant technology, and anti-EGFR / HER3 antibody polypeptides or fragments thereof prepared by recombinant technology.

[0243] As used herein, a “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when introduced into that host cell. An “expression vector” is capable of delivering one or more target polynucleotides and expressing them as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the target polynucleotide is positioned in the vector for expression by means of regulatory elements (such as promoters, enhancers, and / or poly-A tails) operably linked to, or in the genome of, the target polynucleotide at or near, or flanking, the integration site of the target polynucleotide within the vector or in the host cell, such that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0244] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection and infection, and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0245] In some embodiments, the use of a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses) to introduce the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) may involve the use of a non-pathogenic (defective), replicative virus, or a replication-defective virus may be used. In the latter case, viral replication typically occurs only in complementary viral packaging cells. Suitable systems are disclosed in, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; US ​​Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; US Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 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 integrating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating DNA onto biodegradable beads that are efficiently transported into cells.

[0246] For expression, a DNA insert comprising a polynucleotide encoding an antibody or polypeptide disclosed herein may be operatively linked to a suitable promoter (e.g., a heterologous promoter), such as the phage λPL promoter, E. coli (…). E. coliThe expression construct may include promoters such as lac, trp, and tac, early and late SV40 promoters, and promoters of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may also contain transcription initiation and termination sites, and ribosome binding sites for translation within the transcription region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon at the beginning of the polypeptide to be translated and a stop codon (UAA, UGA, or UAG) at an appropriate position at the end of the polypeptide to be translated.

[0247] As described above, the expression vector may include at least one optional marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and *S. fall armyworm* Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0248] Non-restricted vectors for use with bacteria include pQE70, pQE60, and pQE-9 obtained from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A obtained from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 obtained from Pharmacia. Non-restricted eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG obtained from Stratagene; and pSVK3, pBPV, pMSG, and pSVL obtained from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0249] Suitable non-restrictive bacterial promoters include the Escherichia coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters (such as the Roussarcoma virus (RSV) promoter), and metallothionein promoters (such as the mouse metallothionein-I promoter).

[0250] In *Saccharomyces cerevisiae*, many vectors containing constitutive or inducible promoters can be used, such as α-factor, alcohol oxidase, and PGH. For reviews, see Ausubel et al., (1989) *Current Protocols in Molecular Biology*, John Wiley & Sons, New York, NY, and Grant et al. Methods Enzymol., 153: 516-544 (1997).

[0251] Introducing constructs into host cells can be achieved through calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), the full text of which is incorporated herein by reference.

[0252] Transcription of DNA encoding the disclosed anti-EGFR / HER3 antibody in higher eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10–300 bp in size, that 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 behind the origin of replication (OCR), the cytomegalovirus early promoter enhancer, and polyomavirus and adenovirus enhancers located behind the OCR.

[0253] To induce the translated protein to be secreted into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. This signal can be an endogenous or heterologous signal from the polypeptide.

[0254] Peptides (e.g., anti-EGFR / HER3 antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusion proteins) or those tagged with histidine, and can include not only secretion signals but also additional heterologous functional regions. For example, regions of additional amino acids (especially charged amino acids) can be added to the N-terminus of the peptide to improve stability and durability in host cells, during purification, or during subsequent processing and storage. Furthermore, peptide moieties can be added to the peptide to facilitate purification. These regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to induce secretion or excretion, improve stability, and facilitate purification are conventional techniques well known in the art.

[0255] This disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with any nucleotide sequence described herein, and 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% identity with any amino acid sequence described herein.

[0256] This disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any nucleotide sequence described herein, and 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 with any amino acid sequence described herein.

[0257] 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 fewer 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 fewer 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.

[0258] In some embodiments, the amino acid sequence (i) includes an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0259] In some implementations, the nucleic acid sequence (i) includes a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0260] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are considered identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). Considering the number of vacancies that need to be introduced to achieve optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences is a function of the number of common positions shared by the sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0261] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine the percentage of sequence homology is known in the art. In some embodiments, conserved amino acid residues with similar physicochemical properties (homology percentage), such as leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percentage of homology is higher than the percentage of identity.

[0262] This disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding the heavy chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding the light chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding the scFv polypeptide described herein.

[0263] In some embodiments, the vector may have both of the nucleic acids described herein, wherein the vector encodes the VL and VH regions of HER3 that bind together. In some embodiments, a pair of vectors is provided, wherein each vector includes one of the nucleic acids described herein, wherein the pair of vectors co-encodes the VL and VH regions of HER3 that bind together.

[0264] In some embodiments, the vector comprises both of the nucleic acids described herein, wherein the vector encodes the VL and VH regions of EGFR that commonly bind. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, wherein the pair of vectors commonly encodes the VL and VH regions of EGFR.

[0265] Treatment

[0266] The methods described herein include approaches for treating cancer-related conditions. Typically, the methods involve administering a therapeutically effective amount of the anti-EGFR / HER3 antibody or anti-EGFR / HER3 antibody-drug conjugate described herein to a subject who requires or has been determined to require such treatment.

[0267] As used herein, “treatment” means improvement of at least one symptom of a cancer-related condition. Cancer typically leads to death; therefore, treatment can increase life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). Administration of a therapeutically effective amount of the medication described herein to treat a cancer-related condition will reduce the number of cancer cells and / or alleviate symptoms.

[0268] As used herein, the term “cancer” refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid proliferative cell growth. This term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformation of cells, tissues, or organs, regardless of their histopathological type or stage of invasion. As used herein, the term “tumor” refers to cancer cells, such as clusters of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of various organ systems, such as malignancies affecting the lungs, breast, thyroid, lymph nodes, gastrointestinal tract, and genitourinary tract; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell lung cancer, small bowel cancer, and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term “cancer” is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some implementations, the cancer is kidney cancer or melanoma. Exemplary cancers include those arising from tissues of the esophagus, cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, for example, which includes malignant tumors composed of carcinomatous and sarcomatous tissue. “Adenocarcinoma” refers to cancer originating from glandular tissue or in which tumor cells form identifiable glandular structures. The term “sarcoma” is recognized in the art and refers to a mesenchymal-derived malignant tumor.

[0269] In some implementations, the cancer is chemotherapy-resistant cancer.

[0270] In one aspect, this disclosure also provides methods for treating cancer in a subject, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment may terminate, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may reduce the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0271] In one aspect, this disclosure includes methods of administering a therapeutically effective amount of the anti-EGFR / HER3 antibody or anti-EGFR / HER3 antibody drug conjugate of this disclosure to a subject in need, such as a subject who has, has been identified as, or has been diagnosed with cancer, for example, a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma), gastric cancer, skin cancer, colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal carcinoma, brain cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, cervical cancer, or esophageal cancer. In some embodiments, the cancer is esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial cancer, lung cancer, melanoma, ovarian cancer, bladder cancer, non-Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, and cervical cancer.

[0272] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals (human or non-human) for whom treatment is provided according to the method of the invention. The invention is contemplated for both veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0273] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Cancer patients can be identified using a variety of methods known in the art.

[0274] As used herein, “effective amount” means an amount or dose sufficient to achieve beneficial or desired results, including terminating, slowing, delaying, or inhibiting the progression of a disease (e.g., cancer). Effective amounts will vary based on, for example, the age and weight of the subject to be administered an anti-EGFR / HER3 antibody, an anti-EGFR / HER3 antigen-binding fragment, an anti-EGFR / HER3 antibody drug conjugate, an anti-EGFR / HER3 antibody-encoded polynucleotide, a carrier comprising that polynucleotide, and / or a combination thereof, as well as the severity of symptoms and route of administration, and therefore can be determined on an individual basis.

