Anti-TROP2 / EGFR antibodies and uses thereof

Abstract

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

Description

[0001] Priority claim

[0002] This application claims priority to PCT / CN2022 / 117496 filed on September 7, 2022 and PCT / CN2023 / 083228 filed on March 23, 2023. The entire contents of the foregoing applications are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to multispecific anti-TROP2 / EGFR antibodies (eg, bispecific antibodies or antigen-binding fragments thereof) and antibody drug conjugates derived therefrom. Background Art

[0004] A bispecific antibody is an artificial protein that can bind to two different types of antigens or two different epitopes at the same time. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to target sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and britumomab (anti-CD19 and anti-CD3) has become an important milestone in the development of bispecific antibodies.

[0005] Since bispecific antibodies have various applications, there is a need to continue to develop various bispecific antibody-based therapeutic agents. Summary of the invention

[0006] 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 have a common light chain. The present disclosure also relates to antibody drug conjugates derived from these anti-TROP2 / EGFR antibodies.

[0007] In one aspect, the present disclosure provides an anti-TROP2 / EGFR antibody or an antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to EGFR; and a second antigen-binding domain that specifically binds to TROP2.

[0008] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2). In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence 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 at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR1, 2, and 3 amino acid sequences, the selected VL1 The amino acid sequences of CDR1, 2 and 3 are one of the following:

[0009] (1) the selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively;

[0010] (2) the selected VH1 CDR1, 2, 3 amino acid sequences are represented by SEQ ID NOs: 10-12, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are represented by SEQ ID NOs: 1-3, respectively;

[0011] (3) the selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16-18, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and

[0012] (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

[0013] 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 at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence 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 at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 The amino acid sequences of CDR1, 2 and 3 are one of the following:

[0014] (1) the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and

[0015] (2) The selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

[0016] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 7-9, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 4-6, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0017] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 7-9, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0018] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 16-18, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 4-6, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0019] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 16-18, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0020] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 10-12, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 4-6, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0021] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 10-12, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0022] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 19-21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 4-6, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0023] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 19-21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown by SEQ ID NOs: 1-3, respectively.

[0024] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:25, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:22.

[0025] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:24, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:25, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:22.

[0026] In some embodiments, VH1 comprises an amino acid sequence at least 90% identical to a selected VH sequence, and VL1 comprises an amino acid sequence at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0027] (1) the selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 22; and

[0028] (2) The selected VH sequence is SEQ ID NO:24, and the selected VL sequence is SEQ ID NO:22.

[0029] In some embodiments, VH1 comprises VH CDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL1 comprises VL CDR1, VL CDR2, and VL CDR3 identical to 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:

[0030] (1) the selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 22; and

[0031] (2) The selected VH sequence is SEQ ID NO:24, and the selected VL sequence is SEQ ID NO:22.

[0032] In some embodiments, VH2 comprises an amino acid sequence at least 90% identical to a selected VH sequence, and VL2 comprises an amino acid sequence 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.

[0033] In some embodiments, VH2 comprises VH CDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises VL CDR1, VL CDR2, and VL CDR3 identical to 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.

[0034] In some embodiments, VH1 comprises the sequence of SEQ ID NO:23, and VL1 comprises the sequence of SEQ ID NO:22.

[0035] In some embodiments, VH1 comprises the sequence of SEQ ID NO:24, and VL1 comprises the sequence of SEQ ID NO:22.

[0036] In some embodiments, VH2 comprises the sequence of SEQ ID NO:25, and VL2 comprises the sequence of SEQ ID NO:22.

[0037] In some embodiments, the first antigen binding domain specifically binds to human or monkey EGFR; and / or the second antigen binding domain specifically binds to human or monkey TROP2.

[0038] In some embodiments, the first antigen binding domain is human or humanized; and / or the second antigen binding domain is human or humanized.

[0039] In some embodiments, the anti-TROP2 / EGFR antibody is a multispecific antibody (eg, a bispecific antibody).

[0040] In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0041] In some embodiments, the first light chain variable region and the second light chain variable region are identical.

[0042] In one aspect, the present disclosure provides an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof that cross-competes with the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as described herein.

[0043] In one aspect, the present disclosure provides a nucleic acid comprising a polynucleotide encoding an anti-TROP2 / EGFR antibody or an antigen-binding fragment thereof as described herein.

[0044] In one aspect, the disclosure provides a vector comprising a nucleic acid as described herein.

[0045] In one aspect, the disclosure provides a cell comprising a vector as described herein. In some embodiments, the cell is a CHO cell.

[0046] In one aspect, the disclosure provides a cell comprising a nucleic acid as described herein.

[0047] In one aspect, the present disclosure provides a method for producing an anti-TROP2 / EGFR antibody or an antigen-binding fragment thereof, the method comprising:

[0048] (a) culturing the cell under conditions sufficient for the cell to produce the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as described herein; and

[0049] (b) collecting the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof produced by the cell.

[0050] On the one hand, the present disclosure provides an anti-TROP2 / EGFR antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as described herein. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor. In some embodiments, the therapeutic agent is MMAE or MMAF.

