Anti-TROP2 / EGFR antibodies and their uses

Abstract

Anti-TROP2 / EGFR antibodies and antibody-drug conjugates derived therefrom specifically bind to at least two different antigens, EGFR and TROP2.

Description

Priority claims

[0001] This application claims priority to PCT / CN2022 / 117496, filed September 7, 2022, and PCT / CN2023 / 083228, filed March 23, 2023, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present disclosure relates to multispecific anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) and antibody-drug conjugates derived therefrom. [Background technology]

[0003] Bispecific antibodies are engineered proteins that can simultaneously bind to two different types of antigens or two different epitopes. Such bispecificity allows for 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 blinatumomab (anti-CD19 and anti-CD3) marks important milestones in the development of bispecific antibodies.

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

[0005] The present disclosure relates to anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof, wherein these antibodies or antigen-binding fragments thereof specifically bind to EGFR and TROP2. 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 share a common light chain. The present disclosure also relates to antibody-drug conjugates derived from these anti-TROP2 / EGFR antibodies.

[0006] In one aspect, the present disclosure provides an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof, which comprises a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to TROP2.

[0007] In some examples, 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). In some embodiments, a first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; and a first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence. and an amino acid sequence that is at least 80% identical to the selected VH1 CDR1, 2, and 3 amino acid sequence, the selected VL1 CDR1, 2, and 3 amino acid sequence, and the selected VH1 CDR1, 2, and 3 amino acid sequence is one of the following: (1) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively;

[0008] (3) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16 to 18, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively;

[0009] (4) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19 to 21, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively.

[0010] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence, and the second light chain variable region (VL2) comprises CDR1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, The CDR1, 2, and 3 amino acid sequences, and their selected VL2 CDR1, 2, and 3 amino acid sequences, are one of the following: (1) These selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively, and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) These selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively.

[0011] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 7 to 9, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively.

[0012] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 7 to 9, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13 to 15, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively.

[0013] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

[0014] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

[0015] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 10 to 12, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4 to 6, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively.

[0016] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 10 to 12, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13 to 15, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively.

[0017] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

[0018] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.

[0019] In some examples, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 25, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0020] In some examples, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 24, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 25, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0021] In some embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 23 and the selected VL sequence is SEQ ID NO: 22; (2) The selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22.

[0022] In some embodiments, the VH1 comprises a VH CDR1, a VH CDR2, and a VH CDR3 that are identical to the VH CDR1, a VH CDR2, and a VH CDR3 of a selected VH sequence, and the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3 that are identical to the VL CDR1, a VL CDR2, and a VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 23 and the selected VL sequence is SEQ ID NO: 22; (2) The selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22.

[0023] In some embodiments, VH2 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 25 and the selected VL sequence is SEQ ID NO: 22.

[0024] In some embodiments, VH2 comprises a VH CDR1, a VH CDR2, and a VH CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and VL2 comprises a VL CDR1, VL CDR2, and VL CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 25 and the selected VL sequence is SEQ ID NO: 22.

[0025] In some embodiments, VH1 comprises the sequence of SEQ ID NO: 23 and VL1 comprises the sequence of SEQ ID NO: 22.

[0026] In some embodiments, VH1 comprises the sequence of SEQ ID NO: 24 and VL1 comprises the sequence of SEQ ID NO: 22.

[0027] In some embodiments, VH2 comprises the sequence of SEQ ID NO: 25 and VL2 comprises the sequence of SEQ ID NO: 22.

[0028] In some examples, 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 TROP2.

[0029] In some examples, the first antigen-binding domain is human or humanized and / or the second antigen-binding domain is human or humanized.

[0030] In some embodiments, the anti-TROP2 / EGFR antibody is a multispecific antibody (eg, a bispecific antibody).

[0031] In some examples, the first antigen-binding domain is a single-chain variable fragment (scFv) and / or the second antigen-binding domain is an scFv.

[0032] In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0033] In one aspect, the disclosure provides an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof, which cross-competes with an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein.

[0034] In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein.

[0035] In one aspect, the disclosure provides a vector comprising a nucleic acid described herein.

[0036] In one aspect, the present disclosure provides a cell comprising a vector described herein. In some embodiments, the cell is a CHO cell.

[0037] In one aspect, the present disclosure provides a cell comprising a nucleic acid described herein.

[0038] In one aspect, the disclosure provides a method of producing an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof, the method comprising: (a) culturing a cell described herein under conditions sufficient to cause the cell to produce the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof; (b) harvesting the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof produced by the cells.

[0039] In one aspect, the present disclosure provides an anti-TROP2 / EGFR antibody-drug conjugate (ADC), which comprises a therapeutic agent covalently attached to an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent. In some embodiments, the therapeutic agent is MMAE or MMAF.

[0040] In some embodiments, the therapeutic agent is [ka] is selected from.

[0041] In some embodiments, the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker is [ka] It has the following structure.

[0042] In some examples, the antibody drug conjugate comprises: [ka] It has the following structure.

[0043] In some embodiments, n=1 to 8, and in some embodiments, "Ab" refers to an antibody or an antigen-binding fragment thereof.

[0044] In some embodiments, the drug-to-antibody ratio (DAR) is about 4 or 8.

[0045] In one aspect, the disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein, or an anti-TROP2 / EGFR antibody-drug conjugate described herein. In some examples, the subject has a cancer that expresses EGFR and / or TROP2.

[0046] In some embodiments, the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung carcinoma), gastric cancer / gastric carcinoma, skin cancer / skin carcinoma, colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, brain cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, cervical cancer, or esophageal cancer.

[0047] In some embodiments, the subject is a human.

[0048] In some embodiments, the method further comprises administering to the subject an anti-PD1 antibody.

[0049] In some embodiments, the method further comprises administering chemotherapy to the subject.

[0050] In one aspect, the disclosure provides a method of reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of a composition comprising an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein, or an anti-TROP2 / EGFR antibody-drug conjugate described herein.

[0051] In one aspect, the disclosure provides a method of killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein, or an anti-TROP2 / EGFR antibody-drug conjugate described herein.

[0052] In one aspect, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier agent and (a) an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein, and / or (b) an anti-TROP2 / EGFR antibody-drug conjugate described herein.

[0053] In one aspect, the disclosure provides an anti-TROP2 / EGFR antibody-drug conjugate (ADC) comprising a therapeutic agent covalently attached to a bispecific antibody or antigen-binding fragment thereof, wherein the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to TROP2. In some embodiments, the drug-antibody ratio (DAR) is about 4.

[0054] As used herein, the term "antigen-binding domain" refers to one or more protein domains (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens (e.g., effector antigens or control antigens). In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some examples, the antigen-binding domain may be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed from a VH-VL dimer. In some examples, the antigen-binding domain may comprise an alternative scaffold. In some examples, the antigen-binding domain is a VHH. This specification describes non-limiting examples of antigen-binding domains. Other examples of antigen-binding domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other examples, the antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen).

[0055] The term "antibody" is used herein in its broadest sense and includes several types of immunoglobulin molecules, which contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies include, among others, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. One example of an antibody is a protein complex containing two heavy chains and two light chains. Other examples of antibodies have been described herein.

[0056] As used herein, the term "multispecific antibody" refers to an antibody comprising two or more different antigen-binding domains that commonly specifically bind to two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or different antigens (e.g., different proteins present on the same or different cell surfaces). In some embodiments, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some embodiments, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody"). In some embodiments, a multispecific antibody binds to four different epitopes (i.e., a "tetraspecific antibody"). In some embodiments, a multispecific antibody binds to five different epitopes (i.e., a "pentaspecific antibody"). Each binding specificity may be present in any suitable titer. This specification describes non-limiting examples of multispecific antibodies.

[0057] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes, which may be on the same antigen or on different antigens.

[0058] 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, where these different antigen-binding sites can specifically bind to different antigens or epitopes. Similarly, the term "common light chain variable region" refers to a light chain variable region that can interact with two or more different heavy chain variable regions to form different antigen-binding sites, where these different antigen-binding sites can specifically bind to different antigens or epitopes. In some examples, antibodies or antigen-binding fragments thereof may share a common light chain. In some examples, anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof may share a common light chain variable region.

[0059] As used herein, the term "anti-TROP2 / EGFR antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that binds to both TROP2 and EGFR.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; in addition, other suitable methods and materials known in the art may be used. These materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) will control.

[0061] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0062] [Figure 1] FIG. 1 is a schematic diagram showing bispecific anti-TROP2 / EGFR antibodies with knob-and-hole architecture and a common light chain. [Figure 2] FIG. 1 shows the mean tumor volume of different groups of B-NDG mice injected with A431 cells and treated with phosphate-buffered saline (PBS), ADC, or antibody. [Figure 3] FIG. 1 shows the mean tumor volume of different groups of B-NDG mice injected with Panc 02.03 cells and treated with PBS or ADC. [Figure 4] FIG. 1 shows a list of the heavy chain variable region CDR sequences of the anti-EGFR antigen-binding domains (E-1G11 and E-6C4) and the anti-TROP2 antigen-binding domain (T-6F7) of anti-TROP2 / EGFR antibodies, as defined by Kabat numbering. [Figure 5] FIG. 1 shows a list of the heavy chain variable region CDR sequences of the anti-EGFR antigen-binding domains (E-1G11 and E-6C4) and the anti-TROP2 antigen-binding domain (T-6F7) of anti-TROP2 / EGFR antibodies, as defined by Chothia numbering. [Figure 6] FIG. 1 lists common light chain CDR sequences as defined by Kabat and Chothia numbering. [Figure 7] FIG. 1 lists the heavy and light chain variable region sequences of the anti-TROP2 / EGFR antibodies discussed in this disclosure. [Figure 8A] FIG. 1 lists additional amino acid sequences discussed in this disclosure. [Figure 8B] FIG. 1 lists additional amino acid sequences discussed in this disclosure. [Figure 9A] FIG. 1 shows the killing effect of T-6F7-E-6C4-ADC on BxPC-3 cells+NCI-H520 cells, BxPC-3 cells, or NCI-H520 cells. [Figure 9B] FIG. 10 shows the killing effect of T-6F7-E-6C4-ADC (0.1 μg / mL) on BxPC-3 cells+NCI-H520 cells (after a total of 72 hours of incubation). [Figure 10]FIG. 1 shows the mean tumor volume of different groups of B-NDG mice injected with NCI-H292 cells and treated with PBS, antibody or ADC. [Figure 11] FIG. 1 shows the mean tumor volume of different groups of B-NDG mice injected with NUGC-4 cells and treated with PBS, antibody or ADC. [Figure 12A] Figure 12 shows serum concentrations of ADC and total antibody after dosing B-hFcRn mice with ISO-ADC (Figure 12A) or T-6F7-E-6C4-ADC (Figure 12B). [Figure 12B] Figure 12 shows serum concentrations of ADC and total antibody after dosing B-hFcRn mice with ISO-ADC (Figure 12A) or T-6F7-E-6C4-ADC (Figure 12B). [Figure 13] FIG. 1 shows the ratio of free MMAE to ADC in human, cynomolgus monkey (Macaca fascicularis), or SD rat plasma 0, 1, 2, 6, 8, 11, and 14 days after addition of T-6F7-E-6C4-ADC to the plasma. [Figure 14A] Figure 14 shows the endocytic activity of anti-TROP2 / EGFR bispecific antibodies and ADCs in A431 cells (Figure 14A) or NCI-H292 cells (Figure 14B). ISO-CPT2 (DAR8) was used as an isotype control. Sacituzumab and cetuximab were used as controls. [Figure 14B] Figure 14 shows the endocytic activity of anti-TROP2 / EGFR bispecific antibodies and ADCs in A431 cells (Figure 14A) or NCI-H292 cells (Figure 14B). ISO-CPT2 (DAR8) was used as an isotype control. Sacituzumab and cetuximab were used as controls. [Figure 15] FIG. 1 shows the mean tumor volumes of different groups of B-NDG mice injected with patient-derived mammary tumor fragments and treated with PBS or ADC. [Figure 16] FIG. 1 shows the mean tumor volume of different groups of B-NDG mice injected with SKOV-3 cells and treated with PBS or ADC. [Figure 17]FIG. 1 shows the mean tumor volume of different groups of B-NDG mice injected with A431 cells and treated with PBS, antibody or ADC. [Figure 18A] Figure 1 shows the mean tumor volumes of different groups of B-NDG mice injected with tumor fragments derived from patients with head and neck squamous cell carcinoma and treated with T-6F7-E-6C4-CPT2(DAR8). Saline was used as a control. [Figure 18B] Figure 1 shows the mean tumor volumes of different groups of B-NDG mice injected with tumor fragments derived from esophageal cancer patients and treated with T-6F7-E-6C4-CPT2(DAR8). Saline was used as a control. [Figure 18C] Figure 1 shows the mean tumor volume of different groups of B-NDG mice injected with tumor fragments derived from colorectal cancer patients and treated with T-6F7-E-6C4-CPT2(DAR8). Saline was used as a control. [Figure 18D] Figure 1 shows the mean tumor volume of different groups of B-NDG mice injected with tumor fragments derived from colorectal cancer patients and treated with T-6F7-E-6C4-CPT2(DAR8). Saline was used as a control. [Figure 18E] Figure 1 shows the mean tumor volume of different groups of B-NDG mice injected with tumor fragments derived from gastric cancer patients and treated with T-6F7-E-6C4-CPT2(DAR8). Saline was used as a control. [Figure 18F] Figure 1 shows the mean tumor volume of different groups of B-NDG mice injected with tumor fragments derived from gastric cancer patients and treated with T-6F7-E-6C4-CPT2(DAR8). Saline was used as a control. [Figure 19A] Figure 19A shows serum concentrations of total antibody (Figure 19A) and CPT2 (Figure 19B) after administration of T-6F7-E-6C4-CPT2(DAR4) or T-6F7-E-6C4-CPT2(DAR8) to B-NDG mice. [Figure 19B]Figure 19A shows serum concentrations of total antibody (Figure 19A) and CPT2 (Figure 19B) after administration of T-6F7-E-6C4-CPT2(DAR4) or T-6F7-E-6C4-CPT2(DAR8) to B-NDG mice. [Figure 19C] Figure 19C shows tumor tissue concentrations of total antibody (Figure 19C) and CPT2 (Figure 19D) after administration of T-6F7-E-6C4-CPT2 (DAR4) or T-6F7-E-6C4-CPT2 (DAR8) to B-NDG mice. [Figure 19D] Figure 19C shows tumor tissue concentrations of total antibody (Figure 19C) and CPT2 (Figure 19D) after administration of T-6F7-E-6C4-CPT2 (DAR4) or T-6F7-E-6C4-CPT2 (DAR8) to B-NDG mice. [Figure 20A] Figure 20A shows the ratio of free CPT2 to total ADC in human, cynomolgus monkey, or SD rat plasma 0, 1, 2, 6, 8, 11, and 14 days after addition of T-6F7-E-6C4-CPT2(DAR4) (Figure 20A) or T-6F7-E-6C4-CPT2(DAR8) (Figure 20B) to the plasma. [Figure 20B] Figure 20A shows the ratio of free CPT2 to total ADC in human, cynomolgus monkey, or SD rat plasma 0, 1, 2, 6, 8, 11, and 14 days after addition of T-6F7-E-6C4-CPT2(DAR4) (Figure 20A) or T-6F7-E-6C4-CPT2(DAR8) (Figure 20B) to the plasma. DETAILED DESCRIPTION OF THE INVENTION

[0063] A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens). In some embodiments, a bispecific antibody or antigen-binding fragment thereof may have two arms. Each arm may have one heavy chain variable region and one light chain variable region to form an antigen-binding domain (or antigen-binding region). In some embodiments, a bispecific antibody may share a common light chain.

[0064] The present disclosure relates to anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to EGFR and TROP2, and antibody-drug conjugates derived from these anti-TROP2 / EGFR antibodies.

[0065] Anti-TROP2 / EGFR antibody The epidermal growth factor receptor (EGFR, ErbB1, or HER1) is a 170-kDa type 1 transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. Epidermal growth factor receptor is a member of the ErbB receptor family, which is a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In many cancer types, mutations affecting EGFR expression or activity can lead to cancer progression. EGFR signaling is initiated by ligand binding, which subsequently induces conformational changes, homodimerization or heterodimerization of the receptor with other ErbB family members, and trans-autophosphorylation of the receptor, resulting in a signal transduction cascade that ultimately affects various 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. Increased EGFR gene copy number and protein expression in non-small cell lung cancer are associated with the EGFR tyrosine kinase inhibitor IRESSA. TM associated with a favorable response to (gefitinib).