[0275] An effective amount may be administered in a single or multiple doses. For example, an effective amount of an anti-EGFR / HER3 antibody, an anti-EGFR / HER3 antigen-binding fragment, or an anti-EGFR / HER3 antibody drug conjugate is an amount sufficient to improve, terminate, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to improve, terminate, stabilize, reverse, slow, and / or delay the in vitro proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, an effective amount of an anti-EGFR / HER3 antibody, an anti-EGFR / HER3 antigen-binding fragment, or an anti-EGFR / HER3 antibody drug conjugate may vary depending on, in particular, the patient's medical history and other factors such as the type (and / or dosage) of the composition used.

[0276] The effective amounts and dosing regimens of the anti-EGFR / HER3 antibodies, anti-EGFR / HER3 antigen-binding fragments, anti-EGFR / HER3 antibody-encoded polynucleotides, anti-EGFR / HER3 antibody drug conjugates and / or compositions disclosed herein can be determined empirically, and such determinations are within the scope of the art. Those skilled in the art will understand that the necessary dosage will vary depending on, for example, the mammal to which the disclosed anti-EGFR / HER3 antibodies, anti-EGFR / HER3 antigen-binding fragments, anti-EGFR / HER3 antibody-encoded polynucleotides, anti-EGFR / HER3 antibody drug conjugates and / or compositions will be received, the route of administration, the specific type of antibody, antibody-encoded polynucleotide, antigen-binding fragment, antibody drug conjugate and / or composition used, and other drugs administered to the mammal.

[0277] The typical daily dose of an effective amount of anti-EGFR / HER3 antibody or anti-EGFR / HER3 antibody conjugate is from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dosage is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0278] In any of the methods described herein, at least one anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding fragment, anti-EGFR / HER3 antibody-drug conjugate, or pharmaceutical composition (e.g., any one of the anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding fragment, or anti-EGFR / HER3 ADC described herein) and optionally at least one additional therapeutic agent may be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0279] In some embodiments, one or more additional therapeutic agents may be administered to the subject before or after administration of at least one anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, anti-EGFR / HER3 antibody drug conjugate, or pharmaceutical composition (e.g., comprising any anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, or anti-EGFR / HER3 ADC). In some embodiments, administration of one or more additional therapeutic agents and at least one anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, or anti-EGFR / HER3 antibody drug conjugate to the subject results in an overlap in the biological activity periods of the one or more additional therapeutic agents and at least one anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, or anti-EGFR / HER3 ADC.

[0280] In some implementations, at least one anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, anti-EGFR / HER3 antibody-drug conjugate, or pharmaceutical composition (e.g., any one of the anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, or anti-EGFR / HER3 ADC described herein) may be administered to a subject over an extended 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 use any of the methods described herein to determine the length of the treatment period for diagnosis or to track treatment effectiveness (e.g., to observe at least one symptom of cancer). As described herein, skilled medical professionals may also vary the type and amount (e.g., increase or decrease) of the anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, or anti-EGFR / HER3 ADC (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 at least one anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding antibody fragment, or anti-EGFR / HER3 ADC (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of treatment effectiveness (e.g., using any of the methods described herein and known in the art).

[0281] In some embodiments, one or more additional therapeutic agents may be administered to the subject. These 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, EGFR inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0282] In some implementations, 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, hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0283] In some implementations, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, and sorafenib. (sorafenib), Votrient, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0284] 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, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and select protein agonists.

[0285] In some implementation schemes, subjects are administered carboplatin, albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0286] In some implementations, additional therapeutic agents are anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-TIGIT antibodies, anti-CTLA4 antibodies, anti-CD40 antibodies, anti-OX40 antibodies, anti-4-1BB antibodies, anti-TIM3 antibodies, or anti-GITR antibodies.

[0287] Pharmaceutical Compositions and Routes of Administration

[0288] This document also provides pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the anti-EGFR / HER3 antibodies described herein (e.g., bispecific antibodies), anti-EGFR / HER3 antigen-binding fragments, or anti-EGFR / HER3 antibody pharmaceutical conjugates. The pharmaceutical compositions may be formulated in any manner known in the art.

[0289] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), fixative oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetates, citrates, or phosphates), and isotonic agents (such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). Formulations of the compositions may be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. When needed (e.g., in injectable formulations), appropriate flowability can be maintained by, for example, using a coating (such as lecithin) or a surfactant. The absorption of anti-EGFR / HER3 antibodies, anti-EGFR / HER3 antigen-binding fragments, or anti-EGFR / HER3 ADCs can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved via implants and microencapsulated delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza and Nova Pharma).

[0290] Compositions containing one or more of the anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding fragment, and anti-EGFR / HER3 antibody-drug conjugate described herein can be formulated for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) in dose units (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and dose uniformity).

[0291] The toxicity and efficacy of the composition can be determined in cell cultures or laboratory animals (e.g., monkeys) using standard pharmaceutical methods. The LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. When an agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and efficacy can be determined using other standard pharmaceutical methods.

[0292] Data obtained from cell culture assays and animal studies can be used to formulate appropriate doses of any given agent for a subject (e.g., a human). A therapeutically effective amount of an anti-EGFR / HER3 antibody, anti-EGFR / HER3 antigen-binding fragment, or anti-EGFR / HER3 ADC would be the amount used to treat a subject (e.g., a human subject identified as having cancer) of a disease (e.g., killing cancer cells) or a subject identified as being at risk of developing a disease (e.g., a subject who previously had cancer but is now cured), reducing the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., in humans). The effectiveness and dosage of any anti-EGFR / HER3 antibody, its anti-EGFR / HER3 antigen-binding fragment, or anti-EGFR / HER3 ADC described herein can be determined by a healthcare professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors may influence the dose and duration required for effective treatment of a subject (e.g., the severity of the disease or disorder, previous treatments, the subject's general health condition and / or age, and the presence of other diseases).

[0293] Exemplary doses include milligrams or micrograms of any anti-EGFR / HER3 antibody, its anti-EGFR / HER3 antigen-binding fragment, or anti-EGFR / HER3 ADC per kilogram of subject weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg). While these doses cover a wide range, those skilled in the art will understand that the potency of therapeutic agents (including antigen-binding protein constructs, antibodies, and their antigen-binding fragments) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered initially, which may then be gradually increased by the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or by researchers (when still working in the development phase) until an appropriate response is obtained. Furthermore, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health condition, sex and diet, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in the body.

[0294] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use. This disclosure also provides methods for preparing the anti-EGFR / HER3 antibodies, anti-EGFR / HER3 antigen-binding fragments, or anti-EGFR / HER3 ADCs described herein for various applications.

[0295] Example

[0296] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0297] Example 1. Preparation of anti-EGFR / HER3 bispecific antibody

[0298] This invention provides bispecific antigen-binding molecules that target EGFR and HER3. These antigen-binding molecules are referred to below as anti-EGFR / HER3 bispecific antibodies.

[0299] Anti-EGFR antibodies (9F3, VH SEQ ID NO: 41, VL SEQ ID NO: 40; 9D2, VH SEQ ID NO: 42, VL SEQ ID NO: 40 and 9A6, VH SEQ ID NO: 43, VL SEQ ID NO: 40) and anti-HER3 antibodies (1B2, VH SEQ ID NO: 44, VL SEQ ID NO: 40; 3E1, VH SEQ ID NO: 45, VL SEQ ID NO: 40 and 3G6, VH SEQ ID NO: 46, VL SEQ ID NO: 40) can pair to form bispecific antibodies. Vectors encoding antibody light and heavy chains were constructed. CHO-S cells were co-transfected with three vectors, including a first vector encoding the anti-EGFR antibody heavy chain, a second vector encoding the anti-HER3 antibody heavy chain, and a third vector encoding the co-light chain. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography. Exemplary bispecific antibodies obtained include 9F3-1B2, 9F3-3E1, 9F3-3G6, 9D2-1B2, 9D2-3E1, 9D2-3G6, 9A6-1B2, 9A6-3E1, and 9A6-3G6.