[0051] In some embodiments, the therapeutic agent is selected from

[0052]

[0053]

[0054] In some embodiments, the therapeutic agent is connected to the antibody or its antigen-binding fragment via a linker. In some embodiments, the linker has the following structure:

[0055]

[0056] In some embodiments, the antibody drug conjugate has the following structure:

[0057]

[0058] In some embodiments, n=1-8; in some embodiments, "Ab" means an antibody or an antigen-binding fragment thereof.

[0059] In some embodiments, the drug to antibody ratio (DAR) is about 4 or 8.

[0060] In one aspect, the present disclosure provides a method for treating a subject suffering from 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 as described herein, or an anti-TROP2 / EGFR antibody drug conjugate as described herein. In some embodiments, the subject suffers from a cancer that expresses EGFR and / or TROP2.

[0061] 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.

[0062] In some embodiments, the subject is a human.

[0063] In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject.

[0064] In some embodiments, the method further comprises administering chemotherapy to the subject.

[0065] In one aspect, the present disclosure provides a method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as described herein, or an anti-TROP2 / EGFR antibody drug conjugate as described herein.

[0066] In one aspect, the present disclosure provides a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an anti-TROP2 / EGFR antibody or an antigen-binding fragment thereof as described herein, or an anti-TROP2 / EGFR antibody-drug conjugate as described herein.

[0067] In one aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) an anti-TROP2 / EGFR antibody or an antigen-binding fragment thereof as described herein, and / or (b) an anti-TROP2 / EGFR antibody drug conjugate as described herein.

[0068] In one aspect, the present disclosure provides an anti-TROP2 / EGFR antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or its antigen-binding fragment comprising: 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.

[0069] As used herein, the term "antigen binding domain" refers to one or more protein domains (e.g., formed by amino acids from a single polypeptide or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that are capable of specifically binding to one or more different antigens (e.g., effector antigens or control antigens). In some examples, the antigen binding domain can bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or a fragment thereof. An example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. In some embodiments, the antigen binding domain may include an alternative scaffold. In some embodiments, the antigen binding domain is a VHH. Non-limiting examples of antigen binding domains are described herein. Other examples of antigen binding domains are known in the art. In some examples, the antigen binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other examples, the antigen binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen).

[0070] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen binding domains that specifically bind to an antigen or epitope. Antibodies particularly include, for example, complete antibodies (e.g., complete immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a protein complex comprising two heavy chains and two light chains. Other examples of antibodies are described herein.

[0071] As used herein, the term "multispecific antibody" is an antibody comprising two or more different antigen-binding domains that specifically bind to two or more different epitopes. Two or more different epitopes can be epitopes on the same antigen (e.g., a single polypeptide present on a cell surface) or different antigens (e.g., present on the same cell surface or on different cell surfaces). In some aspects, a multispecific antibody binds to two different epitopes (i.e., "bispecific antibodies"). In some aspects, a multispecific antibody binds to three different epitopes (i.e., "trispecific antibodies"). In some aspects, a multispecific antibody binds to four different epitopes (i.e., "tetraspecific antibodies"). In some aspects, a multispecific antibody binds to five different epitopes (i.e., "five specific antibodies"). Each binding specificity can exist with any suitable titer. Non-limiting examples of multispecific antibodies are described herein.

[0072] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

[0073] As used herein, the term "common light chain" refers to a light chain that can interact with two or more different heavy chains to form different antigen binding sites, wherein these different antigen binding sites can specifically bind to different antigens or epitopes. Similarly, the term "common light chain variable region" refers to a light chain variable region that can interact with two or more different heavy chain variable regions to form different antigen binding sites, wherein these different antigen binding sites can specifically bind to different antigens or epitopes. In some embodiments, antibodies or antigen-binding fragments thereof may have a common light chain. In some embodiments, anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof may have a common light chain variable region.

[0074] 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.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also 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 the event of a conflict, the present specification (including definitions) shall prevail.

[0076] Other features and advantages of the invention will become apparent from the following detailed description and from the accompanying drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is a schematic diagram showing a bispecific anti-TROP2 / EGFR antibody having a knob-in-hole structure and a common light chain.

[0078] Figure 2 Shown are the mean tumor volumes of different groups of B-NDG mice injected with A431 cells and treated with phosphate-buffered saline (PBS), ADC or antibodies.

[0079] Figure 3 Shown are the mean tumor volumes of different groups of B-NDG mice injected with Panc 02.03 cells and treated with PBS or ADC.

[0080] Figure 4 The heavy chain variable region CDR sequences of the anti-EGFR antigen binding domain (E-1G11 and E-6C4) and the anti-TROP2 antigen binding domain (T-6F7) in the anti-TROP2 / EGFR antibodies are listed as defined by Kabat numbering.

[0081] Figure 5 The heavy chain variable region CDR sequences of the anti-EGFR antigen binding domain (E-1G11 and E-6C4) and the anti-TROP2 antigen binding domain (T-6F7) in the anti-TROP2 / EGFR antibodies are listed as defined by Chothia numbering.