[0066] Binding of a ligand, such as EGF (epidermal growth factor), to EGFR stimulates receptor dimerization, autophosphorylation, activation of the receptor's internal cytoplasmic tyrosine kinase domain, and initiation of various signal transduction and transactivation pathways involved in the control of DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of EGFR signaling can result in the inhibition of one or more EGFRs. In some embodiments, EGFR ligands include EGF, TGFα, heparin-binding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI).

[0067] A detailed review of EGFR can be found in Sabbah, Dima A., Rima Hajjo, and Kamal Sweidan. "Review on epidermal growth factor receptor (EGFR) structure, signaling pathways, interactions, and recent updates of EGFR inhibitors." Current topics in medicinal chemistry (2020), which is incorporated herein by reference in its entirety.

[0068] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signaling factor 2 (TACSTD2), is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. It shares high structural sequence similarity with the epithelial adhesion molecule Epcam. TROP2 is a protein closely associated with tumors. It promotes tumor cell growth, proliferation, and metastasis, primarily through calcium ion signaling pathways, regulating cyclin expression, and reducing fibronectin adhesion. Studies have shown that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma, and ovarian cancer. This protein can promote tumor cell proliferation, invasion, metastasis, and dissemination. Furthermore, high TROP2 expression in breast cancer and other cancers has been found to be closely associated with more aggressive disease and poor clinical prognosis.

[0069] TROP2 is an intracellular calcium signaling factor differentially expressed in many cancers. It signals cells for self-renewal, proliferation, invasion, and survival. It has stem cell-like properties. TROP2 is expressed in many normal tissues, but in contrast, it is overexpressed in many cancers, and overexpression of TROP2 is important for prognosis. Several ligands that interact with TROP2 have been proposed. TROP2 signals cells through different pathways and is transcribed and regulated by a complex network of several transcription factors. TROP2 expression in cancer cells is associated with drug resistance.

[0070] A detailed review of TROP2 and its overexpression in cancer can be found in Shvartsur, Anna, and Benjamin Bonavida. "TROP2 and its overexpression in cancers: regulation and clinical / therapeutic implications." Genes & Cancer 6.3-4 (2015): 84, which is incorporated herein by reference in its entirety.

[0071] In some embodiments, the bispecific anti-TROP2 / EGFR antibodies described herein may be designed to have an IgG1 subtype structure containing a knob-and-hole (KIH) mutation, which can promote heterodimerization and avoid mismatches between the two heavy chains. In some embodiments, the bispecific anti-TROP2 / EGFR antibodies have a higher endocytosis rate than the corresponding monoclonal antibody or a control bispecific antibody.

[0072] In some embodiments, the bispecific anti-TROP2 / EGFR antibodies described herein can be conjugated with a therapeutic agent to form an antibody-drug conjugate (ADC). In some embodiments, the drug-antibody ratio (DAR) of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4.5. 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 0.2 to about 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.2, about 3.8 to about 4.2, about 3.9 to about 4.2, about 4.0 to about 4.2, about 4.1 to about 4.2, about 3.5 to about 4.1, about 3.6 to about 4.1, about 3.7 to about 4.1, about 3.8 to about 4.1, about 3.9 to about 4.1, about 4.0 to about 4.1, about 3.5 to about 4. 0, about 3.6 to about 4.0, about 3.7 to about 4.0, about 3.8 to about 4.0, about 3.9 to about 4.0, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7, or about 3.5 to about 3.6.In some embodiments, the DAR of the ADCs described herein is about 7.5 to about 8.5, about 7.6 to about 8.5, about 7.7 to about 8.5, about 7.8 to about 8.5, about 7.9 to about 8.5, about 8.0 to about 8.5, about 8.1 to about 8.5, about 8.2 to about 8.5, about 8.3 to about 8.5, about 8.4 to about 8.5, about 7.5 to about 8.4, about 7.6 to about 8.4, about 7.7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8 .2 to approximately 8.3, approximately 7.5 to approximately 8.2, approximately 7.6 to approximately 8.2, approximately 7.7 to approximately 8.2, approximately 7.8 to approximately 8.2, approximately 7.9 to approximately 8.2, approximately 8.0 to approximately 8.2, approximately 8.1 to approximately 8.2, approximately 7.5 to approximately 8.1, approximately 7.6 to approximately 8.1, approximately 7.7 to approximately 8.1, approximately 7.8 to approximately 8.1, approximately 7.9 to approximately 8.1, approximately 8.0 to approximately 8.1, approximately 7.5 to approximately 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.

[0073] In some examples, the anti-TROP2 / EGFR ADCs described herein can effectively inhibit cancer cell growth in vitro at concentrations of less than 10 μg / mL, less than 3.33 μg / mL, less than 1.11 μg / mL, less than 0.37 μg / mL, less than 0.12 μg / mL, less than 0.04 μg / mL, or less than 0.01 μg / mL. In some examples, the anti-TROP2 / EGFR ADCs described herein can inhibit cancer cell growth (e.g., lung cancer, gastric cancer, or skin cancer) in vivo in a xenograft mouse model at dose levels of less than 30 mg / kg, 25 mg / kg, 20 mg / kg, 15 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, or 1 mg / kg.

[0074] In some examples, the anti-TROP2 / EGFR antibodies described herein share a common light chain. In some examples, the anti-TROP2 / EGFR antibodies comprise an anti-EGFR antigen-binding domain (e.g., E-1G11 ("1G11"), E-6C4 ("6C4")), or an anti-TROP2 antigen-binding domain (e.g., T-6F7 ("6F7")). In some examples, the anti-TROP2 / EGFR antibodies have a heavy chain variable region that targets EGFR (e.g., any one of the VHs that target EGFR described herein), a heavy chain variable region that targets TROP2 (e.g., any one of the VHs that target TROP2 described herein), and two identical common light chain variable regions.

[0075] As defined by Kabat numbering, the CDR sequences of the 1G11 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 7-9) and the CDRs of the light chain variable domain (SEQ ID NOs: 1-3). The CDRs may also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are as set forth in SEQ ID NOs: 16-18, and the CDR sequences of the light chain variable domain are as set forth in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region of 1G11 are as set forth in SEQ ID NOs: 22 and 23, respectively.

[0076] As defined by Kabat numbering, the CDR sequences of the 6C4 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 10-12) and the CDRs of the light chain variable domain (SEQ ID NOs: 1-3). According to Chothia numbering, the CDR sequences of the heavy chain variable domain are as set forth in SEQ ID NOs: 19-21, and the CDR sequences of the light chain variable domain are as set forth in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region of 6C4 are as set forth in SEQ ID NOs: 22 and 24, respectively.

[0077] In some embodiments, the anti-TROP2 / EGFR antibodies described herein may contain one, two, or three heavy chain variable region CDRs (selected from the group consisting of SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, and SEQ ID NOs: 19-21) and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3.

[0078] In some examples, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof may have a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence; and the light chain variable region comprises CDRs 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence. The CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the selected VL CDR2 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, and 3 amino acid sequences and selected VL CDR1, 2, and 3 amino acid sequences are shown in Figures 4 to 6.

[0079] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 9 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0080] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 12 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0081] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0082] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0083] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a light chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0084] The insertions, deletions and substitutions may be within the CDR sequence or at one or both ends of the CDR sequence.

[0085] In some embodiments, the anti-TROP2 / EGFR antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and the light chain variable region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 23 or 24, and the selected VL sequence is SEQ ID NO: 22.

[0086] As defined by Kabat numbering, the CDR sequences of the 6F7 antigen-binding domain include the CDRs of the heavy chain variable domain (SEQ ID NOs: 4-6) and the CDRs of the light chain variable domain (SEQ ID NOs: 1-3). The CDRs may also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are as set forth in SEQ ID NOs: 13-15, and the CDR sequences of the light chain variable domain are as set forth in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region of 6F7 are as set forth in SEQ ID NOs: 22 and 25, respectively.

[0087] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein may further contain one, two, or three heavy chain variable region CDRs (selected from SEQ ID NOs: 4 to 6 and SEQ ID NOs: 13 to 15) and / or one, two, or three light chain variable region CDRs selected from SEQ ID NOs: 1 to 3.

[0088] In some examples, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof may have a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence; and the light chain variable region comprises CDRs 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence. The CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the selected VL CDR2 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in Figures 4 and 6 (Kabat CDRs) and Figures 5 and 6 (Chothia CDRs).

[0089] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 6 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0090] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0091] In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may comprise a light chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0092] The insertions, deletions and substitutions may be within the CDR sequence or at one or both ends of the CDR sequence.

[0093] In some embodiments, the anti-TROP2 / EGFR antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and the light chain variable region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22.

[0094] In some embodiments, an anti-TROP2 / EGFR antibody or antigen-binding fragment may have three VH CDRs that are identical to the CDRs of any VH sequence described herein. In some embodiments, an anti-TROP2 / EGFR antibody or antigen-binding fragment may have three VL CDRs that are identical to the CDRs of any VL sequence described herein.

[0095] The present disclosure further provides nucleic acids comprising polynucleotides encoding anti-TROP2 / EGFR antibodies. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-TROP2 / EGFR antibody comprises the CDRs shown in Figure 4, Figure 5, or Figure 6, or has the sequence shown in Figure 7. When a polypeptide pairs with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to TROP2 and / or EGFR.

[0096] The anti-TROP2 / EGFR antibody may be an anti-TROP2 / EGFR antibody variant (including derivatives and conjugates) or antibody fragment of the anti-TROP2 / EGFR antibody. Other anti-TROP2 / EGFR antibodies according to the present specification include polyclonal antibodies, monoclonal antibodies, multispecific (multimeric, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The anti-TROP2 / EGFR antibody may 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 examples, the anti-TROP2 / EGFR antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or antigen-binding fragment thereof.

[0097] Fragments of anti-TROP2 / EGFR antibodies are suitable for use in the provided methods, provided that these antibodies retain the desired affinity and specificity for both TROP2 and EGFR, and thus retain the ability to bind to TROP2 and EGFR.

[0098] Antibodies and antigen-binding fragments thereof In some examples, multispecific anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies) comprise an antigen-binding domain derived from an anti-EGFR antibody and an antigen-binding domain derived from an anti-TROP2 antibody. These anti-TROP2 / EGFR antibodies and antigen-binding fragments thereof may have a variety of forms.

[0099] Typically, antibodies (also called immunoglobulins) may consist of two types of polypeptide chains (light chains and heavy chains). Non-limiting anti-TROP2 / EGFR antibodies of the present disclosure may be intact four-immunoglobulin chain antibodies, comprising two heavy chains and two light chains. The heavy chain of the anti-TROP2 / EGFR antibody may be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a κ light chain or a λ light chain.

[0100] The hypervariable regions (called complementarity-determining regions (CDRs)) form loops that comprise the primary antigen-binding surface of an antibody. With the four framework regions primarily adopting a β-sheet conformation, the CDRs form loops that connect to, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, form the antigen-binding domain.

[0101] Methods for identifying CDR regions of an antibody by analysis of the antibody's amino acid sequence are well known, and a number of definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, while the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. Biophysical Chemistry 68 (1-3): 9-16 (October 1997), Morea et al., J Mol Biol. 275 (2): 269-94 (January 1998), Chothia et al., Nature 342 (6252): 877-83 (December 1989), and Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated herein by reference in its entirety.

[0102] CDRs are important for recognizing antigen epitopes. As used herein, "epitope" refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be approximately 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be located in a continuous linear sequence in the primary structure of the antigen, as the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structure of the antigen.

[0103] In some embodiments, the anti-TROP2 / EGFR antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, with differences occurring in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described, for example, in Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in immunology 5 (2014); Irani et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases," Molecular immunology 67.2 (2015): 171-182; and 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.

[0104] Anti-TROP2 / EGFR antibodies may also be immunoglobulin molecules derived from any species (e.g., human, rodent, mouse, rat, or camelid). An antigen-binding domain or antigen-binding fragment is any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., that can specifically bind to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof may include, for example, scFv, Fv, Fd, dAb, diabody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide containing an antibody domain or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs of a heavy or light chain from an intact antibody.

[0105] In some embodiments, the scFv in the anti-TROP2 / EGFR antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the anti-TROP2 / EGFR scFv has two antigen-binding regions (antigen-binding regions: A and B), and the two antigen-binding regions can bind to corresponding target antigens with different affinities.

[0106] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof may comprise one, two, or three heavy chain variable region CDRs selected from Figures 4 and 5. In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof may comprise one, two, or three light chain variable region CDRs selected from Figure 6.

[0107] In some embodiments, the anti-TROP2 / EGFR antibodies described herein can be conjugated to a therapeutic agent. The anti-TROP2 / EGFR antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently linked to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids (e.g., DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker (e.g., a VC linker). Details of linkers used in ADCs can be found, for example, in Su, Z. et al., "Antibody-drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0108] In some embodiments, the anti-TROP2 / EGFR antibody is a bispecific antibody. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may comprise at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine) creates a compensatory "cavity" on the interface of the second antibody molecule that is identical or similar in size to the large side chain. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products, such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0109] Any of the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stable molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stable molecule can increase the half-life of the anti-TROP2 / EGFR antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture or in the form of a pharmaceutical composition) or in vivo (e.g., in the human body) or extend its biological activity in vitro or in vivo.

[0110] Anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof may have various forms. Many different forms of bispecific antibodies or antigen-binding fragments thereof are known in the art and are described, for example, in Suurs, et al., "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges," Pharmacology & therapeutics (2019), which is incorporated herein by reference in its entirety.

[0111] In some embodiments, the anti-TROP2 / EGFR antibody is a BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scdiabody, scdiabody-CH3, scFv-CH-CL-scFv, HSAbody, scdiabody-HAS, or tandem-scFv. In some embodiments, the anti-TROP2 / EGFR antibody is selected from the group consisting of VHH-scAb, VHH-Fab, bis-scFab, F(ab')2, diabody, crossMab, DAF (2-in-1), DAF (4-in-1), DutaMab, DT-IgG, knob-hole common light chain, knob-hole component, charge pair, Fab-arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-antibody, 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, triabody, minibody, small molecule antibody, TriBi small molecule antibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG complex, Cov-X-Body, or scFv1-PEG-scFv2.

[0112] In some embodiments, the anti-TROP2 / EGFR antibody may be a TrioMab, in which the two heavy chains are derived from different species, where different sequences define the pairing of the heavy and light chains.

[0113] In some embodiments, the anti-TROP2 / EGFR antibody has two different heavy chains and one common light chain. The heavy chain heterodimerization may be based on a knob-and-hole architecture or some other heavy chain pairing technique.

[0114] In some embodiments, CrossMAb technology can be used to generate bispecific anti-TROP2 / EGFR antibodies. CrossMAb technology can be used to enforce correct light chain binding in bispecific heterodimeric IgG antibodies, enabling the production of a variety of bispecific antibody formats, including 2-valent (1 + 1), 3-valent (2 + 1), and 4-valent (2 + 2) bispecific antibodies, and non-Fc tandem antigen-binding fragment (Fab)-based antibodies. These formats can be derived from any conventional antibody pair using domain crossing, without the need to enforce correct light chain binding by identifying a common light chain, post-translational processing / in vitro chemical assembly, or the introduction of a set of mutations. This method is described in Klein et al., "The use of CrossMAb technology for the generation of bi- and multi-specific antibodies." MAbs. Vol. 8, Issue 6. Taylor & Francis, 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 domain in the heavy chain and the CL domain in the light chain are interchangeable.

[0115] Anti-TROP2 / EGFR antibodies are duobodies. In IgG1 antibodies, the Fab exchange mechanism naturally occurring in IgG4 antibodies is mimicked by controlled substances, a mechanism known as controlled Fab exchange. This form ensures specific pairing between the heavy and light chains.

[0116] In dual variable domain antibodies (DVD-Ig), a separate VH and variable light (VL) domain is added to each N-terminus to achieve dual specific targeting. This configuration is similar to IgG-scFv, but instead of the scFv binding to the N-terminus of each heavy chain, the additional binding domains bind separately to the N-terminus of each.

[0117] In scFv-IgG, two scFvs are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG form is also called tetravalent because it has two different bivalent binding sites. scFv-IgG does not have the problem of pairing heavy and light chains.

[0118] In some examples, the anti-TROP2 / EGFR antibody may have an IgG-IgG format, in which two intact IgG antibodies are conjugated by chemically linking them to the C-terminus of the heavy chains.