[0300] Several methods can be used to reduce the chance of mispairing between the two heavy chains. For example, knocks-in-hole mutations can be introduced into the Fc regions of the anti-EGFR arm heavy chain and the anti-HER3 arm heavy chain. For instance, in 9F3-1B2, the heavy chain constant region of 9F3 includes a knocks-in-hole mutation, and the heavy chain constant region of 1B2 includes a knocks-in-hole mutation. As another example, in 9F3-3E1, the heavy chain constant region of 9F3 includes a knocks-in-hole mutation, and the heavy chain constant region of 3E1 includes a knocks-in-hole mutation.

[0301] The sequences of the light chain constant region, the heavy chain constant region containing the mortar mutation, and the heavy chain constant region containing the mortar mutation are shown in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively.

[0302] Four different reference antibodies were synthesized based on published amino acid sequence information: Ref1 and Ref2 (specific to EGFR), Ref3 (specific to HER3), and Ref4 (specific to EGFR / HER3) for the following experiments. Specifically, the VH and VL sequences shown in SEQ ID NO: 53-58 were linked to the constant region of human IgG1, respectively, to form Ref1, Ref2, and Ref3. The heavy and light chain sequences of Ref4 are shown in SEQ ID NO: 59-60.

[0303] Example 2. Species cross-binding activity of anti-EGFR / HER3 bispecific antibody

[0304] CHO-fasEGFR cells, CHO-hHER3 cells, or CHO-fasHER3 cells were respectively treated with 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 1 / 2 well in 96-well plates. Anti-EGFR / HER3 bispecific antibody was added to the 96-well plates and incubated at 4°C for 30 min. Cells were then incubated with the secondary antibody anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearchLaboratories, Inc., Cat#: 109-606-170) at 4°C in the dark for 15 min, followed by flow cytometry analysis. Human IgG1 was used as an isotype control (ISO).

[0305] CHO-fasEGFR cells, CHO-hHER3 cells, and CHO-fasHER3 cells were synthesized using monkeys expressing (… Macaca fascicularis ) EGFR (fasEGFR, SEQ ID NO: 50), human HER3 (hHER3, SEQ ID NO: 51) and monkey ( Macaca fascicularis The HER3 (fasHER3, SEQ ID NO: 52) vector was transfected into CHO-S cells to obtain the cells.

[0306] The test results are shown in the table below. All anti-EGFR / HER3 antibodies can bind to human HER3, monkey HER3 and monkey EGFR.

[0307] Table 1

[0308] Example 3. Endocytosis of anti-EGFR / HER3 bispecific antibody

[0309] 2.5 μg / mL of anti-EGFR antibody, anti-HER3 antibody, or anti-EGFR / HER3 bispecific antibody was added to NCI-H292 or NCI-H226 cells along with pHAb-AffiniPure Fab goat anti-human IgG secondary antibody and incubated for 6 hours. Cells were centrifuged and washed with FACS buffer. MFI was measured by flow cytometry. Antibody endocytosis rate was calculated. Human IgG1 protein was used as an isotype control (ISO). The results are shown in the table below. In both NCI-H292 and NCI-H226 cells, the endocytosis rate of the anti-EGFR / HER3 bispecific antibody was equal to or higher than that of its parental monoclonal antibody.

[0310] Table 2

[0311] In another experiment, the endocytosis of 9F3-3E1 (2.5 μg / mL) in NUGC-4, A431, HCC827, or SW620 cells was detected, and the results are shown in the table below. 9F3-3E1 exhibited better endocytic activity than the reference antibody Ref4.

[0312] Table 3

[0313] Example 4. Binding affinity of anti-EGFR / HER3 bispecific antibody

[0314] Using the Biacore™ (Biacore, Inc., Piscataway NJ) 8K biosensor equipped with a pre-immobilized protein A sensor chip, the binding affinity of the anti-EGFR / HER3 bispecific antibody to human EGFR, human HER3, monkey EGFR, and monkey HER3 was verified by surface plasmon resonance (SPR).

[0315] Specifically, hEGFR-His (ACROBiosystems Inc., Cat#: EGR-H5222), hHER3-His (ACROBiosystems Inc., Cat#: ER3-H5223), fasEGFR-His (ACROBiosystems Inc., Cat#: EGR-C52H1), and fasHER3-His (Sino Biological, Inc., Cat#: 90043-K08H) were diluted to 200 nM or 400 nM with 1× HBS-EP+ buffer (pH 7.4) and then injected into the Biacore™ 8K biosensor at a flow rate of 10 μL / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 50 response units (RU) or 200 RU). Subsequently, purified antibody at a concentration of 2 μg / mL prepared with 1× HBS-EP+ buffer (pH 7.4) was injected at a flow rate of 10 μL / min for 50 seconds. The dissociation process was monitored for 400 seconds. After the last injection of each titration, glycine (pH 1.5) was injected at a flow rate of 30 μL / min for 30 seconds to regenerate the chip.

[0316] The data were fitted to a 1:1 Langmuir binding model using Biacore™ 8K evaluation software 3.0 (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994, Methods Enzymology 6. 99-110) to simultaneously obtain the kinetic binding rate (kon) and dissociation rate (koff). Affinity was derived from the quotient of the kinetic rate constant (KD = koff / kon). As will be understood by those skilled in the art, the same method was used for each tested antibody, with appropriate adjustments to parameters (e.g., antibody concentration).

[0317] The antibody test results are shown in the table below. The anti-EGFR / HER3 bispecific antibody has good binding affinity for human EGFR, monkey EGFR, human HER3 and monkey HER3.

[0318] Table 4

[0319] Example 5. Stability of anti-EGFR / HER3 bispecific antibody

[0320] The buffer for the anti-EGFR / HER3 bispecific antibody was changed to pH 6.0 (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween 80). The antibodies were loaded into sealed Eppendorf tubes and incubated at 40 ± 2 °C, 60% ± 5% RH, and 4 ± 3 °C for 7 days to evaluate their thermostability. Alternatively, the bispecific antibody was loaded onto a Protein A column and eluted with a pH 3.5 buffer (0.1 mol / L HAc). Immediately, half of the antibody was added to 2M Tris buffer, and the pH was adjusted to 7.5. The remaining half was incubated at pH 3.5 for 6 hours, and then the pH was adjusted to 7.5. The diluted antibody was incubated at pH 3.5 ± 0.1, 25 ± 2 °C (hereinafter referred to as pH 3.5) in sealed Eppendorf tubes for 6 hours to test its stability at low pH.

[0321] After the above treatment, the following tests were performed: (1) the purity of the antibody was determined by size exclusion high performance liquid chromatography (SEC-HPLC) (represented as the percentage of the main peak area to the total area of ​​all peaks (purity, %)); (2) the hydrophobicity of the antibody was determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (represented as the retention time of the main peak (HIC, min)); (3) the pI (isoelectric point) and charge change of the antibody were determined by capillary isoelectric focusing (cIEF) (represented as the percentage of the main component, acidic component and basic component); (4) the change in antibody purity under non-reducing (CE-SDS(NR)) or reducing (CE-SDS(R)) conditions was determined by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) (represented as the percentage of the main peak area to the total area of ​​all peaks (purity, %)); (5) appearance and presence of visible insoluble matter.

[0322] In SEC-HPLC experiments, antibody samples were diluted to 1 mg / mL with purified water and an Agilent 1290 chromatography 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% CAN, 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; run time: 7 minutes.

[0323] In the HIC-HPLC experiments, an Agilent 1260 chromatography system (connected to a ProPac HIC-10 column (4.6 x 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; Gradients: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; Column temperature: 30 °C; Detection wavelength: 280 nm, 220 nm; Injection volume: 10 μg; Sample tray temperature: approximately 10 °C; Run time: 50 min.

[0324] In cIEF experiments, the Maurice cIEF method development kit (Protein Simple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 8 μL of 30 μg protein sample was mixed with the following reagents from the kit: 1 μL Maurice cIEF pI label-7.05, 1 μL Maurice cIEF pI label-10.10, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500 mM arginine, 1.33 μL ampholyte (Pharmalyte pH range 3–10), 6.66 μL ampholyte (Pharmalyte pH range 8–10.5), and water (to a final volume of 100 μL). Imaging capillary isoelectric focusing spectra were generated using the Maurice cIEF cartridge (PS-MC02-C) on a Maurice analyzer (Protein Simple, Santa Clara, CA). Sample focusing took a total of 10 minutes. The analysis software installed on the instrument is used to analyze the absorbance of proteins focused at 280 nm.

[0325] In CE-SDS experiments, Maurice (Protein Simple, Maurice™) and the Maurice CE-SDS Size Application Kit (Protein Simple, Cat#: PS-MAK02-S) were used for either CE-SDS(NR) or CE-SDS(R). For CE-SDS, 30 μL of sample buffer, 30 μL of 30 μg antibody sample, 1.5 μL of 25x internal standard, and 3 μL of 250 nM iodoacetamide (SIGMA, Cat#: 16125) were added to a microcentrifuge tube. The tube was then centrifuged at 3000 rpm for 1 minute and 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. The supernatant was then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.

[0326] Detailed results are shown in the table below. The results show that all anti-EGFR / HER3 antibodies 9F3-3E1, 9D2-1B2, 9A6-3E1, and 9A6-3G6 have good stability and physicochemical properties.

[0327] Table 5

[0328] In another experiment, further exploitation analyses of the anti-EGFR / HER3 bispecific antibody were performed. Specifically, the following tests were conducted: (1) observation of the solution appearance and presence of visible insoluble matter; (2) detection of antibody specificity using cross-interaction chromatography (CIC) (expressed as retention time (CIC, min)); (3) detection of antibody colloidal stability using vertical single-layer chromatography (SMAC) (expressed as retention time (SMAC, % / min)); (4) detection of antibody thermal stability using an UNcle system (expressed as melting temperature (Tm) and aggregation temperature (Tagg)); and (5) detection of antibody purity using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (expressed as retention time of the main peak (HIC, min)).

[0329] In CIC analysis, human polyclonal IgG (Sigma, Cat#: I4506) was coupled to HiTrap NHS activated resin (GE Healthcare, Cat#: 17-0716-01), and a CIC column was prepared by passivation with ethanolamine according to a published procedure. The column was then connected to an Agilent 1260 chromatography system and run at 0.1 mL / min using 1× PBS as the mobile phase until a flat baseline was reached. Then, 10 μg of 1 mg / mL antibody solution in PBS was injected. Peak retention times on the column were monitored at 280 nm and 220 nm; run time: 50 min.

[0330] In SMAC analysis, the Zenix column (4.6 mm × 30 cm, Sepax, Cat#: 213300-4630) was connected to the column oven, and appropriate tubing was placed in the mobile phase. The column was equilibrated with mobile phase buffer at a flow rate of 0.350 mL / min for 60 min. The antibody (2 μg) was loaded into the injection sequence. Mobile phase: 150 mM sodium phosphate, pH 7.0; flow rate: 0.35 mL / min; run time: 25 min; column temperature: 30°C; detection wavelengths: 280 nm, 220 nm.

[0331] In the UNcle experiment, 8 μL of protein sample was loaded into a uni tube, and the system was run at a heating rate of 1 °C / min at a thermal temperature ranging from 25 °C to 95 °C. Particle size and polydispersity were detected by dynamic light scattering (DLS) before heating. Protein stability was characterized by differential scanning spectral fluorescence (DSF), static light scattering (SLS), and dynamic light scattering (DLS).

[0332] In the HIC experiment, an Agilent 1260 chromatography system (connected to a ProPac HIC-10 column (4.6 x 100 mm 5 μm)) 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, 25 M phosphate buffer (PB), pH 7.0; Mobile phase B: 25 M phosphate buffer (PB), pH 7.0; Flow rate: 1.0 mL / min; Gradients: 0 min 100% A, 2 min 100% A, 5 min 50% B, 25 min 100% B, 28 min 100% B, 29 min 100% A, and 35 min 100% A; Column temperature: 30 °C; Detection wavelength: 280 nm, 220 nm; Injection volume: 20 μg; Run time: 35 min. This experiment was named HIC. 02 .

[0333] In another HIC experiment, an Agilent 1260 chromatography system (connected to a Proteomix HIC-Phenyl-NP5 column (4.6 x 100 mm 5 μm)) 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, 25 M phosphate buffer (PB), pH 7.0; Mobile phase B: 25 M phosphate buffer (PB), pH 7.0; Flow rate: 0.8 mL / min; Gradients: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; Column temperature: 30 °C; Detection wavelength: 280 nm, 220 nm; Injection volume: 20 μg; Run time: 35 min. This experiment was named HIC. 03 .

[0334] Detailed results are shown in the table below. The results show that the anti-EGFR / HER3 antibody 9F3-3E1 has good stability and physicochemical properties.

[0335] Table 6

[0336] In another experiment, 9F3-3E1 was placed in a sealed Eppendorf tube and kept at 25°C for 30 days to assess its exploitability.

[0337] Detailed results are shown in the table below. The results show that the anti-EGFR / HER3 antibody 9F3-3E1 has good stability and physicochemical properties.

[0338] Table 7

[0339] Following the above treatment, flow cytometry was used to analyze the binding activity of 9F3-3E1 cells to NUGC-4 cells. MFI values ​​were used to plot antibody concentration fitting curves. EC was determined. 50 Values. The test results are shown in the table below. No significant changes in binding activity were observed after different treatments.

[0340] Table 8

[0341] In another experiment, flow cytometry was used to analyze the binding activity of 9F3-3E1 cells to NUGC-4 cells. MFI values ​​were used to plot antibody concentration-fitted curves. EC was determined. 50 Values. The test results are shown in the table below. No significant changes in binding activity were observed after different treatments.

[0342] Table 9

[0343] Example 6. Anti-EGFR / HER3 antibody-drug conjugate (ADC)

[0344] Each purified antibody (9F3, 9D2, 9A6, 1B2, 3E1, 3G6, 9F3-3E1, 9D2-1B2, 9A6-1B2, 9A6-3E1, or 9A6-3G6) was conjugated to MMAE (monomethylaurestatin E) via a maleiminohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. For the antibody-drug conjugate name, "MMAE" was simply added after the antibody name. For example, if 9F3-3E1 was conjugated to MMAE, it was named 9F3-3E1-MMAE. Antibody-drug conjugates generated using a similar method also included Ref1-MMAE, Ref2-MMAE, and Ref3-MMAE. For the isotype control, human IgG1 was conjugated to MMAE to form ISO-MMAE. The conjugation of the antibody to the drug molecule was detected using HIC-HPLC. The results showed that the drug-antibody ratio (DAR) for each ADC was approximately 4.

[0345] In another example, purified antibodies are also conjugated to CPT-1, CPT-2, CPT-3, or CPT-4 via the CPT-L linker. For the ADC name, CPTx is simply added after the antibody name. For example, if 9F3-3E1 is conjugated to CPT-1, it is named 9F3-3E1-CPT1. Similarly, if 9F3-3E1 is conjugated to CPT-2, it is named 9F3-3E1-CPT2. Antibody-drug conjugates generated using a similar method also include Ref4-CPT2. For the isotype control, human IgG1 is conjugated to CPT-2 to form ISO-CPT2. The conjugation of the antibody and drug molecule is detected using MS (mass spectrometry). MS results indicate that the drug-antibody ratio (DAR) of the ADC is approximately 8.