[0119] The anti-TROP2 / EGFR antibody may also have a Fab-scFv-Fc format, which comprises a light chain, a heavy chain, an Fc region, and a third chain containing an scFv, which can be efficiently produced and purified.

[0120] In some embodiments, the anti-TROP2 / EGFR antibody may be TF. Three Fab fragments are linked via disulfide bonds. Two fragments target tumor-associated antigens (TAA) and one fragment targets a hapten. The TF form lacks an Fc region.

[0121] ADAPTIR has two scFvs that bind either side of the Fc region. It abandons intact IgG as the basis for its construction, but retains the Fc region to extend half-life and facilitate purification.

[0122] Dual affinity retargeting proteins (DARTs) have two peptide chains connecting opposing fragments (thus, VLA and VHB and VLB and VHA) and a sulfide bond fusing them at their C-termini. The sulfide bond in DARTs can improve stability, making them more stable than BiTEs.

[0123] In DART-Fc, the Fc region is linked to the DART structure. It can be produced by assembling three chains, where two chains are assembled via disulfide bonds, as in DART. One chain contains half of the Fc region, which dimerizes with the third chain and expresses only the Fc region. The addition of the Fc region enhances half-life, prolongs effective concentration, and avoids continuous IV administration.

[0124] In a tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are produced by dimerizing half of the Fc region. This form binds to two targets bivalently, making it a tetravalent molecule.

[0125] A tandem diabody (TandAb) contains two diabodies, each consisting of a covalently linked VHA and VLB fragment and a VHA and VLB fragment. The two diabodies are linked via a peptide chain. This improves stability and may be superior to a diabody consisting of two scFvs. It has two bivalent binding sites.

[0126] The scFv-scFv-toxin comprises a toxin and two scFvs with a stable linker, which can be used for specific delivery of payloads.

[0127] In some embodiments, the anti-TROP2 / EGFR antibody is a bispecific antibody. In some embodiments, the bispecific antibody of the present disclosure is designed to be 1+1 (monovalent for each target) and have an IgG1 subtype structure. This can reduce avidity for cells with low expression levels of EGFR and TROP2 and increase avidity for cells co-expressing EGFR and TROP2, thereby achieving enhanced targeting function.

[0128] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof has a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 26, and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 27 and 28.

[0129] In some embodiments, the anti-TROP2 / EGFR antibody comprises a KIH mutation. In some embodiments, the anti-TROP2 / EGFR antibody comprises a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to TROP2. In some embodiments, the first antigen-binding domain comprises a heavy chain comprising one or more knob mutations (knob heavy chain), and the second antigen-binding domain comprises a heavy chain comprising one or more hole mutations (hole heavy chain). In some embodiments, the first antigen-binding domain comprises a heavy chain comprising one or more hole mutations (hole heavy chain), and the second antigen-binding domain comprises a heavy chain comprising one or more knob mutations (knob heavy chain). In some embodiments, the anti-TROP2 / EGFR antibody comprises a knob heavy chain, wherein the knob heavy chain comprises a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27. In some embodiments, the anti-TROP2 / EGFR antibody comprises a hole heavy chain, wherein the hole heavy chain comprises a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28.

[0130] Antibody and ADC characteristics An anti-TROP2 / EGFR antibody may comprise an anti-EGFR antigen-binding domain and any anti-TROP2 antigen-binding domain described herein.

[0131] The present disclosure provides anti-TROP2 / EGFR antibodies and antigen-binding fragments thereof capable of specifically binding to EGFR. These anti-TROP2 / EGFR antibodies may be agonists or antagonists. The anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein can bind to EGFR and block the binding between EGFR and EGF and / or between EGFR and TGFα. By blocking the binding between EGFR and EGF and / or between EGFR and TGFα, the anti-TROP2 / EGFR antibodies can suppress EGFR-related signaling pathways and treat cancer (e.g., NSCLC). In some examples, the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof can initiate CMC or ADCC.

[0132] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof exhibits a kinetic affinity of 0.1 s -1 Less than 0.01 s -1 Less than 0.001 s -1 Less than 0.0001 s -1 Less than or equal to 0.00001 seconds -1 In some embodiments, the koff is less than 0.01 s. -1 Super, 0.001 s -1 Super, 0.0001 s -1 Super, 0.00001 s -1 Greater than or equal to 0.000001 s -1 It's super.

[0133] In some embodiments, the kinetic association rate (k) is greater than or equal to 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 × 10 7 / Ms is less than.

[0134] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is administered in a concentration of 1×10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 More than M or 1 x 10 -10 It's over M.

[0135] The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof may further comprise an antigen-binding domain capable of specifically binding to TROP2. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein can block the binding between TROP2 and its ligands (e.g., claudin-1, claudin-7, cyclin D1, and IGF-1). In some embodiments, by binding to TROP2, the anti-TROP2 / EGFR antibody can also inhibit cell proliferation, differentiation, and / or metastasis by suppressing TROP2-associated signaling pathways. Thus, in some embodiments, the anti-TROP2 / EGFR antibody described herein is a TROP2 agonist. In some embodiments, the anti-TROP2 / EGFR antibody is a TROP2 antagonist.

[0136] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is administered at 0.1 s -1 Less than 0.01 s -1 Less than 0.001 s -1 Less than 0.0001 s -1 Less than or equal to 0.00001 seconds -1 It can bind to TROP2 (e.g., human TROP2, monkey TROP2, mouse TROP2, and / or chimeric TROP2) with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001 s -1 Super, 0.0001 s -1 Super, 0.00001 s -1 Greater than or equal to 0.000001 s -1 It's super.

[0137] In some embodiments, the kinetic association rate (k) is greater than or equal to 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×105 / Ms less than 1 × 10 6 / Ms or less than 1 × 10 7 / Ms is less than.

[0138] The affinity (KD=koff / kon) can be derived from the quotient of the kinetic rate constants. In some embodiments, KD is 1×10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 More than M or 1 x 10 -10 It's over M.

[0139] Because anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies) bind to both TROP2 and EGFR, they have higher binding affinity to cells expressing both TROP2 and EGFR. Avidity can be used to measure the binding affinity of an antibody to these cells. Avidity is the cumulative strength of the affinities of multiple single non-covalent interactions.

[0140] In some examples, the anti-TROP2 / EGFR antibodies or ADCs described herein can bind to cells expressing TROP2 and / or EGFR (e.g., A431 cells or human lung cancer HCC827 cells) with an EC50 value of less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.9 nM, less than 1.8 nM, less than 1.7 nM, less than 1.6 nM, or less than 1.5 nM.

[0141] Additionally, thermal stability can be determined. The Tm of the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein may be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. Because IgG may be described as a multidomain protein, the melting curve may show two transitions: a first denaturation temperature, Tm D1, and a second denaturation temperature, Tm D2. The presence of these two peaks typically indicates denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When two peaks are present, the Tm typically refers to Tm D2. Thus, in some embodiments, the Tm D1 of an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein is greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the Tm D2 of the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein is greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the Tm, Tm D1, and Tm D2 are less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0142] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is capable of binding to human EGFR or monkey EGFR. In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is incapable of binding to human EGFR or monkey EGFR. In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is capable of binding to human TROP2 or monkey TROP2. In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is incapable of binding to human TROP2 or monkey TROP2.

[0143] In some embodiments, the purity of the anti-TROP2 / EGFR antibody, antigen-binding fragment, or ADC is greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC. In some embodiments, the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC.

[0144] In some embodiments, the anti-TROP2 / EGFR antibody, antigen-binding fragment, or ADC exhibits a rate or percent tumor growth inhibition (TGI%) of more 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-TROP2 / EGFR antibody, antigen-binding fragment, or ADC exhibits a percent tumor growth inhibition of 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 initiation of treatment. As used herein, the following formula is used to calculate tumor growth inhibition rate or percent (TGI%): TGI (%)=[1-(Ti-T0) / (Vi-V0)] ×100% Ti is the mean tumor volume on day i of the treatment group. TO is the mean tumor volume on day 0 of the treatment group. Vi is the mean tumor volume on day i of the control group. V0 is the mean tumor volume on day 0 of the control group.

[0145] In some embodiments, the anti-TROP2 / EGFR antibody, antigen-binding fragment, or ADC comprises a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0146] In some embodiments, the anti-TROP2 / EGFR antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein has an Fc region that does not have effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (EU numbering L234A and L235A mutations) or LALA-PG mutations (EU numbering L234A, L235A, P329G mutations).

[0147] Several other modifications may also be made to the Fc region, for example, by introducing cysteine ​​residue(s) in the Fc region, which allows interchain disulfide bond formation in this region, resulting in a homodimeric fusion protein which may have any additional in vitro and / or in vivo half-life.

[0148] In some embodiments, the IgG4 has an S228P mutation (EU numbering), which prevents IgG4 Fab arm exchange in vivo and in vitro.

[0149] In some embodiments, an Fc Region is provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc Region. For example, the amount of fucose in such an Fc Region composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all glycan structures (e.g., complexed, heteroconjugated, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues, or position 314 in Kabat numbering); however, due to minor sequence variations in the Fc region sequence, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., positions 294-300. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region may be further engineered to replace asparagine at position 297 (N297A) with alanine to reduce glycan heterogeneity.

[0150] In some embodiments, after purification by Protein A-based affinity chromatography and / or size exclusion chromatography, the main HPLC-SEC peak represents at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the protein complex described herein.

[0151] In some examples, the anti-TROP2 / EGFR ADCs described herein have an IC50 for killing cancer cells in vitro (e.g., human epidermoid carcinoma cell line A431, human breast cancer cell line MCF-7, human lung cancer cell line NCI-H226 or NCI-H520) of less than 5 μg / mL, less than 4.5 μg / mL, less than 4 μg / mL, less than 3.5 μg / mL, less than 3 μg / mL, less than 2.5 μg / mL, less than 2 μg / mL, less than 1.5 μg / mL, less than 1 μg / mL Less than 0.9 μg / mL, less than 0.8 μg / mL, less than 0.7 μg / mL, less than 0.6 μg / mL, less than 0.5 μg / mL, less than 0.4 μg / mL, less than 0.3 μg / mL, less than 0.2 μg / mL, less than 0.1 μg / mL, less than 0.05 μg / mL, less than 0.025 μg / mL, less than 0.0125 μg / mL, 0.005 μg / mL or less than 0.0025 μg / mL. In some examples, the anti-TROP2 / EGFR ADCs described herein have an IC50 for killing cancer cells (e.g., HCC827 cells, NCI-H292 cells, A431 cells, or Panc 02.03 cells) in vitro of less than 15 μg / mL, less than 10 μg / mL, less than 5 μg / mL, less than 1 μg / mL, less than 0.9 μg / mL, less than 0.8 μg / mL, less than 0.7 μg / mL, less than 0.6 μg / mL, or less than 0.5 μg / mL.

[0152] In some embodiments, the anti-TROP2 / EGFR antibodies or ADCs described herein have a higher endocytosis rate than the corresponding monoclonal antibodies and / or control bispecific antibodies described herein. In some embodiments, the anti-TROP2 / EGFR antibodies or ADCs described herein have a higher endocytosis rate than a cetuximab analog. In some embodiments, the anti-TROP2 / EGFR antibodies or ADCs described herein have a higher endocytosis rate than a DS-1062 analog and / or a sacituzumab analog. In some embodiments, the bispecific anti-TROP2 / EGFR antibodies or ADCs described herein have a higher endocytosis rate than an amivantamab analog. In some embodiments, the anti-TROP2 / EGFR ADCs described herein have a higher endocytosis rate than an isotype control ADC (e.g., ISO-CPT2).

[0153] In some embodiments, the anti-TROP2 / EGFR ADCs described herein have a half-life of at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, or at least 13 days when administered at 1 to 20 mg / kg (e.g., about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg) and detected 15 minutes, 2 hours, 6 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, or 21 days after administration. In some embodiments, the half-life of an anti-TROP2 / EGFR ADC described herein is at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, or at least 140% compared to an isotype control ADC (e.g., an ISO-ADC). In some embodiments, the clearance of an anti-TROP2 / EGFR ADC described herein is less than 25 mL / day / kg, 24 mL / day / kg, 23 mL / day / kg, 22 mL / day / kg, 21 mL / day / kg, 20 mL / day / kg, 19 mL / day / kg, 18 mL / day / kg, 17 mL / day / kg, 16 mL / day / kg, 15 mL / day / kg, 14 mL / day / kg, 13 mL / day / kg, 12 mL / day / kg, 11 mL / day / kg, or 10 mL / day / kg. In some examples, the PK pattern of an anti-TROP2 / EGFR ADC described herein is determined based on the serum concentration of the administered anti-TROP2 / EGFR ADC or total antibody from the anti-TROP2 / EGFR ADC.

[0154] In some embodiments, at least 1, 2, 6, 8, 11, 14, 18, or 21 days after addition of the anti-TROP2 / EGFR ADC to plasma (e.g., human, monkey, or rat plasma), the ratio of free therapeutic agent (e.g., MMAE or CPTx) from an administered anti-TROP2 / EGFR ADC described herein is less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the final concentration of the ADC is about 10-500 μg / mL (e.g., 100 μg / mL).

[0155] Antibody Drug Conjugates (ADCs) The anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent (drug). The therapeutic agent can be covalently or non-covalently bound to the anti-TROP2 / EGFR antibody. In some embodiments, the anti-TROP2 / EGFR antibody is an anti-TROP2 / EGFR bispecific antibody. In some embodiments, the bispecific antibody may share a light chain.

[0156] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, maytansines (e.g., DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs). Useful classes of cytotoxic, cytostatic, or immunomodulatory agents include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0157] In some embodiments, the therapeutic agent may include, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antibacterial agent. In some embodiments, the conjugated therapeutic agent may be selected from, but is not limited to, MMAE (monomethylauristatin E), MMAD (monomethylauristatin D), or MMAF (monomethylauristatin F).

[0158] The following provides a more detailed description of the definitions of certain functional groups and chemical terms. For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, 75th Edition, inside cover, and certain functional groups are generally defined as set forth therein. General principles of organic chemistry and specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sokhsari, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987.

[0159] Unless expressly stated otherwise, all ranges cited herein are inclusive. When a range of numerical values ​​is recited, it is intended to encompass all values ​​and subranges within that range. For example, "C 1-6 ” is C1, C2, C3, C4, C5, C6, C1-6 , 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 is intended to encompass:

[0160] The compounds of the present disclosure, or any formulas used to depict and describe the compounds, may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the compounds of the present invention, or any formulas used to depict and describe the compounds. Asymmetric centers present in the compounds of the present invention, or any formulas used to depict and describe the compounds, may, independently of one another, have either the (R) or (S) configuration. When a bond to a chiral carbon is depicted as a straight line in a structural formula, or when the compound name does not include an (R) or (S) chiral designation for the chiral carbon, it should be understood that both the (R) and (S) configurations of each chiral carbon are included in the formula or name, and therefore each enantiomer or diastereomer and mixtures thereof are also included in the formula or name.

[0161] The present disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example, mixtures of enantiomers and / or diastereomers in all ratios. Enantiomers are therefore the subject of the present disclosure, in enantiopure form (both levorotatory and dextrorotatory enantiomers), racemic form, and mixtures of two enantiomers in all ratios. In the case of cis / trans isomerism, the present disclosure includes both cis and trans forms and mixtures of these forms in all ratios. If necessary, mixtures can be separated by conventional methods (e.g., chromatography or crystallization) and single stereoisomers can be prepared by synthesis from stereochemically uniform starting materials or by stereoselective synthesis. Optionally, derivatization can be performed before separating stereoisomers. Separation of stereoisomeric mixtures can be performed at an intermediate step during compound synthesis or on the final racemic product. Absolute stereochemistry can be established by X-ray crystallography of crystallized products or intermediates, and, if necessary, derivatizing these products or intermediates with a reagent containing a stereocenter of known configuration. Alternatively, absolute stereochemistry can be established by vibrational circular dichroism (VCD) spectroscopy.