[0346] For reference purposes, Ref3 is coupled to Dxd via the GGFG linker to form Ref3-Dxd, with a DAR of approximately 4.

[0347] Example 7. Antitumor activity in the NUGC-4 xenograft model

[0348] The effect of ADCs on in vivo tumor growth was tested in a NUGC-4 xenograft model of gastric cancer. Specifically, approximately 5 × 10⁻⁶ ADCs were used. 6 NuGC-4 cells were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: B-CM-002). EGFR and HER3 expression levels, as measured by RNA-seq, were 8.58 and 154.99, respectively. When the mouse tumor volume reached approximately 200 mm... 3 Mice were randomly assigned to different groups (n=6 per group) based on tumor volume. Subsequently, mice were administered phosphate-buffered saline (PBS), ISO-MMAE, Ref1-MMAE, Ref3-MMAE, 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, or 9D2-1B2-MMAE via intravenous (iv) injection. Administration was once weekly (twice in total).

[0349] Tumor volume was measured twice weekly, and mouse body weight was recorded simultaneously. The tumor volume was recorded when it reached 3000 mm². 3 At that time, euthanasia was performed.

[0350] Measure the lengths of the tumor's major and minor axes, and calculate the tumor volume using the following formula: 0.5 × (major axis) × (minor axis) 2Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero. Statistical analysis was performed using the t-test. P < 0.05 was the threshold for statistical significance.

[0351] like Figure 4 As shown, all five anti-EGFR / HER3 bispecific ADCs (G5-G9) exhibited better antitumor activity in the gastric cancer model than the positive controls Ref1-MMAE (G3) and Ref3-MMAE (G4).

[0352] Example 8. Antitumor activity in the NCI-H292 xenograft model

[0353] The effect of ADCs on in vivo tumor growth was tested in a lung cancer NCI-H292 cell xenograft model. Specifically, approximately 5 × 10⁻⁶ cells were used. 6 NCI-H292 cells were subcutaneously injected into B-NDG mice. EGFR and HER3 expression levels, measured by RNA-seq, were 55.75% and 4.33%, respectively. When the mouse tumor volume reached approximately 200 mm... 3 Mice were randomly assigned to different groups (n=6 per group) based on tumor volume. Subsequently, mice were administered PBS, ISO-MMAE, Ref1-MMAE, Ref3-MMAE, 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, or 9D2-1B2-MMAE via intravenous (iv) injection. Administration was once weekly (twice in total).

[0354] Tumor volume was measured twice a week. Figure 5 Tumor volume measurements were presented, showing that all anti-EGFR / HER3 ADCs exhibited significant tumor suppression. The TGI% (e.g., day 27) in the G5-G9 groups was higher than that in the control groups G2 and G4, indicating that 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, and 9D2-1B2-MMAE have good tumor suppression effects in the lung cancer model.

[0355] Example 9. Antitumor activity in a xenograft model derived from a gastric cancer patient.

[0356] The effect of ADCs on in vivo tumor growth was tested in a patient-derived xenograft (PDX) model of gastric cancer. Immunohistochemical staining of patient-derived gastric tumor fragments revealed that these fragments were tissues with high co-expression of EGFR and HER3. Specifically, patient-derived gastric tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right dorsal side of B-NDG mice. When the tumor volume in the mice reached approximately 250 mm², the ADCs were used to target tumor growth. 3 Mice were randomly divided into groups of 6 based on tumor volume. Subsequently, mice were administered PBS, ISO-MMAE, Ref3-MMAE, 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, or 9D2-1B2-MMAE via intravenous (iv) injection. Administration was once weekly (twice in total).

[0357] Measure tumor volume twice a week. Figure 6 As shown, the tumor volume in the G3-G8 treatment groups was smaller than that in the control groups G1 and G2. The results also showed that all five anti-EGFR / HER3 ADCs (G4-G8) exhibited better anti-tumor activity than the positive control Ref3-MMAE (G3). Specifically, on day 21, the TGI% of the G4 to G8 groups were 86.2%, 86.4%, 85.4%, 87.2%, and 87.4%, respectively, while the value in the G3 group was lower at 68.2%, indicating that 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, and 9D2-1B2-MMAE have good tumor-suppressive effects in the gastric cancer model.

[0358] In another similar experiment, PBS, Ref2-MMAE, Ref3-Dxd, 9A6-3E1-CPT2, 9A6-1B2-CPT2, 9F3-3E1-CPT2, or 9F3-1B2-CPT2 were administered intravenously (iv) to PDX model mice with gastric cancer. Dosing was once weekly, except for Ref3-Dxd, which was administered twice. Results are as follows: Figure 7 As shown, in groups G1 and G3, the tumor volume of mice reached 3000 mm on day 11 after grouping. 3 Euthanasia was performed. Furthermore, compared to the positive controls Ref2-MMAE and Ref3-Dxd, 9A6-3E1-CPT2, 9A6-1B2-CPT2, 9F3-3E1-CPT2, and 9F3-1B2-CPT2 showed better tumor suppression effects.

[0359] Example 10. Antitumor activity in a colorectal cancer PDX model

[0360] The in vivo antitumor activity of the ADC was tested in a PDX model of colorectal cancer with high EGFR / HER3 expression. Specifically, tumor fragments (2 mm × 2 mm × 2 mm) derived from patients were implanted into the right side of the back of B-NDG mice. When the tumor volume of the mice reached approximately 250 mm, the in vivo antitumor activity was tested. 3 Mice were randomly assigned to different groups (n=5 per group) based on tumor volume, and then administered PBS, ISO-MMAE, Ref2-MMAE, 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, or 9D2-1B2-MMAE intravenously (iv). Tumor volume was measured twice weekly.

[0361] like Figure 8 As shown, 9A6-3E1-MMAE, 9A6-3G6-MMAE, 9A6-1B2-MMAE, 9F3-3E1-MMAE, and 9D2-1B2-MMAE exhibited better antitumor activity than the positive control Ref2-MMAE.

[0362] In a similar experiment, PBS, ISO-CPT2, Ref2-MMAE, Ref3-Dxd, 9A6-3E1-CPT2, 9A6-1B2-CPT2, 9F3-3E1-CPT2, or 9F3-1B2-CPT2 were administered intravenously (iv) to PDX model mice with colorectal cancer. Tumor volume was measured twice weekly. Results are as follows: Figure 9 As shown, all four anti-EGFR / HER3 bispecific ADCs exhibited better anti-tumor activity than the positive controls Ref2-MMAE and Ref3-Dxd.

[0363] In another similar experiment, mice with colorectal cancer PDX model were administered PBS (G1), Ref2-MMAE (G2), Ref3-Dxd (G3), Ref4-CPT2 (G4 and G5), 9F3-3E1-CPT2 (G6 and G7), 9F3-CPT2 (G8), 3E1-CPT2 (G9), or a combination of 9F3-CPT2 and 3E1-CPT2 (G10) via intravenous (iv) injection. Tumor volume was measured twice weekly. Tumor volumes in different groups of mice treated with ADC or PBS are shown below. Figure 10As shown, the treatment groups (G2-G10) exhibited different tumor-suppressive effects compared to the control group G1. Among the treatment groups, the 6 mg / kg 9F3-3E1-CPT2 treatment group (G7) showed the best tumor-suppressive effect, followed by the parental monoclonal antibody ADC treatment groups (G8 and G9) and the positive control group (G2-G5). Furthermore, 9F3-3E1-CPT2 demonstrated a dose-dependent tumor-suppressive effect.