[0162] Unless otherwise stated, the structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. Such compounds are referred to as "isotopic variants." The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds of the present invention, or any formulas in which the compounds are depicted and described. Examples of isotopes suitable for inclusion in the compounds of the present invention include hydrogen isotopes, e.g., 2 H (i.e. D) and 3 H, carbon, e.g. 11 C. 13 C and 14 C, chlorine, e.g., 36 Cl, fluorine, e.g. 18 F, iodine, e.g.123 I and 125 I, nitrogen, e.g. 13 N and 15 N, oxygen, e.g. 15 O. 17 O and 18 O, phosphorus, e.g. 32 P, and sulfur, e.g. 35 Some isotopic variations of the compounds of the present disclosure or of any formula depicted and illustrated compounds, for example, those incorporating radioactive isotopes, may be used in drug and / or substrate tissue distribution studies. In particular, compounds having the depicted structures (e.g., replacement of hydrogen with heavy isotopes (e.g., deuterium ( 2 H or D) may offer certain therapeutic advantages (e.g., increased metabolic stability, increased in vivo half-life, or reduced dose requirements) and therefore may be used in certain specific cases. Isotopic variants of the compounds of the present disclosure or of any formula illustrated and described compounds are generally prepared by synthesis using methods known to those skilled in the art and analogous to those described in the accompanying examples, and using an appropriate isotopically labeled reagent in place of the previously used unlabeled reagent.

[0163] The compounds according to the present disclosure are described with reference to both general formulas and specific compounds. It should be noted that the compounds of the present disclosure can exist in a variety of different forms or derivatives, all of which are within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0164] As used herein, unless otherwise specified, the term "pharmaceutically acceptable salt" includes salts that retain the biological effectiveness of the free acid / base form of the particular compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic and organic bases or acids. When a compound of the present disclosure contains one or more acidic or basic groups, the present disclosure further includes the corresponding pharmaceutically acceptable salts. Therefore, compounds of the present invention that contain an acidic group (e.g., a carboxyl group) can exist in the form of a salt and can be used in accordance with the present invention, for example, as an alkali metal salt, alkaline earth metal salt, aluminum salt, or ammonium salt. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines (e.g., ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids). These salts are readily available, for example, by reacting a compound having an acidic group with a suitable base (e.g., lithium hydroxide, sodium hydroxide, propanol, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide). Other base salts of the compounds of the present disclosure include, but are not limited to, copper(I), copper(II), iron(II), iron(III), manganese(II), and zinc salts. Compounds of the present disclosure that contain one or more basic groups (e.g., protonatable groups) may exist in the form of salts and can be used in accordance with the present disclosure in the form of their addition salts with inorganic or organic acids.Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, puritic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, pamoic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid or aspartic acid, and other acids known to those skilled in the art. The salts formed are, inter alia, hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate / mesylate, toluenesulfonate, carbonate, bicarbonate, formate, acetate, sulfoacetate, trifluoromethanesulfonate, oxalate, malonate, maleate, succinate, tartrate, malate, pamoate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate, and glutamate. It should be noted that the stoichiometry of the salts formed with the compounds of the present disclosure may be an integer or non-integer multiple of 1.

[0165] Compounds of the present disclosure containing a basic nitrogen-containing group can be quaternized with agents, and these agents are 1-4 Alkyl halides, such as methyl, ethyl, isopropyl and t-butyl chlorides, bromides and iodides, di-C 1-4 Alkyl sulfates, such as dimethyl, diethyl and dipentyl sulfates, C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides, and aryl C 1-4 Alkyl halides, for example, benzyl chloride and phenethyl bromide.

[0166] When the compounds of the present disclosure contain both acidic and basic groups in the molecule, in addition to the salt forms mentioned, the present disclosure also includes internal salts or betaines (zwitterions). The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion or cation exchange with other salts. The present disclosure also includes all salts of the compounds of the present disclosure, which, due to their poor physiological compatibility, are not directly suitable for pharmaceutical use, but can be used, for example, as intermediates in chemical reactions or in 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).

[0167] The compounds of the present disclosure or any formulas shown and described for compounds and their pharmaceutically acceptable salts can exist in unsolvated and solvated forms. As used herein, the term "solvate" refers to a molecular complex comprising a compound having formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules. For example, when the solvent is water, the term "hydrate" is used.

[0168] Pharmaceutically acceptable solvates in accordance with the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, eg, D2O, d6-acetone, d6-DMSO.

[0169] Linker (Linker Compound) In some examples, the therapeutic agent is conjugated via a linker (or linker compound). As used herein, the term "linker" or "linker compound" refers to a compound that can react with a pair of ligand compound and therapeutic compound, e.g., by a coupling reaction, to link the ligand (e.g., an antibody or antigen-binding fragment thereof described herein) and the therapeutic agent (e.g., any therapeutic agent described herein) to form a ligand-drug conjugate.

[0170] In some examples, the linkers described herein are compounds having the formula: [ka] or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein Q represents a linking moiety capable of coupling to a ligand via a bond (selected from the group consisting of a carbonyl bond, a thioether bond, an amide bond, a disulfide bond, and a hydrazone bond), and L refers to a linker moiety capable of connecting Q to a therapeutic agent.

[0171] In some embodiments, the linking moiety (Q in Formula (I)) is: [ka] It has the following structure.

[0172] In some embodiments, the linker moiety (L in Formula (I)) is [ka] and wherein L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, which includes at least one amino acid residue having a side chain carboxyl group, such as a glutamic acid residue or an aspartic acid residue, and wherein "-COOH" represents the carboxyl group at the C-terminus of the amino acid residue of the polypeptide residue; L2 is absent or is bonded to a monodentate, bidentate or tridentate hydrophilic group of a side chain carboxyl group in an amino acid residue of the polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c ), where R L2a , R L2b and R L2c are each independently H, -(CHO)(CHCHO) m (CH2) p C(O)OH, and -(CH2O) (CH2CH2O) m (CH2) p C(O)NHR L2d and R L2d is H or C optionally substituted with 1 to 6 hydroxy groups 1-6 an alkyl group, each m is independently an integer of 0 to 10, preferably an integer of 0 to 4, for example, 0, 1, 2, 3, or 4, particularly preferably, m is 0, and each p is independently an integer of 1 to 4, for example, 1, 2, 3, or 4; and [ka] represents the N-terminal side of the polypeptide residue that is covalently attached to the linking moiety Q.

[0173] In some embodiments, the polypeptide residue L1 is NH -Glu-Val-Ala- COOH In some embodiments, the hydrophilic group L2 has the following structure: [ka] where "*" indicates the site of covalent attachment to polypeptide residue L1, e.g., NH -Glu-Val-Ala- COOH represents the side chain of the Glu residue in

[0174] In some embodiments, the linkers described herein are [ka] It is a compound having the following structure.

[0175] In some embodiments, the linker is a VC linker. Details of linkers used in ADCs can be found, for example, in Su, Z. et al., "Antibody-drug conjugates: Recent advances in linker chemistry," Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0176] therapeutic agent In some examples, the therapeutic agent conjugated to an antibody or antigen-binding fragment thereof described herein is as described below.

[0177] In some embodiments, a therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, an analog, or a derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the structure: [ka] wherein X is selected from the group consisting of —CH 2 —, O, and S, and Y is selected from the group consisting of H, D, and F.

[0178] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione) (CPT-1). The structure of CPT-1 is shown below: [ka]

[0179] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(2H)-dione (CPT-2). The structure of CPT-2 is shown below: [ka]

[0180] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below: [ka]

[0181] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(2H)-dione (CPT-4). The structure of CPT-4 is shown below: [ka]

[0182] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also referred to in the art as a derivative of dolastatin-10), or a derivative thereof. For example, the auristatin may be an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structures of exemplary auristatins are described in U.S. Patent Application Publication No. 2003-0083263, International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 04 / 010958, and U.S. Pat. Appl. Pub. No. 2004 / 010959. No. 02 / 088172 and U.S. Patents 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,2 Nos. 84, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, each of which is incorporated herein by reference in its entirety for all purposes.

[0183] Auristatins interfere with microtubule dynamics and nuclear and cellular division and have been shown to have anti-cancer activity. Auristatins can bind to tubulin and exert cytotoxic or cytostatic effects on cancer cells. Many different assays known in the art can be used to determine whether an auristatin or the resulting antibody-drug conjugate exerts a cytostatic or cytotoxic effect on the desired cells.

[0184] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN). TMalkanesulfonates, e.g., busulfan, improsulfan, and piperosulfan; aziridines, e.g., benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine; nitrogen mustards, e.g., chlorambucil, chlornaphazine, colofsphamide, estramustine, isocyclophosphamide, chlormethine, mechlorethamine oxide hydrochloride hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycin , actinomycin C, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogues, e.g., denopterin, methotrexate, pteropterin, trimetrexate; purine analogues, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., pregnenolone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; adrenergic antagonists, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; Bestravsil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid (mitoguazone); mitoxantrone; mopidamol; nitrerin; pentostatin; phenamet; pirarubicin; losoxantrone (podophyllinic acid); 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tenuazonic acid acid); triaziquone; 2',2',2'-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromogalactinol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide;Taxanes, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Anthony, France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; vinorelbine (navelbine); mitoxantrone (novantrone); teniposide; donomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); tretinoin; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives thereof. This definition further includes antihormonal agents for regulating or inhibiting the action of hormones on tumors, such as anti-androgens including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, troboxifene, raloxifene (keoxifene), LY117018, onapristone, and toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin, and any pharmaceutically acceptable salts, acids, or derivatives thereof. A detailed description of chemotherapy agents can be found, for example, in US 20180193477 A1, which is incorporated by reference in its entirety.

[0185] Linker-Therapeutic Compound In some examples, a linker (e.g., any linker described herein) and a therapeutic agent (e.g., any therapeutic agent described herein) can be linked to form a "linker-therapeutic agent" compound.

[0186] In some embodiments, the linker-therapeutic agent compound comprises: [ka] It has the following structure.

[0187] In some embodiments, the linker-therapeutic agent compound comprises: [ka] It has the following structure.

[0188] In some examples, an antibody ("Ab") (e.g., any antibody or antigen-binding fragment thereof described herein) can be linked to a linker-therapeutic compound (e.g., any linker-therapeutic compound described herein) to produce an antibody drug conjugate. In some examples, the antibody drug conjugate has the structure: [ka] Here, n=1 to 8. In some embodiments, n=1 to 8. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In some embodiments, n is about 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8. In some embodiments, n is an integer or non-integer multiple of 1.

[0189] In some embodiments, the anti-TROP2 / EGFR antibody is conjugated to the drug via a cleavable linker (e.g., an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker). In some embodiments, the anti-TROP2 / EGFR antibody is conjugated to the drug via a non-cleavable linker (e.g., an MCC linker formed with SMCC or sulfo-SMCC). Those skilled in the art can easily select an appropriate linker for a given ADC based on knowledge in the art and taking into account relevant factors, such as the binding site of the anti-TROP2 / EGFR antibody, any structural constraints of the drug, and the hydrophobicity of the drug (see, for example, the review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer, Sprager Publishers). In some embodiments, several specific linker-toxin combinations are described and used in conjunction with the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein to prepare ADCs. Examples include, but are not limited to, cleavable peptide-based linkers and auristatins (e.g., MMAE and MMAF), camptothecin (e.g., SN-38), duocarmycin and PBD dimers, non-cleavable MC-based linkers and auristatins MMAF and MMAE, hydrazone-based acid-labile linkers and calicheamicin and doxorubicin, disulfide-based linkers and maytansines (e.g., DM1 and DM4), and bis-maleimide-trioxyethylene glycol (BMPEO)-based linkers and maytansine DM1.Some of these therapeutic agents and linkers are described, for example, in Peters and Brown, (2015) Biosci. Rep. e00225, Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65, U.S. Patent Publication Nos. US 2015 / 0374847 and US 20180193477 A1, which are incorporated herein by reference in their entireties.

[0190] Based on the required drug and the selected linker, those skilled in the art can select an appropriate method for coupling them. For example, several conventional coupling methods (e.g., amine coupling methods) can be used to form the required drug-linker conjugate, which still contains a reactive group and can be used to covalently conjugate with an anti-TROP2 / EGFR antibody or its antigen-binding fragment. In some embodiments, a drug-maleimide conjugate (i.e., a maleimide-linked drug) can be used for the payload having a reactive group in the present disclosure. In ADC production, the most common reactive group that can be conjugated to a sulfhydryl group is maleimide. Organic bromides and iodides are also commonly used.

[0191] Anti-TROP2 / EGFR ADCs can be prepared by one of several routes known in the art using organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). For example, conjugation can be achieved by: (1) reacting a nucleophilic or electrophilic group on an antibody with a bivalent linker reagent to 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 on a drug moiety with a linker reagent to covalently form a drug-linker intermediate DL, which is then reacted with a nucleophilic or electrophilic group on an antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the anti-TROP2 / EGFR ADCs described herein. Various engineered linkers, linker moieties, and toxins are commercially available or can be prepared by standard synthetic organic chemistry techniques, as described, for example, in March's Advanced Organic Chemistry (Smith and March, 2006, 6th ed., Wiley), Toki et al. (2002) J. Org. Chem. 67:1866-1872, Frisch et al. (1997) Bioconj. Chem. 7:180-186, Bioconjugate Techniques (GT Hermanson, 2013, Academic Press), US 20210379193 A1, and US 20180193477 A1, which are incorporated herein by reference in their entireties.Additionally, many preformed drug-linkers suitable for reaction with a selected anti-TROP2 / EGFR antibody or antigen-binding fragment are commercially available; for example, linker-toxins (including DM1, DM4, MMAE, MMAF, or duocarmycin SA) are available from Creative BioLabs (Shirley, NY).

[0192] Some specific examples of methods for producing anti-TROP2 / EGFR ADCs are known in the art and are described in U.S. Patent No. 8,624,003 (the Pott method), U.S. Patent No. 8,163,888 (the one-step method), U.S. Patent No. 5,208,020 (the two-step method), and U.S. Patent No. 20180193477 A1, which are incorporated herein by reference in their entireties. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer [Sprager Publishers].

[0193] Drug loading is expressed by the number of drug moieties per antibody in an ADC molecule. For some antibody-drug conjugates, drug loading may be limited by the number of binding sites on the antibody. For example, when the linkage is a cysteine ​​mercapto group, in some exemplary embodiments described herein, drug loading may be in the range of 0 to 8 drug moieties per antibody. In some embodiments, high drug loading (e.g., p≧5) may cause aggregation, insolubility, toxicity, or loss of cell permeability of some antibody-drug conjugates. In some embodiments, the average drug loading of anti-TROP2 / EGFR antibody-drug conjugates ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In fact, it has been shown that for some antibody-drug conjugates, the optimal ratio of drug moieties per antibody may be about 4. In some embodiments, the DAR of an anti-TROP2 / EGFR ADC composition is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the anti-TROP2 / EGFR ADC composition has a mean DAR of about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0194] In some embodiments, anti-TROP2 / EGFR antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all glycostructures (e.g., complexed, heterozygous, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues, or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some examples, the Fc region of an anti-TROP2 / EGFR antibody may be further engineered to replace asparagine at position 297 (N297A) with alanine to reduce glycan heterogeneity.

[0195] In some embodiments, to improve production efficiency by avoiding Fab-arm exchange, the Fc region of an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof is further engineered to replace serine at position 228 (EU numbering) of IgG4 (S228P) with proline. A detailed description of the S228 mutation is provided, for example, in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation," Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated by reference in its entirety.

[0196] In some embodiments, the method design described herein is used to produce bispecific anti-TROP2 / EGFR antibodies. Bispecific anti-TROP2 / EGFR antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may comprise at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine) creates a compensatory "cavity" on the interface of a second antibody molecule that is identical or similar in size to the large side chain. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products, such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0197] In some embodiments, knob-into-hole (KIH) technology may be used, which involves engineering CH3 domains to create one "knob" or one "hole" on each heavy chain to promote heterodimerization. KIH technology is described, for example, in Xu, Yiren, et al., "Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system," MAbs. [Monoclonal Antibodies] Vol. 7, Issue 1, Taylor & Francis, 2015, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has T366W and / or S354C (knob) substitutions (EU numbering), and another heavy chain has Y349C, T366S, L368A, and / or Y407V (hole) substitutions (EU numbering). In some examples, one heavy chain has one or more of the following substitutions: Y349C and T366W (EU numbering). Another heavy chain may have one or more of the following substitutions: E356C, T366S, L368A, and Y407V (EU numbering). Furthermore, a substitution (-ppcpScp-->-ppcpPcp-) may be introduced in the hinge region of the two substituted IgGs.

[0198] Recombinant vector The present disclosure further provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., the polynucleotides and / or vectors that cause these host cells to contain the polynucleotides), and the production of anti-TROP2 / EGFR antibody polypeptides or fragments thereof by recombinant techniques.

[0199] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering one or more polynucleotides of interest and expressing them as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, the polynucleotides of interest are located and expressed in the vector by being operably linked to regulatory elements, such as a promoter, enhancer, and / or polyA tail, which are located in the genome of the vector or host cell, at or near the integration site of the polynucleotides of interest, or on both sides of the integration site, thereby allowing the polynucleotides of interest to be translated in a host cell into which the expression vector is introduced.

[0200] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), nude DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0201] In some embodiments, a viral expression system (e.g., cowpox or other poxvirus, retrovirus, or adenovirus) is used to introduce a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-defective virus. In the latter case, viral propagation typically occurs only in complementary virus-packaging cells. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 86:317-321, Flexner et al., 1989, Ann. NY Acad. Sci. [Annual Comment of the New York Academy of Sciences] 569:86-103, Flexner et al., 1990, Vaccine 8:17-21, U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487, WO 89 / 01973, U.S. Pat. No. 4,777,127, GB 2200651, EP 0345242, WO 91 / 02805, Berkner-Biotechniques Biotechnology, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may be "nude," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692.The uptake of nude DNA can be increased by coating the DNA onto biodegradable beads, which can be efficiently delivered to cells.

[0202] For expression, a DNA insert containing a polynucleotide encoding a polypeptide disclosed herein may be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac promoter, the E. coli trp promoter and the E. coli tac promoter, the SV40 early and late promoters, and promoters of retroviral long terminal repeats (to name just a few). Other suitable promoters are known to those of skill in the art. The expression construct may further contain sites for transcription initiation, termination, and (in the transcribed region) a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the construct may include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0203] As indicated, expression vectors may contain at least one selectable marker. Such markers include genes for dihydrofolate reductase or neomycin resistance for culturing eukaryotic cells, and tetracycline or ampicillin resistance for culturing in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as E. coli cells, Streptomyces cells and Salmonella typhimurium cells, fungal cells, such as yeast cells, insect cells, such as Drosophila S2 cells and Spodoptera Sf9 cells, animal cells, such as CHO cells, COS cells, Bowes melanoma cells and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

[0204] Non-limiting examples of vectors that can be used in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, and pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting examples of eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0205] Suitable bacterial promoters include, but are not limited to, the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the λ PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, promoters of retroviral LTRs (e.g., the Rous sarcoma virus (RSV) promoter), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0206] In the yeast Saccharomyces cerevisiae, a variety of vectors containing constitutive or inducible promoters, such as α-factor, alcohol oxidase, and PGH, can be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0207] Constructs can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0208] Insertion of an enhancer sequence into a vector can increase transcription of DNA encoding the anti-TROP2 / EGFR antibody of the present disclosure in higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act to increase promoter transcriptional activity in a given host cell type. Examples of enhancers include the SV40 enhancer (located late on the replication origin at base pairs 100 to 270), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer on the late side of the replication origin, and adenovirus enhancers.

[0209] For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or heterologous signals.

[0210] Polypeptides (e.g., anti-TROP2 / EGFR antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and may contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and persistence in host cells during purification or subsequent processing and storage. Similarly, peptide moieties can be added to polypeptides to facilitate purification. Such regions can also be removed before final polypeptide production. Adding peptide moieties to polypeptides to induce secretion or excretion improves stability and facilitates purification, among other things, and is a conventional technique well known in the art.

[0211] The disclosure further provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein. and provides an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence described herein.

[0212] The disclosure further provides nucleic acid sequences that have at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the nucleotide sequences described herein. and providing an amino acid sequence having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any amino acid sequence described herein.

[0213] In some embodiments, the disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is smaller 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 smaller 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.

[0214] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence, or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0215] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence, or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0216] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap introduced to achieve optimal alignment of the two sequences. For example, sequence comparison and percent identity determination between two sequences can use the Blosum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0217] Additionally, the percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can 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 (percent homology), e.g., leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties are known in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent identity is higher than the percent identity.

[0218] The present disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some examples, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a heavy chain polypeptide described herein. In some examples, the nucleic acid comprises a polynucleotide encoding a light chain polypeptide described herein. In some examples, the nucleic acid comprises a polynucleotide encoding an scFv polypeptide described herein.

[0219] In some embodiments, a vector may comprise two nucleic acids described herein, wherein the vectors encode a VL region and a VH region that together bind to EGFR. In some embodiments, vectors are provided, wherein each vector comprises one of the nucleic acids described herein, and wherein the vector pair together encodes a VL region and a VH region that together bind to EGFR.

[0220] In some embodiments, a vector comprises two nucleic acids described herein, wherein the vectors encode a VL region and a VH region that together bind to TROP2. In some embodiments, vectors are provided, wherein each vector comprises one of the nucleic acids described herein, wherein the vector pair together encodes a VL region and a VH region that together bind to TROP2.

[0221] Treatment method The methods described herein include methods for treating cancer-related disorders. Typically, these methods involve administering to a subject in need of, or determined to be in need of, such treatment, a therapeutically effective amount of an anti-TROP2 / EGFR antibody or anti-TROP2 / EGFR antibody-drug conjugate described herein.

[0222] As used in this context, "treatment" means improving at least one symptom of a cancer-related disorder. Cancer usually causes death, so treatment can extend life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years). Administration of a therapeutically effective amount of an agent described herein for treating a cancer-related condition results in a reduction in the number of cancer cells and / or a reduction in symptoms.

[0223] As used herein, the term "cancer" refers to an abnormal condition or disease characterized by cells capable of autonomous proliferation, i.e., rapidly proliferating cell growth. The term is intended to include all types of cancerous growth or oncogenic activity, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. As used herein, the term "tumor" refers to cancer cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of various organ systems (e.g., those affecting the lung, breast, thyroid, lymphatic, gastrointestinal, and genitourinary tracts), and adenocarcinomas (including malignancies such as most colon, renal cell, prostate, and / or testicular tumors, non-small cell lung, small intestine, and esophageal cancers). In some embodiments, the agents described herein are designed for use in treating or diagnosing cancer in a subject. The term "cancer" is well known in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory system cancer, digestive system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary cancers include cancers of cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term further includes carcinosarcomas, which include malignant tumors of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to a cancer of fibrous tissue, or a cancer in which the tumor cells form a distinct linear structure. The term "sarcoma" is well known in the art and refers to a malignant tumor of mesenchymal origin.

[0224] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0225] In one aspect, the present disclosure further provides methods for treating cancer in a subject, for reducing the rate of increase in tumor volume over time in a subject, for reducing the risk of developing metastasis, or for reducing the risk of developing further metastases in a subject. In some examples, the treatment can stop, slow, delay, or inhibit the progression of the cancer. In some examples, the treatment can cause a decrease in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0226] In one aspect, the disclosure features methods of administering a therapeutically effective amount of an anti-TROP2 / EGFR antibody or anti-TROP2 / EGFR antibody-drug conjugate disclosed herein to a subject in need thereof, e.g., a subject identified or diagnosed as suffering from, or having, cancer, such as a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung carcinoma), 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 cancer, brain cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, cervical cancer, or esophageal cancer.

[0227] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to a human or non-human animal to which treatment is provided according to the methods of the present invention. The present invention encompasses veterinary and non-veterinary applications. A human patient may be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, miniature pigs), equines, canines, felines, bovines, and other domestic, farm, and zoo animals.

[0228] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified by various methods known in the art.

[0229] As used herein, an "effective amount" refers to an amount or dosage sufficient to achieve beneficial or desired effects (including halting, slowing, delaying, or inhibiting the progression of a disease (e.g., cancer)). The effective amount depends, for example, on the age and weight of the subject to be administered the anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment, anti-TROP2 / EGFR antibody-drug conjugate, polynucleotide encoding the anti-TROP2 / EGFR antibody, vector comprising the polynucleotide, and / or composition thereof, as well as the severity of symptoms and the administration route, and therefore, administration can be determined according to individual conditions.

[0230] An effective amount may be administered once or multiple times. For example, an effective amount of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment, or anti-TROP2 / EGFR antibody-drug conjugate is an amount sufficient to ameliorate, terminate, stabilize, reverse, inhibit, and / or slow the progression of a patient's autoimmune disease or cancer, or to ameliorate, arrest, stabilize, reverse, slow, and / or slow the proliferation of cells in vitro (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)). As is understood in the art, an effective amount of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment, or anti-TROP2 / EGFR antibody-drug conjugate may vary and may depend, inter alia, on the patient's medical history and other factors, such as the type (and / or dosage) of the agent used.

[0231] Effective amounts and schedules for administering the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, polynucleotides encoding the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antibody-drug conjugates, and / or compositions disclosed herein can be determined empirically, and such determination is within the skill of one in the art. As one of skill in the art will appreciate, the dosage that must be administered will vary depending, for example, on the mammal receiving the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, polynucleotides encoding the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antibody-drug conjugates, and / or compositions disclosed herein, the route of administration, the particular type of agent or composition disclosed herein used, and other agents being administered to the mammal.

[0232] A typical daily dose of an effective amount of an anti-TROP2 / EGFR antibody or anti-TROP2 / EGFR ADC is 0.01 mg / kg to 100 mg / kg. In some examples, 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 examples, the dose may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0233] In any of the methods described herein, the subject may be administered at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment thereof, anti-TROP2 / EGFR antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any one of an anti-TROP2 / EGFR antibody, an anti-TROP2 / EGFR antigen-binding antibody fragment, or an anti-TROP2 / EGFR ADC), and optionally at least one additional therapeutic agent (e.g., once weekly, twice weekly, three times weekly, four times weekly, once daily, twice daily, or three times daily).

[0234] In some examples, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, anti-TROP2 / EGFR antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any one of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, or anti-TROP2 / EGFR ADC). In some examples, the one or more additional therapeutic agents and the at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, or anti-TROP2 / EGFR antibody-drug conjugate are administered to a subject such that the periods of biological activity of the one or more additional therapeutic agents and the at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment, or anti-TROP2 / EGFR ADC in the subject overlap.

[0235] In some examples, a subject may be administered at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, anti-TROP2 / EGFR antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any one of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, or anti-TROP2 / EGFR ADC) for 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 can determine the length of the treatment period by any of the methods for diagnosing or tracking the effectiveness of treatment (e.g., observing at least one cancer symptom) described herein. As described herein, a skilled medical professional can also change (e.g., increase or decrease) the type and number of anti-TROP2 / EGFR antibodies or anti-TROP2 / EGFR antigen-binding antibody fragments, anti-TROP2 / EGFR antibody-drug conjugates (and / or one or more additional therapeutic agents) administered to a subject, and can adjust the dosage or frequency of administration of at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, or anti-TROP2 / EGFR ADC (and / or one or more additional therapeutic agents) to a subject based on an evaluation of therapeutic efficacy (e.g., using any method known in the art as described herein).

[0236] In some embodiments, the subject may be administered one or more additional therapeutic agents. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of a B-Raf inhibitor, an EGFR inhibitor, a MEK inhibitor, an ERK inhibitor, a K-Ras inhibitor, a c-Met inhibitor, a TROP2 inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, an Akt inhibitor, an mTOR inhibitor, a PI3K / mTOR dual inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, and an isocitrate dehydrogenase 1 (IDH1) inhibitor and / or an isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epalrestat).

[0237] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of a HER3 inhibitor, an LSD1 inhibitor, an MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an activated Hedgehog signaling pathway inhibitor, and an agent that selectively degrades the estrogen receptor.

[0238] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, rerafenib, Reolysin, Alimta, Zykadia, Sutent, trolimus, axitinib, erolimus, sorafenib, vemurafenib (Votrient), pazopanib, IMA-901, AGS-003, kazopanib, ketamine, ketamine-1, ketamine-2, ketamine-3, ketamine-4, ketamine-5, ketamine-6, ketamine-7, ketamine-8, ketamine-9, ketamine-10, ketamine-11, ketamine-12, ketamine-13, ketamine-14, ketamine-15, ketamine-16, ketamine-17, ketamine-18, ketamine-19, ketamine-20, ketamine-21, ketamine-22, ketamine-23, ketamine-24, ketamine-25, ketamine-26, ketamine-27, ketamine-28, ketamine-29, ketamine-30, ketamine-31, ketamine-32, ketamine-33, ketamine-34, ketamine-35, ketamine-36, ketamine-37, ketamine-38, ketamine-39, ketamine-39- The treatment may include one or more therapeutic agents selected from the group consisting of bozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomid, amrubicin, carbenzumib, pralatrexate, and enzastaurin.

[0239] In some embodiments, the additional therapeutic agents may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapeutic agent, a CXCL9-targeted therapeutic agent, a CXCL10-targeted therapeutic agent, a CCL5-targeted therapeutic agent, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0240] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexet, gemcitabine, FOLFOX, or FOLFIRI.

[0241] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0242] Pharmaceutical Compositions and Routes of Administration The present specification further provides pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies), anti-TROP2 / EGFR antigen-binding fragments, or anti-TROP2 / EGFR antibody-drug conjugates described herein. Pharmaceutical compositions can be prepared in any manner known in the art.

[0243] Pharmaceutical compositions are prepared to be compatible with their desired route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may contain a sterile diluent (e.g., sterile water or sterile saline), fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid, buffers (e.g., acetates, citrates, or phosphates), and isotonic agents (e.g., sugars, e.g., dextrose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Combinations of these may also be included. Liposomal suspensions may be used as medicament-acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The composition may be prepared and enclosed in ampoules, disposable syringes, or multiple-dose vials. If necessary (e.g., in the form of an injectable formulation), proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin or a surfactant). Absorption-delaying agents (e.g., aluminum monostearate and gelatin) can be included to prolong absorption of the anti-TROP2 / EGFR antibody, its anti-TROP2 / EGFR antigen-binding fragment, or anti-TROP2 / EGFR ADC. Alternatively, controlled-release can be achieved by implants and microencapsulated delivery systems, which may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, Alza Corporation and Nova Pharmaceutical, Inc.).

[0244] Compositions containing one or more of any of the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments, and anti-TROP2 / EGFR antibody-drug conjugates described herein may be prepared for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., a physically discrete unit containing a predetermined amount of active compound for uniformity of administration and dosage).

[0245] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0246] Data obtained from cell culture assays and animal studies can be used to prepare an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment thereof, or anti-TROP2 / EGFR ADC is that amount that treats the disease in a subject (e.g., a human subject identified as having cancer) or identified as being at risk for developing the disease (e.g., a subject who previously had cancer but has now been cured) (e.g., a subject who has previously had cancer but has now been cured) and reduces the severity, frequency, and / or duration of one or more symptoms in the subject (e.g., a human). The efficacy and dosing of any anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment thereof, or anti-TROP2 / EGFR ADC described herein can be determined by a medical or veterinary professional using methods known in the art and by observing one or more symptoms in the subject (e.g., a human). Several factors can affect the dose and duration required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0247] Exemplary doses include milligram or microgram amounts of any anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment thereof, or anti-TROP2 / EGFR ADC described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg). In some embodiments, the dosage level is 5-30 mg / kg, 5-25 mg / kg, 5-20 mg / kg, 5-15 mg / kg, 5-10 mg / kg, 10-30 mg / kg, 10-25 mg / kg, 10-20 mg / kg, 10-15 mg / kg, 15-30 mg / kg, 15-25 mg / kg, 15-20 mg / kg, 20-30 mg / kg, 20-25 mg / kg, or 25-30 mg / kg. In some embodiments, the dose levels are about 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, or 30 mg / kg. In some embodiments, the dose levels described herein do not induce serious toxic effects in the subject. While these doses fall within a wide range, one of skill in the art will appreciate that therapeutic agents vary in efficacy, and effective amounts can be determined using methods known in the art. Typically, a relatively low dose is administered initially, and the dose can then be gradually increased by the attending physician or veterinary professional (for therapeutic uses) or researcher (if still in development) until an appropriate response is obtained. 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 particular compound used, the subject's age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the in vivo half-life of the therapeutic agent.

[0248] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. The present disclosure further provides methods of producing the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, or anti-TROP2 / EGFR ADCs for the various uses described herein.

[0249] example The following examples further illustrate the invention and do not limit the scope of the invention as described in the claims.

[0250] Example 1. Production and analysis of anti-TROP2 / EGFR bispecific antibodies Provided herein are bispecific antigen-binding molecules that target TROP2 and EGFR. These antigen-binding molecules are hereinafter referred to as anti-TROP2 / EGFR bispecific antibodies.