[0364] In another similar experiment, PBS (G1), ISO-MMAE (G2), 9F3-3E1-MMAE (G3), 9F3-MMAE (G4), or 3E1-MMAE (G5) were administered intravenously (iv) to PDX model mice with colorectal cancer. Tumor volume was measured twice weekly. Tumor volumes of mice in different groups treated with ADC or PBS are shown below. Figure 13 As shown, the treatment groups (G3-G5) exhibited different tumor-suppressive effects compared to the control groups G1 and G2. Among the treatment groups, the 9F3-3E1-MMAE treatment group (G3) showed better tumor-suppressive effects than the parental monoclonal antibody ADC treatment groups (G4 and G5), and also had a synergistic effect.

[0365] In another similar experiment, PDX-type mice with colorectal cancer were administered PBS (G1), Ref2-MMAE (G2), Ref3-Dxd (G3), Ref4-CPT2 (G4 and G5), and 9F3-3E1-CPT2 (G6 and G7) via intravenous (iv) injection. Tumor volume was measured twice weekly. Tumor volumes of mice in different groups treated with ADC or PBS are shown in the figure. Figure 14 As shown, the treatment groups (G2-G7) exhibited different tumor-suppressive effects compared to the control group G1. Among the treatment groups, the 6 mg / kg 9F3-3E1-CPT2 treatment group (G7) showed the best tumor-suppressive effect compared to the positive control group (G2-G5). Furthermore, 9F3-3E1-CPT2 demonstrated a dose-dependent tumor-suppressive effect.

[0366] Example 11. Antitumor activity in a lung cancer patient-derived xenograft model

[0367] The effect of ADCs on tumor growth in a lung cancer patient-derived xenograft model was tested. Immunohistochemical staining of patient-derived lung tumor fragments revealed that the fragments were EGFR-high and HER3-low expression tissue. Specifically, patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were implanted into the right dorsal side of B-NDG mice. When the tumor volume in the mice reached approximately 200 mm², the ADCs were implanted. 3Mice were randomly divided into groups of 5 based on tumor volume. Subsequently, mice were administered PBS (G1), Ref2-MMAE (G2), Ref3-Dxd (G3), 9A6-3E1-CPT2 (G4), 9A6-1B2-CPT2 (G5), 9F3-3E1-CPT2 (G6), or 9F3-1B2-CPT2 (G7) via intravenous (iv) injection.

[0368] Tumor volume in different groups of mice as follows Figure 11 As shown, 9A6-3E1-CPT2 (G4), 9A6-1B2-CPT2 (G5), 9F3-3E1-CPT2 (G6), or 9F3-1B2-CPT2 (G7) achieved better results than the positive control group (G2 or G3).

[0369] In another similar experiment, based on similar molar amounts, PBS (G1), Ref2-MMAE (G2), Ref3-Dxd (G3), Ref4-CPT2 (G4), or 9F3-3E1-CPT2 (G5) were administered intravenously (iv) to PDX-type mice with lung cancer. However, due to the lower toxicity of Dxd, the dosage of G3 was doubled. Tumor volume was measured twice weekly. Tumor volumes of mice in different groups treated with ADC or PBS are shown below. Figure 12 As shown, 9F3-3E1-CPT2 exhibited the best tumor suppression effect compared with the positive controls Ref2-MMAE, Ref3-Dxd and Ref4-CPT2.

[0370] Example 12. The effect of anti-EGFR / HER3 antibody and its ADC in blocking ligand-receptor binding.

[0371] The effects of anti-EGFR / HER3 antibodies or anti-EGFR / HER3 ADCs on the binding of His-labeled human NRG1 (hNRG1-His, ACROBiosystems Inc., Cat#: NR1-H5246) to human HER3 were detected by flow cytometry using NUGC-4 cells.

[0372] NUGC-4 cells were loaded at 2×10 5Cells were seeded in 96-well plates. 50 µL of Fixable Viability DyeeFluor™ 506 (eBioscience, Cat#: 65-0866-14) was added to each well, and the plates were incubated at 2–8°C in the dark for 30 minutes. After washing with PBS, 50 µL of Human TruStrain FcX (Biolegend, Cat#: 422302) was added to each well, and the plates were incubated at room temperature in the dark for 15 minutes. After washing with PBS, 50 µL of serially diluted test samples (maximum concentration 100 ug / mL) were added to each well, followed by 50 µL of hNRG1-His at a final concentration of 50 ng / mL in each well. After incubating at 2–8°C in the dark for 30 minutes, the 96-well plates were washed twice with PBS. Cells were incubated with secondary antibody (APC anti-His labeled antibody, (BioLegend, Cat#: 362605)) at 2-8°C in the dark for 30 minutes, followed by flow cytometry analysis.

[0373] Geometric mean fluorescence intensity (gMFI) was measured. A fitted curve was obtained with antibody concentration (µg / mL) as the X-axis and NRG1 binding percentage as the Y-axis. NRG1 binding percentage (%) = (gMFI value of corresponding well - gMFI value of secondary antibody control well) / (gMFI value of reference well - gMFI value of secondary antibody control well) × 100%, where the reference well contains only NRG1 and no sample, the secondary antibody control well contains only secondary antibody, and the corresponding well contains both NRG1 and different concentrations of sample. IC50 was determined. 50 value.

[0374] The results are shown in the table below. 9F3-3E1 and 9F3-3E1-CPT2 were able to inhibit the binding of NRG1 to NUGC-4 cells, indicating that the above-mentioned anti-EGFR / HER3 antibody and anti-EGFR / HER3 ADC can block the binding of NRG1 to HER3.

[0375] Table 10

[0376] In another similar experiment, NCI-H1975 cells were used to detect the blocking effect of anti-EGFR / HER3 antibody or anti-EGFR / HER3 ADC on the binding of His-labeled human EGF (hEGF-His, ACROBiosystems Inc., Cat#: EGF-H52H3) to human EGFR by flow cytometry.

[0377] The results showed that 9F3-3E1 and 9F3-3E1-CPT2 could inhibit the binding of EGF to NCI-H1975 cells, indicating that the above-mentioned anti-EGFR / HER3 antibody and anti-EGFR / HER3 ADC could block the binding of EGF to EGFR (data not shown).

[0378] Example 13. Blocking effect of anti-EGFR / HER3 antibody and ADC on EGFR / HER3 signal transduction

[0379] The blocking effect of anti-EGFR / HER3 antibodies and their ADCs on NRG1 or EGF-induced signal transduction was detected by Western blotting.

[0380] Specifically, NCI-H1975 cells were used at a rate of 2 × 10⁻⁶. 6 Cells were seeded in 96-well plates. 500 µL of test sample (final concentration 100 µg / mL) was added to each well and incubated for 60 minutes. 250 µL of NRG1 (ACROBiosystems Inc., Cat#: C1572P1-23CWW1-1HT) or EGF (ACROBiosystems Inc., Cat#: EGF-H52H3) (final concentration 100 ng / mL) was added to each well and incubated for 5 minutes. Cells were collected into 2 mL centrifuge tubes and centrifuged at 500 × g for 5 minutes at 4°C, discarding the supernatant. 100 μL of RIPA lysis buffer was added to each tube and the tubes were incubated on ice for 30 minutes. The tubes were centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant (total protein solution) was collected. Protein concentration was determined using a BCA protein assay kit (Beyotime, Cat#: P0010). 5x reduction sample buffer was added to the protein solution, which was then denatured in a metal bath for 10 minutes. The expression of phosphorylated and total HER3, as well as phosphorylated and total MAPK, was detected by Western blotting.

[0381] Calculate the fluorescence intensity of each sample. Relative intensity (%) = target fluorescence intensity / reference (e.g., GAPDH or β-tubulin) fluorescence intensity × 100%.