[0251] Production of anti-TROP2 / EGFR bispecific antibodies Anti-TROP2 / EGFR bispecific antibodies may have an anti-TROP2 antigen-binding domain (T-6F7, VH:SEQ ID NO: 25, VL:SEQ ID NO: 22) and an anti-EGFR antigen-binding domain (E-1G11, VH:SEQ ID NO: 23, VL:SEQ ID NO: 22, or E-6C4, VH:SEQ ID NO: 24, VL:SEQ ID NO: 22). These antigen-binding domains can be paired to form bispecific antibodies. Vectors encoding the light and heavy chains of anti-TROP2 / EGFR antibodies were constructed. CHO-S cells were co-transfected with three vectors, including a first vector encoding the anti-TROP2 binding arm heavy chain, a second vector encoding the anti-EGFR binding arm heavy chain, and a third vector encoding the common light chain. After 14 days of culture, cell supernatants were collected and purified by protein A affinity chromatography.

[0252] The possibility of mismatches between the two heavy chains can be reduced in various ways. For example, knob-and-hole mutations can be introduced into the Fc regions of the anti-TROP2 arm heavy chain and the anti-EGFR arm heavy chain. Exemplary bispecific antibodies obtained include T-6F7-E-1G11 and T-6F7-E-6C4. To verify the binding affinity of the bispecific antibody, an anti-TROP2 or anti-EGFR control bispecific antibody is further produced, where one arm of the control bispecific antibody recognizes TROP2 or EGFR, and the other arm recognizes CD28. These control bispecific antibodies are produced in a similar manner and tested, for example, using RenLite. TM VH sequences were obtained by immunizing mice. Exemplary control bispecific antibodies were designated T-6F7-CD28, CD28-T-6F7, CD28-E-1G11, CD28-E-6C4, E-1G11-CD28, and E-6C4-CD28.

[0253] Knob-hole structure mutations were introduced into all bispecific antibodies. For example, in T-6F7-E-1G11, the heavy chain constant region of T-6F7 contains a knob mutation and the heavy chain constant region of E-1G11 contains a hole mutation. In T-6F7-CD28, the heavy chain constant region of T-6F7 contains a knob mutation and the heavy chain constant region of CD28 contains a hole mutation. Exemplary antibody structures are shown in Figure 1, where target 1 and target 2 can be TROP2 and EGFR, respectively, EGFR and TROP2, TROP2 and CD28, CD28 and TROP2, EGFR and CD28, or CD28 and EGFR.

[0254] The constant region may further comprise one or more mutations. For example, when SI mutations (EU numbers: S239D and I332E mutations) were introduced into the Fc region of T-6F7, the resulting antibody was designated T-6F7-SI.

[0255] The sequences of the light chain constant region, the heavy chain constant region with a knob mutation, and the heavy chain constant region with a hole mutation are shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively.

[0256] Internalization of antibodies targeting TROP2 and / or EGFR Anti-TROP2 antibody, anti-EGFR antibody, anti-TROP2 / EGFR bispecific antibody, or anti-TROP2 / CD28 bispecific antibody and pHAb goat anti-human IgG secondary antibody were added to NCI-H292 cells (ATCC, catalog number: CRL-1848) and incubated for 1 hour. The cells were centrifuged and washed with FACS buffer. Mean fluorescence intensity (MFI) was measured using flow cytometry. The endocytosis rate of the antibody was calculated. Human IgG1 protein (hIgG1) was used as an isotype control (ISO). The results are shown in the table below.

[0257] [Table 1]

[0258] Cetuximab is a chimeric monoclonal IgG1 antibody targeting EGFR, originally developed by ImClone Systems and marketed by Merck KGaA in Switzerland in 2003 under the name Erbitux. TM It was first marketed as a monotherapy using cetuximab and was used in combination with irinotecan to treat irinotecan-refractory metastatic colorectal cancer. The heavy and light chain sequences of the cetuximab analog are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

[0259] Sacituzumab-SI analog is a humanized anti-TROP2 monoclonal IgG1 antibody with SI mutations in the constant region. The heavy and light chain sequences of sacituzumab-SI analog are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0260] The results showed that the endocytosis rates of the bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 were higher than those of the corresponding monoclonal antibodies T-6F7, E-6C4, or E-1G11, whereas the endocytosis rates of the control bispecific antibodies T-6F7-CD28, E-6C4-CD28, and E-1G11-CD28 were lower than those of the corresponding bispecific antibodies or monoclonal antibodies.

[0261] Antibody purity analysis The purified anti-TROP2 / EGFR bispecific antibody was analyzed by non-reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and SEC-HPLC (size exclusion chromatography-high performance liquid chromatography).

[0262] Non-reducing SDS-PAGE was performed on 4% to 12% acrylamide gels. These protein samples were prepared as follows: First, 2.4 μL of protein sample was mixed with 6 μL of Tris-glycine SDS sample buffer (2×) (Invitrogen, catalog number: LC2676) and 3.6 μL of distilled water. The mixture was then boiled for 2 minutes and immediately centrifuged before loading. 4 μg of each sample was loaded onto the gel.

[0263] For the SEC-HPLC method, antibody samples were diluted to 1 mg / mL in PBS (pH 7.2-7.4, 0.01 M) and analyzed using an Agilent 1290 chromatography system (Xbridge). TM A Protein BEH SEC column (200A, connected to Waters Corporation) was used. The following parameters were used: mobile phase: 25 mmol / L phosphate buffer (PB) + 300 mmol / L NaCl, pH 6.8, flow rate: 1.8 mL / min, column temperature: 25°C, detection wavelength: 280 nm, injection volume: 10 mL, sample tray temperature: approximately 4°C, and run time: 7 min. The results are summarized in the table below.

[0264] [Table 2]

[0265] Binding affinity of anti-TROP2 / EGFR bispecific antibody Biacore with pre-immobilized protein A sensor chip TM The binding affinity of the anti-TROP2 / EGFR bispecific antibody to human TROP2, human EGFR, monkey TROP2, and monkey EGFR was verified by surface plasmon resonance (SPR) using an 8K biosensor (Biacore, Inc., Piscataway, NJ).

[0266] Specifically, hTROP2-His (ACROBiosystems Inc., Catalog No. TR2-H5223), hEGFR-His (ACROBiosystems Group, Catalog No. EGR-H5222), fasTROP2-His (ACROBiosystems Group, Catalog No. TR2-R52H3), and fasEGFR-His (ACROBiosystems Group, Catalog No. EGR-C52H1) were diluted to 200 nM in 1x HBS-EP+ buffer (pH 7.4). Purified antibodies were filtered at 10 mL / min on a Biacore™ filter. TM The 8K biosensor was injected for approximately 50 seconds to achieve the desired protein density (e.g., approximately 350 response units (RU)), and diluted antigen protein at a concentration of 200 nM was injected for 180 seconds at 30 mL / min. Dissociation was monitored for 400 seconds. After the last injection of each titration, the chip was regenerated with glycine solution (pH 1.5) at 30 mL / min for 30 seconds.

[0267] Biacore TMThe kinetic association rate (k) and dissociation rate (k) were obtained simultaneously by globally fitting the data to a 1:1 Langmuir binding model using 8K Evaluation Software 3.0 (Karlsson, R., Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6. 99-110). The affinity (K = k / k) was derived from the quotient of the kinetic rate constants.

[0268] As will be appreciated by those skilled in the art, the same method was followed for each antibody tested, with parameters (e.g., antibody concentration) adjusted appropriately. The results for the antibodies tested are summarized in the table below.

[0269] [Table 3]

[0270] The results showed that the anti-TROP2 / EGFR bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 have good binding affinity to human TROP2, monkey TROP2, human EGFR, and monkey EGFR.

[0271] Stability of anti-TROP2 / EGFR bispecific antibodies Anti-TROP2 / EGFR bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 were incubated in a pH 6.0 buffer (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween TM The antibody was diluted to 5 mg / mL using 100% ethanol (80). The diluted antibody was stored in a sealed Eppendorf tube at 4°C ± 3°C (hereinafter referred to as 4°C) for 7 days or at 40°C ± 3°C (hereinafter referred to as 40°C) for 7 days to evaluate its thermal stability. Alternatively, the bispecific antibody was further incubated under low pH conditions. In particular, the antibody was incubated in 1 mol / L acetic acid at pH 3.5 for 0 or 6 hours to determine its stability under acidic conditions.

[0272] After the above treatment, the following tests were performed: (1) observing the appearance of the solution and the presence of visible insoluble matter; (2) detecting changes in antibody purity by size-exclusion ultra-performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area relative to the sum of all peak areas (purity, %)); (3) detecting changes in the hydrophobicity of the antibody appearance by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (expressed as the retention time of the main peak (HIC, min)); (4) detecting changes in antibody purity by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under non-reducing (CE-SDS(NR)) conditions (expressed as the percentage of the main peak area relative to the sum of all peak areas (purity, %)); and (5) detecting charge variants in the antibody by capillary isoelectric focusing (Cief) (expressed as the percentage of the main component, acidic component, and basic component).

[0273] For SEC-UPLC experiments, antibody samples were diluted to 1 mg / mL with purified water and analyzed using an Agilent 1290 chromatography system (Xbridge TM The column was connected to a Protein BEH SEC column (200 A, Waters). The following parameters were used: mobile phase: 100 mmol / L phosphate buffer ("PB") (pH 7.4) + 0.2 mol / L NaCl + 10% acetonitrile, flow rate: 1.8 mL / min, column temperature: 25°C, detection wavelength: 280 nm, injection volume: 10 mL, sample tray temperature: approximately 6°C, and run time: 7 minutes.

[0274] For HIC-HPLC experiments, an Agilent 1260 chromatography system (ProPac TMA HIC-10 column (4.6 x 250 mm, connected to Thermo Scientific) was used, and the sample was diluted to 0.5 mg / mL with mobile phase A. The following parameters were used: mobile phase A: 1.0 M PB, 10% acetonitrile pH 6.5, mobile phase B: 0.1 M PB, 10% acetonitrile pH 6.5, flow rate: 0.8 mL / min, gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A, column temperature: 30°C, detection wavelength: 280 nm, injection volume: 10 mL, sample tray temperature: approximately 6°C, and run time: 45 min.

[0275] For the Cief experiment, samples were prepared using the Maurice Cief Method Development Kit (Protein Simple, catalog number: PS-MDK01-C). Specifically, 40 μg of protein sample was mixed with the following reagents in the kit: 1 mL of Maurice Cief Pi Marker-4.05, 1 mL of Maurice Cief Pi Marker-9.99, 35 mL of 1% methylcellulose solution, 2 mL of Maurice Cief 500 mM arginine, 4 mL of ampholytes (Pharmalyte pH range 3-10), and water (added to a final volume of 100 mL). Imaging capillary isoelectric focusing spectra were generated using Maurice Cief Cartridges (PS-MC02-C) on a Maurice analyzer (Protein Simple, Santa Clara, CA). Samples were focused for a total of 10 min. The instrument's installed analysis software was used to integrate the absorbance of the focused protein at 280 nm.

[0276] For the CE-SDS(NR) experiment, Maurice (Protein Simple, Maurice TM) and a Maurice CE-SDS Size Application Kit (Protein Simple, catalog number: PS-MAK02-S). 54 mL of sample buffer, 6 mL of antibody sample, 2.4 mL of 25x internal standard, and 3 mL of 250 nM iodoacetamide (Sigma, catalog number: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3,000 rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The sample was then cooled to room temperature and centrifuged at 10,000 rpm for 3 minutes. The supernatant sample preparation was then transferred to a 96-well plate and tested in the 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.

[0277] Detailed results of the anti-TROP2 / EGFR bispecific antibodies are shown in the table below. The results show that T-6F7-E-6C4 and T-6F7-E-1G11 have better stability and physicochemical properties than the other tested antibodies.

[0278] [Table 4]

[0279] Example 2. Antibody drug conjugates After purification of Protein A, the bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 were dialyzed and concentrated in PBS buffer by ultrafiltration. The concentrations were determined by UV absorption. These antibodies were used in subsequent antibody-drug coupling reactions.

[0280] Coupling of antibodies to drug molecules The purified antibody was coupled to MMAE (monomethylauristatin E) or MMAF (monomethylauristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0281] When naming antibody-drug conjugates, if the antibody is coupled to MMAE, "ADC" is added directly after the antibody name. For example, if T-6F7-E-6C4, which has an IgG1 constant region, is coupled to MMAE, it is named T-6F7-E-6C4-ADC. Similarly, if a cetuximab analogue, which has an IgG1 constant region, is coupled to MMAE, it is named cetuximab analogue-ADC. If an antibody partial analogue of MRG003, which has an IgG1 constant region, is coupled to MMAE, it is named MRG003-ADC. If the constant region contains an SI mutation, "SI" is added to the name to form names such as sacituzumab-SI analogue-ADC, DS-1062-SI analogue(DXd), and DS-1062-SI analogue(MMAE).

[0282] HIC-HPLC was performed to detect the coupling between antibodies and drug molecules. For HIC-HPLC experiments, an Agilent 1260 chromatography system (ProPac) was used. TM A HIC-10 column (4.6 × 250 mm, Thermo Scientific) was used, and the sample was diluted to 0.5 mg / mL with mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile pH 6.5; mobile phase B: 0.1 M PB, 10% acetonitrile pH 6.5; flow rate: 0.8 mL / min; gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 10 mL; sample tray temperature: approximately 6 °C; and run time: 45 min.

[0283] For the isotype control, human IgG1 was coupled with MMAE to form an isotype-ADC (ISO-ADC). HIC-HPLC detection revealed that the drug-antibody ratio (DAR) of the ADC was approximately 4.

[0284] In vitro killing activity The purified antibodies (10 μg / mL, 3.333 μg / mL, 1.111 μg / mL, 0.370 μg / mL, 0.123 μg / mL, 0.041 μg / mL, 0.014 μg / mL, and 0.005 μg / mL) and their corresponding ADCs were cultured in cell culture plates using human epidermoid carcinoma cell line A431 (ATCC, Catalog No. CRL-1555), human breast cancer cell line MCF-7, and human lung cancer cell line NCI-H226 or NCI-H292 (5 × 10 3 ), and purified by IncuCyte (Sartorius AG, IncuCyte (R) Killing activity was detected after 3 days of incubation in S3) and the results are shown in the table below.

[0285] DS-1062 (datopotamab deruxtecan) is an antibody-drug conjugate containing the drug deruxtecan (DXd) targeting TROP2. SI mutations (EU numbers: S239D and I332E mutations) were introduced into the constant region of DS-1062 to produce the DS-1062-SI analog (DXd). The inventors further replaced the drug DXd with MMAE to obtain the DS-1062-SI analog (MMAE). The heavy and light chain sequences of the DS-1062-SI analog (DXd or MMAE) are shown as SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

[0286] [Table 5]

[0287] According to the above results, T-6F7-E-1G11-ADC and T-6F7-E-6C4-ADC have good in vitro killing activity.

[0288] In another experiment, different concentrations of antibody or ADC (10 μg / mL, 3.333 μg / mL, 1.111 μg / mL, 0.370 μg / mL, 0.123 μg / mL, 0.041 μg / mL, 0.014 μg / mL, 0.004 μg / mL, and 0.0015 μg / mL) were used to stimulate the growth of pancreatic cancer Pan.02.03 cells (TROP2) cultured in cell culture plates. + EGFR + ), human pancreatic adenocarcinoma BxPC-3 cells (TROP2 + EGFR + ), human lung cancer NCI-H292 cells (TROP2 + EGFR + ) and PrestoBlue TM The cell viability reagent was used to detect the killing activity after 72 hours of incubation, and the results are shown in the table below.

[0289] Sacituzumab (from Immunomedics, Inc.) is a humanized anti-TROP2 monoclonal antibody-drug conjugate.

[0290] [Table 6]

[0291] Results show that T-6F7-E-6C4-ADC (G5) exhibits tumor-killing efficacy against several cell lines, comparable to its parent TROP2 or EGFR ADCs (G3, G4).

[0292] In another experiment, 0.1 μg / mL T-6F7-E-6C4-ADC was administered to BxPC-3 cells + NCI-H520 cells in Group 1, BxPC-3 cells in Group 2, and NCI-H520 cells (TROP2 - EGFR - The tumor-killing effect was tested by adding T-6F7-E-6C4-ADC to three groups of cells: the control group, which did not receive T-6F7-E-6C4-ADC. After 72 hours of co-incubation at 37°C and 5% CO2, the cell pellets were stained with a death dye and a viability dye (eBioscience TMFixable viability dye eFluor TM The cells were stained with IgG (Bioscience, Catalog No. 65-0865-14) and analyzed by flow cytometry. The results are shown in Figure 9A, which indicated that T-6F7-E-6C4-ADC exhibited potent tumor-killing activity against TROP2-EGFR double-positive BxPC-3 cells but not against TROP2-EGFR double-negative NCI-H520 cells. However, when BxPC-3 cells and NCI-H520 cells were cocultured in Group 1 (Figures 9A-9B), T-6F7-E-6C4-ADC exhibited potent tumor-killing activity against NCI-H520 cells, demonstrating that T-6F7-E-6C4-ADC exhibited significant in vitro bystander killing.