[0382] The results are as follows Figure 15-17 As shown, the pEGFR, pHER3, and pMAPK levels were decreased in the 9F3-3E1 and 9F3-3E1-CPT2 groups, indicating that the aforementioned anti-EGFR / HER3 antibodies and anti-EGFR / HER3 ADCs can block EGF-induced EGFR signaling and NRG1-induced HER3 signaling.

[0383] Example 14. Developability assessment of anti-EGFR / HER3 ADC

[0384] 9F3-3E1-CPT2 was packaged in sealed Eppendorf tubes and kept at 40°C or 25°C for 7 days. Its development potential was evaluated by SEC-HPLC, CE-SDS(NR), and CE-SDS(R) tests. Detailed results are shown in the table below. The results show that the anti-EGFR / HER3 ADC 9F3-3E1-CPT2 has good stability.

[0385] Table 11

[0386] After the above treatment, the binding activity of 9F3-3E1-CPT2 to EGFR / HER3 antigen was detected by ELISA. An antibody concentration fitting curve was plotted using optical density values. EC 50 Values. The test results are shown in the table below. No significant changes in binding activity were observed after different treatments.

[0387] Table 12

[0388] After the above treatment, the cytotoxic activity of 9F3-3E1-CPT2 cells was detected using HCC827 cells. Specifically, HCC827 cells were cultured at 3 × 10⁻⁶ cells / year. 3 Cells were seeded at the specified density in 96-well plates. Samples treated under different conditions were serially diluted 4-fold starting at 400 nM and then added to the 96-well plates. After incubation, the plates were centrifuged for 5 minutes, and the culture supernatant was discarded. 100 μL of Vazyme Biotech Cell Counting-Lite 2.0 Luminescent CellViability Assay reagent (Vazyme, Cat#: DD1101-02) was added to the corresponding wells, and the plates were incubated at room temperature in the dark for 10 minutes. Cell viability was then measured using a microplate reader.

[0389] Plot antibody concentration fitting curves using cell viability percentage. Determine IC50. 50 The test results are shown in the table below. No significant changes in cell-killing activity were observed after different treatments.

[0390] Table 13

[0391] In another experiment, the anti-EGFR / HER3 ADC 9F3-3E1-CPT2 was oxidized with AAPH (2,2'-azobis(2-methylpropanediamine) dihydrochloride) or TBHP (tert-butyl hydroperoxide) for 6 hours or 24 hours, respectively, and its exploitability was evaluated by SEC-HPLC, CE-SDS(NR) and CE-SDS(R) tests.

[0392] Detailed results are shown in the table below. The results show that the anti-EGFR / HER3 ADC 9F3-3E1-CPT2 exhibits good stability after oxidation treatment.

[0393] Table 14

[0394] After the above treatment, the binding activity of 9F3-3E1-CPT2 with EGFR / HER3 antigen was detected by ELISA. The test results are shown in the table below. No significant changes in binding activity were observed after different treatments.

[0395] Table 15

[0396] After the above treatment, the cytotoxic activity of 9F3-3E1-CPT2 cells was detected using HCC827 cells. The test results are shown in the table below. There was no significant change in cytotoxic activity after different treatments.

[0397] Table 16

[0398] Example 15. Pharmacokinetic Characteristics

[0399] The pharmacokinetic clearance of the anti-EGFR / HER3 ADC was determined in C57BL / 6 mice. Specifically, mice were divided into four groups (n=6 per group) and administered 9F3-3E1-CPT2 (G1, 3 mg / kg; G2, 10 mg / kg) or Ref4-CPT2 (G3, 4 mg / kg; G4, 12 mg / kg) intravenously. Blood samples were collected 3 days before administration and at 15 minutes, 1 day, 3 days, 7 days, 10 days, 14 days, and 21 days after administration.

[0400] Serum levels of antibodies and ADCs were determined using a sandwich ELISA. In short, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., Cat#: 109-005-088) or anti-DXD mIgG (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: BCG108501) was diluted to a final concentration of 2000 ng / mL and added to 96-well plates at 100 µL / well, then incubated overnight at 2–8°C. After incubation, the plates were washed four times with PBS-T buffer (PBS supplemented with Tween™ 20). Unbound areas were blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. After washing, 100 µL of sample was added to each well. The wells were sealed and incubated at 37°C for 1 hour. After washing, Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch Inc., Cat#: 109-036-098) or Anti-Kappa light chain-HRP (Abcam, Cat#: ab202549) was added at 100 µL / well to the corresponding wells and incubated at 37°C for 1 hour. After washing, tetramethylbenzidine (TMB) solution was added at 100 µL / well as substrate to the 96-well plate. After incubation at room temperature in the dark, 100 µL of stop solution (Beyotime, Cat#: P0215) was added to each well. The luminescence signal of the plate was measured at 450 nm and 630 nm. A standard curve with four parameters was constructed using the absorbance values ​​and corresponding concentrations of calibration samples prepared with each test product. The antibody or ADC concentration for each serum sample was calculated using the standard curve. Drug concentration-time curves are constructed using sample concentrations calculated at each time point. Phoenix™ WinNolin 8.3 is used to calculate pharmacokinetic parameters (i.e., T0). 1 / 2 C max AUC 0-21天 (and CL). The results are shown in the table below. The results indicate that the binding of drug molecules does not affect the in vivo pharmacokinetic clearance of the bispecific antibody.

[0401] Table 17

[0402] In another experiment, the pharmacokinetic clearance of the anti-EGFR / HER3 ADC was determined in a PDX mouse model of colorectal cancer. Specifically, a fragment of colorectal tumor (2 mm × 2 mm × 2 mm) derived from a patient was implanted into the right dorsal side of B-NDG mice. Mice were randomly assigned to groups (n=8 per group) based on tumor volume and administered either 9F3-3E1-CPT2 (G1, 6 mg / kg) or Ref4-CPT2 (G2, 8 mg / kg) intravenously. Blood and tumor samples were collected at different time points.

[0403] Serum and tumor levels of antibodies and ADCs were determined using sandwich ELISA, and serum and tumor levels of CPT-2 release were determined using LC-MS / MS. The results are shown in the table below. The results indicate that the half-lives of ADCs, antibodies, and CPT2 in tumor samples from group G1 were longer than those in group G2, and the C50% CPT2 content in tumor samples from group G1 was significantly higher. max The concentration reached 8.120 ng / g, compared to 6.370 ng / g in the G2 group. This indicates that 9F3-3E1-CPT2 can accumulate in tumors, exert its effects, and has a good half-life.

[0404] Table 18

[0405] Table 19

[0406] Humanized mouse models (e.g., dual-humanized EGFR / HER3 mice (B-hEGFR / hHER3 mice)) provide new tools for testing novel treatments in clinical settings by significantly reducing the differences in clinical outcomes between humans and normal mice (expressing mouse EGFR or HER3). In another experiment, the pharmacokinetic clearance of anti-EGFR / HER3 ADCs was determined in B-hEGFR / hHER3 mice. Specifically, B-hEGFR / hHER3 mice were divided into different groups (n=8 per group) and administered 9F3-3E1-CPT2 or Ref4-CPT2 intravenously. Control mice were injected with ISO-CPT2. Blood samples were collected after injection.

[0407] Serum levels of antibodies and ADCs were determined using a sandwich ELISA. Similar to the results above, the 9F3-3E1-CPT2 results were consistent with typical pharmacokinetic characteristics.

[0408] Other implementation plans

[0409] It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An anti-EGFR / HER3 antibody or its antigen-binding fragment, comprising: It contains a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to HER3.