[0293] Example 3. Antitumor activity in A431 xenograft model The effect of antibodies or ADCs on in vivo tumor growth was tested in an epidermoid carcinoma model. 6 A431 cells were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Catalog No.: B-CM-002). The tumor volume of the mice was approximately 300 mm. 3 When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Mice were then injected with phosphate-buffered saline (PBS), ADC, or antibody. The details are shown in the table below.

[0294] The length of the long and short axes of the tumor were measured, and the tumor volume was calculated as 0.5 × (long axis) × (short axis). 2Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100%. Ti is the mean tumor volume on day i of the treatment group. T0 is the mean tumor volume on day 0 of the treatment group. Vi is the mean tumor volume on day i of the control group. V0 is the mean tumor volume on day 0 of the control group. T-test was performed and used for statistical analysis. A TGI greater than 60% indicates significant tumor growth inhibition. P<0.05 indicates a significant difference threshold.

[0295] [Table 7]

[0296] The mice's weights were measured twice a week. On the day of grouping (day 0), the average weight range for each group was 20.7g to 22.1g. At the end of the experiment (day 21), the average weight range for each group was 20.4g to 22.9g. Therefore, the average weight change range for each group was 98.5% to 107.9%. The results showed that the tested antibodies were well tolerated and had no obvious toxicity to the mice.

[0297] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and at the end of the experiment (day 21), mouse survival rate, TGI (%), and statistical differences (P values) in body weight and tumor volume between the treatment and control groups.

[0298] [Table 8]

[0299] Figure 2 shows the tumor volumes of different groups of mice treated with antibody, ADC, or PBS. Compared with the control groups (G1-G2) treated with PBS or ISO-ADC, the treatment groups (G3-G9) showed better tumor suppression.

[0300] Furthermore, compared with sacituzumab-SI analog-ADC, cetuximab analog-ADC, or DS-1062-SI analog (DXd), the anti-TROP2 / EGFR bispecific antibody ADC (G5-G6) and the anti-TROP2 / EGFR bispecific antibody (G8-G9) showed better tumor suppression activity.

[0301] Example 4. Antitumor activity in the Panc 02.03 xenograft model The effect of ADCs on in vivo tumor growth was tested in a xenograft model of pancreatic adenocarcinoma. 6 Pancreatic adenocarcinoma epithelial Panc 02.03 (ATCC, Catalog No. CRL-2553) cells were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 200 mm 3 When the tumor volume reached 1000 mg / kg, the mice were randomly divided into different groups according to tumor volume. Then, the mice were intravenously injected with PBS or ADC once a week (a total of 1 injection). The details are shown in the table below.

[0302] [Table 9]

[0303] There was no significant difference in the body weight of the mice in each group during the experiment.

[0304] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 23 days after grouping (day 23), and at the end of the experiment (day 40), mouse survival rate, TGI (%), and statistical differences (P values) in body weight and tumor volume between the treatment and control groups.

[0305] [Table 10]

[0306] Figure 3 shows tumor size in the ADC-treated groups. The treatment groups exhibited different tumor-inhibitory effects. Overall, at a dose of 10 mg / kg, the anti-TROP2 / EGFR bispecific antibody ADCs (T-6F7-E-1G11-ADC and T-6F7-E-6C4-ADC) exhibited better anti-tumor activity than the controls (ISO-ADC, cetuximab analog-ADC, and DS-1062-SI analog (MMAE)). The anti-TROP2 / EGFR bispecific antibody ADCs (T-6F7-E-1G11-ADC and T-6F7-E-6C4-ADC) exhibited dose-dependent anti-tumor activity.

[0307] Example 5. Antitumor activity in pancreatic adenocarcinoma PDX models The effect of ADC on in vivo tumor growth was tested in a xenograft model of pancreatic adenocarcinoma. Specifically, tumor fragments derived from patients with pancreatic adenocarcinoma were subcutaneously inoculated into B-NDG mice. When tumor volumes in mice reached approximately 250–300 mm, 3 When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Then, mice were injected intravenously (iv) with PBS or ADC. The details are shown in the table below.

[0308] [Table 11]

[0309] There was little difference in the weight of the mice in each group during the experiment.

[0310] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 31 days after grouping (day 31), and at the end of the experiment (day 41), mouse survival rate, TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0311] [Table 12]

[0312] The tumor volumes in all treatment groups (G3-G7) were smaller than those in the control groups (G1 and G2). The treatment groups had different tumor-inhibitory effects. The TROP2 / EGFR bispecific antibody ADCs (G5-G6) at a dose level of 3 mg / kg demonstrated durable and effective tumor-inhibitory effects. T-6F7-E-6C4-ADC (G6) had the highest TGI of 99.3%. The TGI values ​​of all tested TROP2 / EGFR bispecific antibody ADCs (G5-G6) were higher than those of the controls (cetuximab analog-ADC or sacituzumab).

[0313] Example 6. Antitumor activity in patient-derived lung cancer xenograft models The effect of ADC on in vivo tumor growth was tested in a lung cancer xenograft model. Specifically, tumor fragments derived from lung cancer patients were subcutaneously inoculated into B-NDG mice. When tumor volumes in mice reached approximately 250–300 mm, 3 When tumor volume reached 1000 mg / kg, mice were randomly divided into different groups according to tumor volume. Then, mice were intravenously injected with PBS, ADC, or antibody once a week (a total of two injections). The details are shown in the table below.

[0314] [Table 13]

[0315] There was little difference in the weight of the mice in each group during the experiment.

[0316] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 13 days after grouping (day 13), and at the end of the experiment (day 20), mouse survival rate, TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0317] [Table 14]

[0318] The treatment groups have different tumor-suppressing effects. The TGI value (G5) of the TROP2 / EGFR bispecific antibody ADC at the 3 mg / kg dose level was higher than those of the positive control ISO-ADC, cetuximab analog ADC, and sacituzumab.

[0319] Example 7. Antitumor activity in the NCI-H292 xenograft model The effect of antibodies or ADCs on in vivo tumor growth was tested in a lung cancer xenograft model. 6 NCI-H292 cells were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 200 mm 3 When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Mice were then injected intravenously (iv) with PBS, antibody, or ADC. Details are shown in the table below.

[0320] Cetuximab is a chimeric monoclonal IgG1 antibody from Merck that targets EGFR.

[0321] [Table 15]

[0322] MRG003 is an antibody-drug conjugate formed by conjugating a fully human IgG1 monoclonal antibody targeting EGFR with monomethyl auristatin E (MMAE) for the treatment of solid tumors and is currently in early clinical development by Shanghai Miracogen Biotechnology Co., Ltd. The heavy and light chain sequences of the MRG003 antibody are shown in SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0323] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 18 days after grouping (day 18), and at the end of the experiment (day 35), mouse survival rate, TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0324] [Table 16]

[0325] Figure 10 shows tumor size in the antibody- or ADC-treated groups. The anti-TROP2 / EGFR bispecific antibody ADC T-6F7-E-6C4-ADC (G5-G7) demonstrated superior antitumor activity (at a dose level of 10 mg / kg) compared with the controls (cetuximab and MRG003-ADC) and exhibited superior tumor suppression activity (at a dose level of 3 mg / kg) compared with the corresponding parent ADCs (T-6F7-SI-ADC and E-6C4-ADC). Furthermore, T-6F7-E-6C4-ADC demonstrated dose-dependent antitumor activity. Notably, T-6F7-E-6C4-ADC (G7) demonstrated sustained antitumor activity in all six mice, and all mice were tumor-free 35 days after grouping.

[0326] Example 8. Antitumor activity in NUGC-4 xenograft model The effect of antibodies or ADCs on in vivo tumor growth was tested in a xenograft model of gastric cancer. 6 NUGC-4 cells were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 200 mm 3 When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Mice were then injected intravenously (iv) with PBS, antibody, or ADC. Details are shown in the table below.

[0327] [Table 17]

[0328] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 13 days after grouping (day 13), and at the end of the experiment (day 27), as well as TGI (%) and statistical differences (P values) in tumor volumes between treatment and control groups.

[0329] [Table 18]

[0330] Figure 11 shows tumor size in the antibody- or ADC-treated groups. The ADC T-6F7-E-6C4-ADC (G7, G8, G9) showed better antitumor activity than the controls cetuximab (G4), sacituzumab (G3), and MRG003-ADC (G5, G6). Furthermore, T-6F7-E-6C4-ADC showed dose-dependent antitumor activity.

[0331] Example 9. Pharmacokinetic characteristics and plasma stability The pharmacokinetic clearance of anti-EGFR / TROP2 bispecific antibody ADCs was determined in B-hFcRn mice (Biocytogen (Pekin) Pharmaceuticals, catalog number: 110001). Specifically, mice were divided into four groups (six mice per group) and administered ISO-ADC (G1, 3 mg / kg; G2, 10 mg / kg) or T-6F7-E-6C4-ADC (G3, 3 mg / kg; G4, 10 mg / kg) by intravenous injection. Blood samples were collected pre-dose and 15 minutes post-dose, and on days 1, 3, 7, 10, 14, and 21.

[0332] Serum levels of total antibody and ADC were determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., Cat. No. 109-005-088) or anti-MMAE mIgG (Acro Biosystems, Cat. No. MME-M5252) was diluted to a final concentration of 2000 ng / mL, added at 100 μL / well to a 96-well plate (ELISA plate), and incubated overnight at 2°C to 8°C. After incubation, the plate was resuspended in PBS-T buffer (Tween 1). TMThe plates were washed four times with PBS supplemented with 20% BSA (PBS containing 20% ​​ATP). Antibody-free areas were blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. The plates were then washed four times with PBS-T buffer. After washing, 100 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at 37°C for 1 hour. After washing the plates with a plate washer, 100 μL / well of peroxidase AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch, catalog number: 109-036-098) was added to each well of each plate and incubated at 37°C for 1 hour to determine the serum concentration of total antibody. Alternatively, Gh-IgG κ L-HRP (Abcam, catalog number: ab202549) was added to determine the serum concentration of ADC. After washing the plate, 100 μL / well of tetramethylbenzidine (TMB) solution was added to the 96-well plate as a substrate. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, Cat. No. P0215) was added to each well. The luminescence signals of the plate were measured at 450 nm and 630 nm to calculate the concentrations. The absorbance values ​​and corresponding concentrations of the calibration samples prepared for each test product were used to calculate four parameters (i.e., T 1 / 2 , C max , AUC 0-21日 A standard curve with CL (and CL) was prepared. The total antibody or ADC concentration of each serum sample was calculated using the standard curve. Drug concentration-time curves were prepared using the calculated sample concentrations at each time point. Phoenix TM Pharmacokinetic parameters were calculated using WinNolin 8.3.

[0333] The results are shown in the table below and in Figures 12A-12B, and demonstrate that T-6F7-E-6C4-ADC exhibits a half-life similar to the isotype control.

[0334] [Table 19]

[0335] In another experiment, the plasma stability of T-6F7-E-6C4-ADC in human plasma, monkey (cynomolgus monkey) plasma, and rat (SD rat) plasma was determined. Specifically, T-6F7-E-6C4-ADC was added to human, cynomolgus monkey, and SD rat plasma, respectively, to a final concentration of 100 μg / mL. In the control group, plasma was replaced with PBS containing 0.5% BSA. The contents of free MMAE and ADC were determined on days 0, 1, 2, 6, 8, 11, and 14 after T-6F7-E-6C4-ADC addition, and the ratio of free MMAE to ADC was calculated. The results are shown in Figure 13.

[0336] The results showed that after 14 days, the percentage of free MMAE relative to total MMAE in human plasma, monkey plasma, and rat plasma was all less than 2%, indicating that T-6F7-E-6C4-ADC was relatively stable in human, monkey, and rat plasma.

[0337] Example 10. Antibody drug conjugates Coupling of antibodies to drug molecules The purified antibody was coupled to CPT-1, CPT-2, CPT-3, or CPT-4 via a CPT-L linker. The antibody-drug conjugate is named by adding CPTx (x=1, 2, 3, or 4) directly after the antibody name. For example, when T-6F7-E-6C4 is coupled to CPT-1, it is designated as T-6F7-E-6C4-CPT1. As another example, when T-6F7-E-6C4 is coupled to CPT-2, it is designated as T-6F7-E-6C4-CPT2. Exemplary ADCs obtained in this manner include T-6F7-E-6C4-CPT1 and T-6F7-E-6C4-CPT2.

[0338] Mass spectrometry (MS) was used to detect the coupling of the antibody and drug molecules. Human IgG1 molecules were coupled with CPT-2 to form isotype-CPT2 (ISO-CPT2) as an isotype control. According to the MS detection results, the drug-antibody ratio (DAR) of the ADC was approximately 4 or 8. Regarding ADC nomenclature, if the DAR of T-6F7-E-6C4-CPT2 is approximately 4, the ADC is named T-6F7-E-6C4-CPT2(DAR4). If the DAR of T-6F7-E-6C4-CPT2 is approximately 8, the ADC is named T-6F7-E-6C4-CPT2(DAR8).

[0339] In vitro killing activity HCC827 cells, NCI-H292 cells, A431 cells, or Panc 02.03 cells cultured in cell culture plates were treated with different concentrations of purified antibodies or ADCs, and the killing activity was detected after 7 days of incubation using the CellCounting-Lite 2.0 Kit Luminescent Cell Viability Assay (Nanjing Vazyme Biotech Co., Ltd., catalog number: DD1101-02). The results are shown in the table below.

[0340] [Table 20]

[0341] The above results show that T-6F7-E-6C4-CPT2(DAR8) has good in vitro killing activity against HCC827 cells, NCI-H292 cells, A431 cells and Panc 02.03 cells.

[0342] Internalization of anti-TROP2 / EGFR bispecific antibodies and ADCs A431 cells or NCI-H292 cells cultured in cell culture plates were treated with anti-TROP2 / EGFR bispecific antibodies and ADCs (shown in the table below), and then analyzed using IncuCyte (Sartorius, Inc.). (R)Internalization activity was monitored within a 24-hour incubation period using a fluorochrome S3 (Figure 14A-14B), with images taken hourly. The results are shown in Figures 14A-14B, which demonstrated that the endocytic activity of T-6F7-E-6C4-CPT2(DAR4), T-6F7-E-6C4-CPT2(DAR8), and T-6F7-E-6C4 was superior to that of sacituzumab and cetuximab.

[0343] [Table 21]

[0344] Binding activity of anti-TROP2 / EGFR bispecific antibodies and ADCs The purpose of this experiment was to test the binding activity of anti-TROP2 / EGFR bispecific antibodies and ADCs to tumor cell lines.

[0345] In particular, 2 × 10 A431 cells or human lung cancer HCC827 cells (ATCC, Catalog No.: CRL-2868) were used. 5 The cells were transferred to a 96-well plate at a density of 130 nM cells / well. Serially diluted anti-TROP2 / EGFR bispecific antibodies or ADCs (highest concentration: 130 nM, 9 2-fold gradient serial dilutions) were added to the 96-well plate and incubated at 4°C for 25–30 min. Then, the cells and secondary antibody Alexa Fluor ® were incubated for 25–30 min before flow cytometry analysis. (R) 647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch, Catalog No. 109-606-170) was incubated for 25-30 minutes at 4°C in the dark. According to the results shown in the table below, T-6F7-E-6C4-CPT2(DAR4), T-6F7-E-6C4-CPT2(DAR8), and T-6F7-E-6C4 can bind to A431 cells and HCC827 cells with high affinity.

[0346] [Table 22]

[0347] Example 11. Antitumor activity in patient-derived breast cancer xenograft models Tumor tissue fragments (2mm x 2mm x 2mm) derived from breast cancer patients were transplanted into the right flank of B-NDG mice. Immunofluorescence staining of the patient-derived breast tumor fragments was performed, and the images were analyzed using HALO 3.2. Results showed that EGFR-positive cells and TROP2-positive cells accounted for 96.92% and 49.87%, respectively, in the tumor fragments. When the tumor volume of the mice was approximately 200-300mm, the tumor tissue fragments were transplanted into the right flank of B-NDG mice. 3 When tumor volume reached 1000 mg / kg / day, the mice were randomly divided into different groups according to tumor volume. Then, the mice were injected with PBS or ADC by intravenous administration. Detailed information on the administration schedule, route, and frequency is shown in the table below.