2. The anti-EGFR / HER3 antibody or its antigen-binding fragment 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 anti-EGFR / HER3 antibody or its antigen-binding fragment according to claim 2, wherein... The first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region contains an amino acid sequence having at least 80% identity with a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region contains an amino acid sequence having at least 80% identity with a selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) comprises CDR1, 2, and 3, wherein the VL1 CDR1 region contains an amino acid sequence having at least 80% identity with a selected VL1CDR1 amino acid sequence, the VL1 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VL1CDR2 amino acid sequence, and the VL1 CDR3 region contains an amino acid sequence having at least 80% identity with a selected VL1CDR3 amino acid sequence. The selected VH1 CDR1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22-24, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 25-27, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; and (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28-30, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3.

4. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to claim 2 or 3, wherein... The second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence having at least 80% identity with a selected VH2CDR1 amino acid sequence, the VH2 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VH2CDR2 amino acid sequence, and the VH2 CDR3 region contains an amino acid sequence having at least 80% identity with a selected VH2CDR3 amino acid sequence; and The second light chain variable region (VL2) comprises CDR1, 2, and 3, wherein the VL2 CDR1 region contains an amino acid sequence having at least 80% identity with a selected VL2CDR1 amino acid sequence, the VL2 CDR2 region contains an amino acid sequence having at least 80% identity with a selected VL2CDR2 amino acid sequence, and the VL2 CDR3 region contains an amino acid sequence having at least 80% identity with a selected VL2CDR3 amino acid sequence. The selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (2) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (3) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31-33, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; (5) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34-36, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively; and (6) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37-39, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3.

5. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-4, wherein... (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3. (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3; or (5) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3.

6. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 41, the first light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 45, and the second light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

40.

7. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 42, the first light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 44, and the second light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

40.

8. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 43, the first light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 44, and the second light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

40.

9. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 43, the first light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 45, and the second light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

40.

10. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 43, the first light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 40, the second heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 46, and the second light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

40.

11. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-10, wherein the VH1 comprises an amino acid sequence having at least 90% identity with a selected VH sequence, and the VL1 comprises an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 42, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO:

40.

12. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-11, wherein the VH1 comprises VH1 CDR1, VH1 CDR2, and VH1 CDR3 identical to the selected VH sequences; and the VL1 comprises VL1 CDR1, VL1 CDR2, and VL1 CDR3 identical to the selected VL sequences, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 42, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO:

40.

13. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-12, wherein the VH2 comprises an amino acid sequence having at least 90% identity with a selected VH sequence, and the VL2 comprises an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 44, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO:

40.

14. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-13, wherein the VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 identical to the selected VH sequences; and the VL2 comprises VL2 CDR1, VL2 CDR2, and VL2 CDR3 identical to the selected VL sequences, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 44, and the selected VL sequence is SEQ ID NO: 40; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 40; and (3) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO:

40.

15. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-14, wherein the VH1 comprises the sequence of SEQ ID NO: 41 and the VL1 comprises the sequence of SEQ ID NO:

40.

16. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-14, wherein the VH1 comprises the sequence of SEQ ID NO: 42 and the VL1 comprises the sequence of SEQ ID NO:

40.

17. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-14, wherein the VH1 comprises the sequence of SEQ ID NO: 43 and the VL1 comprises the sequence of SEQ ID NO:

40.

18. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-17, wherein the VH2 comprises the sequence of SEQ ID NO: 44 and the VL2 comprises the sequence of SEQ ID NO:

40.

19. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-17, wherein the VH2 comprises the sequence of SEQ ID NO: 45 and the VL2 comprises the sequence of SEQ ID NO:

40.

20. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-17, wherein the VH2 comprises the sequence of SEQ ID NO: 46 and the VL2 comprises the sequence of SEQ ID NO:

40.

21. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 1-20, wherein the first antigen-binding domain specifically binds to human or monkey EGFR; and / or the second antigen-binding domain specifically binds to human or monkey HER3.

22. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 1-21, wherein the first antigen-binding domain is human or humanized; and / or the second antigen-binding domain is human or humanized.

23. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 1-22, wherein the antibody is a multispecific antibody (e.g., a bispecific antibody).

24. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 1-23, wherein the first antigen-binding domain is a single-chain variable fragment (scFv); and / or the second antigen-binding domain is scFv.

25. The anti-EGFR / HER3 antibody or its antigen-binding fragment according to any one of claims 2-24, wherein the first light chain variable region and the second light chain variable region are identical.

26. An anti-EGFR / HER3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof cross-competes with the anti-EGFR / HER3 antibody or antigen-binding fragment thereof according to any one of claims 1-25.

27. A nucleic acid comprising a polynucleotide encoding an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof as described in any one of claims 1-26.

28. A vector comprising the nucleic acid of claim 27.

29. A cell comprising the carrier of claim 28.

30. The cell of claim 29, wherein the cell is a CHO cell.

31. A cell comprising the nucleic acid of claim 27.

32. A method for generating an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof, the method comprising: (a) The cells are cultured under conditions sufficient to cause the cells of any one of claims 29-31 to produce the anti-EGFR / HER3 antibody or its antigen-binding fragment; and (b) Collect the anti-EGFR / HER3 antibody or its antigen-binding fragment produced by the cells.

33. An anti-EGFR / HER3 antibody-drug conjugate (ADC), said antibody-drug conjugate comprising the anti-EGFR / HER3 antibody or its antigen-binding fragment as described in any one of claims 1-26, covalently bound to a therapeutic agent.

34. The anti-EGFR / HER3 antibody drug conjugate according to claim 33, wherein the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

35. The anti-EGFR / HER3 antibody drug conjugate according to claim 33 or 34, wherein the therapeutic agent is MMAE or MMAF.

36. The antibody-drug conjugate of claim 33, wherein the therapeutic agent is selected from... (CPT-1), (CPT-2), (CPT-3) or (CPT-4).

37. The antibody-drug conjugate according to claim 33 or 36, wherein the therapeutic agent is linked to the antibody or its antigen-binding fragment via a linker.

38. The antibody-drug conjugate according to claim 37, wherein the linker has the following structure: 。 39. The antibody-drug conjugate according to any one of claims 33 and 36-38, wherein the antibody-drug conjugate has the following structure: or ， Where n = 1-8; and "Ab" represents the antibody or its antigen-binding fragment.

40. A method for treating a subject suffering from cancer, the method comprising: The subject is given a therapeutically effective amount of the composition, the composition comprising an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof according to any one of claims 1-26, or an anti-EGFR / HER3 antibody drug conjugate according to any one of claims 33-39.

41. The method of claim 40, wherein the subject has cancer expressing EGFR and / or HER3 (e.g., expressing both EGFR and HER3).

42. The method according to claim 40 or 41, wherein the cancer is esophageal cancer, colorectal cancer, gastric cancer, breast cancer, endometrial cancer, lung cancer, melanoma, ovarian cancer, bladder cancer, non-Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, and cervical cancer.

43. The method according to any one of claims 40-42, wherein the subject is a human.

44. The method according to any one of claims 40-43, wherein the method further comprises administering an anti-PD1 antibody to the subject.

45. The method according to any one of claims 40-44, wherein the method further comprises administering chemotherapy to the subject.

46. ​​A method for reducing tumor growth rate, the method comprising contacting the tumor cells with an effective amount of a composition comprising an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-26, or an anti-EGFR / HER3 antibody drug conjugate as claimed in any one of claims 33-39.

47. A method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an anti-EGFR / HER3 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-26, or an anti-EGFR / HER3 antibody drug conjugate as claimed in any one of claims 33-39.

48. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) The anti-EGFR / HER3 antibody or its antigen-binding fragment as described in any one of claims 1-26, and / or (b) The anti-EGFR / HER3 antibody drug conjugate according to any one of claims 33-39.

49. An anti-EGFR / HER3 antibody-drug conjugate (ADC), the antibody-drug conjugate comprising a therapeutic agent covalently bound to a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment comprising a first antigen-binding domain specifically binding to EGFR and a second antigen-binding domain specifically binding to HER3.

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