[0348] [Table 23]

[0349] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 17 days after grouping (day 17), and 35 days after grouping (day 35), TGI (%), the ratio of tumor-free mice on day 35, and statistical differences (P values) in tumor volumes between treatment and control groups.

[0350] [Table 24]

[0351] Figure 15 shows the tumor size in the groups treated with PBS or ADC. The results showed that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 both exhibited effective tumor-suppressing activity in a dose-dependent manner. Furthermore, T-6F7-E-6C4-CPT2 with DAR4 exhibited better tumor-suppressing activity than T-6F7-E-6C4-ADC at a dose of 3 mg / kg.

[0352] Example 12. Antitumor activity in patient-derived pancreatic cancer xenograft models Tumor tissue fragments (2 mm × 2 mm × 2 mm) derived from pancreatic cancer patients were transplanted into the right flank of B-NDG mice. Immunofluorescence staining revealed that EGFR-positive cells and TROP2-positive cells in the pancreatic tumor fragments accounted for 71.08% and 89.09%, respectively. When the tumor volume in the mice was approximately 200–300 mm, the tumor tissue fragments were transplanted into the right flank of B-NDG mice. 3 When tumor volume reached 1000 mg / kg / day, the mice were randomly divided into different groups according to tumor volume. Then, the mice were injected with PBS or ADC by intravenous administration. The details are shown in the table below.

[0353] [Table 25]

[0354] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and 32 days after grouping (day 32), TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0355] [Table 26]

[0356] The results showed that both T-6F7-E-6C4-CPT2 carrying DAR4 and DAR8 exhibited tumor-suppressing activity in a dose-dependent manner.

[0357] Example 13. Antitumor activity in SKOV-3 xenograft model The effect of the ADC on in vivo tumor growth was tested in a xenograft model of ovarian adenocarcinoma. 6 SKOV-3 cells (ATCC, Catalog No. HTB-77) were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 300 mm 3When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Then, mice were injected intravenously (iv) with PBS or ADC. The details are shown in the table below.

[0358] [Table 27]

[0359] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 17 days after grouping (day 17), and 35 days after grouping (day 35), TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0360] [Table 28]

[0361] Figure 16 shows the tumor size in the groups treated with PBS or ADC. The results showed that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 both exhibited dose-dependent tumor-suppressing activity in an ovarian adenocarcinoma model, and T-6F7-E-6C4-CPT2(DAR8) exhibited better tumor-suppressing activity than T-6F7-E-6C4-CPT2(DAR4).

[0362] Example 14. Antitumor activity in A431 xenograft model The effect of the ADC on in vivo tumor growth was tested in a xenograft model of epidermoid carcinoma. 6 A431 cells were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 200 mm 3 When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Mice were then injected intravenously (iv) with PBS, antibody, or ADC. Details are shown in the table below.

[0363] [Table 29]

[0364] Body weight was measured twice a week. During the experiment, mice in all groups gained weight, with no significant differences in weight between groups, indicating that the tested ADCs were well tolerated and had no obvious toxicity to mice.

[0365] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 17 days after grouping (day 17), and 31 days after grouping (day 31), TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0366] [Table 30]

[0367] Figure 17 shows tumor size in the groups treated with PBS, antibody, or ADC. The results showed that T-6F7-E-6C4-CPT2, which has DAR4 and DAR8, exhibited better tumor-suppressing activity than sacituzumab or cetuximab in a dose-dependent manner. The experiment continued for 49 days after grouping, and 6 mg / kg or 10 mg / kg of T-6F7-E-6C4-CPT2, which has DAR4 and DAR8, exhibited tumor-suppressing activity in a dose-dependent manner.

[0368] Example 15. Antitumor activity in the NCI-H292 xenograft model The effect of antibodies or ADCs on in vivo tumor growth was tested in a lung cancer xenograft model. 6 NCI-H292 cells were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 300 mm. 3 When tumor volume reached 1000 mg / kg / day, mice were randomly divided into different groups according to tumor volume. Mice were then injected intravenously (iv) with PBS, antibody, or ADC. Details are shown in the table below.

[0369] [Table 31]

[0370] The table below summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 21 days after grouping (day 21), and at the end of the experiment (day 39), mouse survival rate, TGI (%), and statistical differences (P values) in tumor volumes between treatment and control groups.

[0371] [Table 32]

[0372] The results showed that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 both exhibited effective tumor suppression in a dose-dependent manner in lung cancer models, and that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 both exhibited tumor suppression at a dose level of 10 mg / kg, with a TGI higher than that of sacituzumab or cetuximab.

[0373] Example 16. Antitumor activity in patient-derived xenograft models The effect of T-6F7-E-6C4-CPT2 (DAR8) on tumor growth was tested in head and neck squamous cell carcinoma, esophageal cancer, colorectal cancer, and gastric cancer models. Specifically, patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) were implanted into BALB / c nude mice. When tumor volumes in mice were approximately 100–200 mm, tumors were transplanted into BALB / c nude mice. 3 When tumor volume reached 1000 mg / kg, mice were randomly divided into different groups (3 mice per group) according to tumor volume, and injected with saline (G1, control) or 6 mg / kg T-6F7-E-6C4-CPT2(DAR8) (G2) (a total of 1 injection).

[0374] Immunohistochemistry (IHC) staining was performed on tumor tissues from different patients, and the histochemistry scores (H scores) of EGFR or TROP2 expression levels in tumor tissues from patients are shown in the table below. Table 33 below also shows the TGI (%) of xenograft models from different patients.

[0375] [Table 33]

[0376] Figures 18A to 18F show the tumor size in the groups treated with saline or T-6F7-E-6C4-CPT2 (DAR8), demonstrating that T-6F7-E-6C4-CPT2 (DAR8) exhibits good tumor growth inhibitory effects in head and neck squamous cell carcinoma, esophageal cancer, colorectal cancer, and gastric cancer.

[0377] Example 17. Pharmacokinetic characteristics and plasma stability The pharmacokinetic clearance of the anti-EGFR / TROP2 bispecific ADC was determined in B-NDG mice. Specifically, approximately 1 × 10 6 A431 cells were subcutaneously injected into B-NDG mice. The tumor volume of the mice was approximately 300 mm. 3 When tumor volume reached 100 mg / kg, mice were randomly divided into different groups (3 mice per group) according to tumor volume and administered PBS (G2), T-6F7-E-6C4-CPT2(DAR4) (G3-G10, 10 mg / kg), or T-6F7-E-6C4-CPT2(DAR8) (G11-G18, 10 mg / kg) intravenously (a total of one administration). Group G1 served as a blank control. Blood and tumor tissue samples were collected from mice in groups G3-G10 and G11-G18 at 15 minutes, 2 hours, 6 hours, 1 day, 3 days, 5 days, 7 days, and 14 days after administration. Blood and tumor tissue samples were collected from mice in group G1 1 hour before administration, while those from mice in group G2 were collected 14 days after administration. These collected samples will be used to detect total antibody levels in serum and tumor tissue by sandwich ELISA and to examine free payload by MS (mass spectrometry).

[0378] Total antibody levels were determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch, Cat. No. 109-005-088) was diluted to a final concentration of 2000 ng / mL, added at 100 μL / well to a 96-well plate (ELISA plate), and incubated overnight at 2°C to 8°C. After incubation, the plate was resuspended in PBS-T buffer (Tween 1). TM The plate was then washed four times with PBS supplemented with 20% BSA (PBS containing 20% ​​BSA). The antibody-free areas were blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. The plate was then washed four times with PBS-T buffer. After washing, 100 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at 37°C for 1 hour. After washing the plate with a plate washer, 100 μL of peroxidase AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch, catalog number: 109-036-098) was added to each well of the plate and incubated at 37°C for 1 hour to determine the concentrations of total antibody and payload CPT2. After washing the plate, 100 μL of tetramethylbenzidine (TMB) solution was added to the 96-well plate as a substrate. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, Cat. No. P0215) was added to each well. The luminescence signals of the plate were measured under 450 nm and 630 nm to calculate the concentrations. The absorbance values ​​and corresponding concentrations of the calibration samples prepared for each test product were used to calculate four parameters (i.e., T 1 / 2 , C max , AUC 0-21日 A standard curve with CL (and CL) was prepared. The antibody or ADC concentration of each serum sample was calculated using the standard curve. Drug concentration-time curves were prepared using the calculated sample concentrations at each time point. Phoenix TM Pharmacokinetic parameters were calculated using WinNolin 8.3.

[0379] The results are shown in the table below and in Figures 19A-19D, which demonstrated that T-6F7-E-6C4-CPT2(DAR4) and T-6F7-E-6C4-CPT2(DAR8) exhibited the expected PK behavior.

[0380] [Table 34]

[0381] In another experiment, the plasma stability of T-6F7-E-6C4-CPT2(DAR4) and T-6F7-E-6C4-CPT2(DAR8) in human plasma, monkey (cynomolgus monkey) plasma, and rat (SD rat) plasma was determined. Specifically, T-6F7-E-6C4-CPT2(DAR4) or T-6F7-E-6C4-CPT2(DAR8) was added to human, monkey, or rat plasma, respectively, to a final concentration of 100 μg / mL. In the control group, plasma was replaced with PBS containing 0.5% BSA. The content of free payload CPT2 and ADC was determined on days 0, 1, 2, 6, 8, 11, and 14 after ADC addition, and the ratio of free CPT2 to total ADC was calculated. The results, shown in Figures 20A-20B, indicated that T-6F7-E-6C4-CPT2(DAR4) and T-6F7-E-6C4-CPT2(DAR8) were stable in human, monkey, and rat plasma, with a maximum release rate of free CPT2 of 2.0% or less.

[0382] Example 18. Toxicological evaluation In a preliminary study, T-6F7-E-6C4-CPT2 (DAR8) was administered intravenously to cynomolgus monkeys three times at 3-week intervals (Days 1, 22, and 43) to study its safety and toxicokinetic (TK) characteristics. The dose regimen is shown in the table below. On Day 50, the animals were sacrificed for gross and histopathological examinations. Mortality / moribundity, general observations, body weight, food consumption, clinical pathology (hematology, coagulation, serum chemistry, and urinalysis), and gross lesions were assessed. Blood samples were also collected for TK analysis, and key TK parameters, such as Tmax, Cmax, and AUC(0-t), of the payload, total antibody, and ADC were calculated. Results indicated that T-6F7-E-6C4-CPT2 (DAR8) had a favorable safety profile.

[0383] [Table 35]

[0384] Other Examples It is to be understood that while the invention has been described in connection with the detailed description, the foregoing description is intended to be illustrative, rather than limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An anti-TROP2 / EGFR antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to TROP2.

2. 2. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

3. the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDR1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence; wherein these selected VH1 CDR1, 2, and 3 amino acid sequences, these selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7 to 9, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10 to 12, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (3) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16-18, respectively, and these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (4) The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to claim 2, wherein the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19 to 21, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively.

4. the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDR1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence; wherein these selected VH2 CDR1, 2, and 3 amino acid sequences and these selected VL2 CDR1, 2, and 3 amino acid sequences are one of the following: (1) These selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4 to 6, respectively, and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively; (2) The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13 to 15, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1 to 3, respectively.

5. (1) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7-9, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (2) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7-9, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13-15, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (3) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16-18, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (4) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16-18, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13-15, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (5) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10-12, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (6) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10-12, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13-15, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (7) These selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21, respectively; these selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; these selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively; and these selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; or (8) The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, wherein the selected VH1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19 to 21, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13 to 15, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1 to 3, respectively.

6. 6. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2 to 5, wherein the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 23; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 25; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

22.

7. 6. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2 to 5, wherein the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 24; the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22; the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 25; and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

22.

8. the VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 23 and the selected VL sequence is SEQ ID NO: 22; (2) The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, wherein the selected VH sequence is SEQ ID NO: 24 and the selected VL sequence is SEQ ID NO:

22.

9. the VH1 comprises a VH CDR1, a VH CDR2, and a VH CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 23 and the selected VL sequence is SEQ ID NO: 22; (2) The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 2 to 8, wherein the selected VH sequence is SEQ ID NO: 24 and the selected VL sequence is SEQ ID NO:

22.

10. 10. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2 to 9, wherein the VH2 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 25 and the selected VL sequence is SEQ ID NO:

22.

11. 11. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2 to 10, wherein the VH2 comprises a VH CDR1, a VH CDR2, and a VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and the VL2 comprises a VL CDR1, a VL CDR2, and a VL CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO:

22.

12. 12. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of claim 2, wherein the VH1 comprises the sequence of SEQ ID NO: 23 and the VL1 comprises the sequence of SEQ ID NO:

22.

13. 13. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2 to 12, wherein the VH1 comprises the sequence of SEQ ID NO: 24 and the VL1 comprises the sequence of SEQ ID NO:

22.

14. 14. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2 to 13, wherein the VH2 comprises the sequence of SEQ ID NO: 25 and the VL2 comprises the sequence of SEQ ID NO:

22.

15. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the first antigen-binding domain specifically binds to human or monkey EGFR, and / or the second antigen-binding domain specifically binds to human or monkey TROP2.

16. 16. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 15, wherein the first antigen-binding domain is human or humanized, and / or the second antigen-binding domain is human or humanized.

17. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody is a multispecific antibody (e.g., a bispecific antibody).

18. 18. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 17, wherein the first antigen-binding domain is a single-chain variable fragment (scFv) and / or the second antigen-binding domain is an scFv.

19. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the first light chain variable region and the second light chain variable region are identical.

20. An anti-TROP2 / EGFR antibody or an antigen-binding fragment thereof that cross-competes with the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 19.

21. A nucleic acid comprising a polynucleotide encoding the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 20.

22. A vector comprising the nucleic acid of claim 21.

23. A cell comprising the vector of claim 22.

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

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

26. 1. A method for producing an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof, the method comprising: (a) culturing the cells of any one of claims 23 to 25 under conditions sufficient to cause the cells to produce the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof; (b) harvesting the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof produced by the cell.

27. 21. An anti-TROP2 / EGFR antibody drug conjugate (ADC) comprising a therapeutic agent covalently attached to the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 20.

28. 28. The anti-TROP2 / EGFR antibody-drug conjugate of claim 27, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

29. 29. The anti-TROP2 / EGFR antibody-drug conjugate of claim 27 or 28, wherein the therapeutic agent is MMAE or MMAF.

30. The therapeutic agent is 【Chemical 1】 28. The antibody drug conjugate of claim 27, selected from:

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

32. The linker is 【Chemistry 2】 32. The antibody drug conjugate of claim 31 having the structure:

33. The antibody drug conjugate has the following structure: 【Chemistry 3】 33. The antibody drug conjugate of any one of claims 27 and 30-32, wherein n=1-8, and wherein "Ab" represents the antibody or antigen-binding fragment thereof or the antigen-binding protein construct.

34. 34. A method of treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 20, or the anti-TROP2 / EGFR antibody-drug conjugate of any one of claims 27 to 33.

35. 35. The method of claim 34, wherein the subject has a cancer that expresses EGFR and / or TROP2.

36. The cancer may be solid tumor, lung cancer (e.g. non-small cell lung cancer, lung adenocarcinoma or lung cancer), gastric cancer / gastric cancer, skin cancer / skin cancer.

36. The method according to claim 34 or 35, wherein the cancer is cancer), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, brain cancer, colon cancer, bladder cancer, oral squamous cell cancer, cervical cancer or esophageal cancer.

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

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

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

40. 34. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 20, or the anti-TROP2 / EGFR antibody-drug conjugate of any one of claims 27 to 33.

41. 34. A method of killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1 to 20, or the anti-TROP2 / EGFR antibody-drug conjugate of any one of claims 27 to 33.

42. A pharmaceutical composition comprising a pharmaceutically acceptable carrier agent; (a) an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, and / or (b) the anti-TROP2 / EGFR antibody-drug conjugate of any one of claims 27 to 33. A pharmaceutical composition comprising the above.

43. 1. An anti-TROP2 / EGFR antibody drug conjugate (ADC), comprising a therapeutic agent covalently attached to a bispecific antibody or antigen-binding fragment thereof, wherein the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to TROP2.

44. 44. The anti-TROP2 / EGFR ADC of any one of claims 27 to 33 and 43, wherein the drug-to-antibody ratio (DAR) is about 4 or 8.

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