[0082] Figure 6 The CDR sequences of the common light chain as defined by Kabat and Chothia numbering are listed.

[0083] Figure 7 The anti-TROP2 / EGFR antibody heavy and light chain variable region sequences discussed in this disclosure are listed.

[0084] Figure 8 Additional amino acid sequences discussed in this disclosure are listed.

[0085] Fig.9A The killing efficacy of T-6F7-E-6C4-ADC on BxPC-3 cells+NCI-H520 cells, BxPC-3 cells, or NCI-H520 cells was shown.

[0086] Fig. 9B The killing efficacy of T-6F7-E-6C4-ADC (0.1 μg / mL) on BxPC-3 cells + NCI-H520 cells (after 72 hours of co-incubation) is shown.

[0087] Fig.10 Shown are the mean tumor volumes of different groups of B-NDG mice injected with NCI-H292 cells and treated with PBS, antibodies or ADCs.

[0088] Fig.11 Shown are the mean tumor volumes of different groups of B-NDG mice injected with NUGC-4 cells and treated with PBS, antibodies or ADC.

[0089] Figures 12A-12B Administration of ISO-ADC to B-hFcRn mice ( Fig. 12A ) or T-6F7-E-6C4-ADC( Fig. 12B ) after ADC and total antibody serum concentrations.

[0090] Fig.13 Shown are the ratios of free MMAE to ADC in human, cynomolgus monkey (Macaca fascicularis) or SD rat plasma at 0, 1, 2, 6, 8, 11 and 14 days after addition of T-6F7-E-6C4-ADC to plasma.

[0091] Figures 14A-14B The anti-TROP2 / EGFR bispecific antibody and ADC were shown to be effective in A431 cells ( Fig.14A ) or NCI-H292 cells ( Fig. 14B ). ISO-CPT2 (DAR8) was used as an isotype control. Gosartuzumab and cetuximab were used as controls.

[0092] Fig.15 Shown are the mean tumor volumes of different groups of B-NDG mice injected with patient-derived breast tumor fragments and treated with PBS or ADC.

[0093] Fig.16 Shown are the mean tumor volumes of different groups of B-NDG mice injected with SKOV-3 cells and treated with PBS or ADC.

[0094] Fig.17Shown are the mean tumor volumes of different groups of B-NDG mice injected with A431 cells and treated with PBS, antibody or ADC.

[0095] Fig.18A Shown are the mean tumor volumes of different groups of B-NDG mice injected with head and neck squamous cell carcinoma patient-derived tumor fragments and treated with T-6F7-E-6C4-CPT2 (DAR8). Saline was used as a control.

[0096] Fig.18B Shown are the mean tumor volumes of different groups of B-NDG mice injected with esophageal cancer patient-derived tumor fragments and treated with T-6F7-E-6C4-CPT2 (DAR8). Saline was used as a control.

[0097] Fig.18C and Fig.18D Shown are the mean tumor volumes of different groups of B-NDG mice injected with colorectal cancer patient-derived tumor fragments and treated with T-6F7-E-6C4-CPT2 (DAR8). Saline was used as a control.

[0098] Fig.18E and Fig.18F Shown are the mean tumor volumes of different groups of B-NDG mice injected with gastric cancer patient-derived tumor fragments and treated with T-6F7-E-6C4-CPT2 (DAR8). Saline was used as a control.

[0099] Figures 19A-19B Shown are the total antibody ( Fig.19A ) and CPT2( Fig.19B ) serum concentration.

[0100] Figures 19C-19D Shown are the total antibody ( Fig.19C ) and CPT2( Fig.19D ) in tumor tissue.

[0101] Figures 20A-20B showed that the addition of T-6F7-E-6C4-CPT2 (DAR4) ( Fig. 20A ) or T-6F7-E-6C4-CPT2(DAR8)( Fig. 20B ) at 0, 1, 2, 6, 8, 11 and 14 days after PCR. DETAILED DESCRIPTION

[0102] A bispecific antibody or its antigen-binding fragment is an artificial protein that can bind to two different epitopes (e.g., on two different antigens) simultaneously. In some embodiments, a bispecific antibody or its antigen-binding fragment can have two arms. Each arm can have a heavy chain variable region and a light chain variable region to form an antigen-binding domain (or antigen-binding region). In some embodiments, the bispecific antibody has a common light chain.

[0103] The present disclosure relates to anti-TROP2 / EGFR antibodies (eg, 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.

[0104] Anti-TROP2 / EGFR Antibodies

[0105] The epidermal growth factor receptor (EGFR, ErbBI or HER1) is a 170 kDa type 1 transmembrane glycoprotein encoded by the c-erbBl proto-oncogene. The EGFR 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). Mutations that affect EGFR expression or activity may lead to cancer in many cancer types. EGFR signaling is initiated by ligand binding, which subsequently induces conformational changes, homo- or heterodimerization of the receptor with other ErbB family members, and trans-autophosphorylation of the receptor, which initiates a signal transduction cascade that ultimately affects various cellular functions, including cell proliferation and survival. Increased EGFR expression or kinase activity has been associated with a range of human cancers, making EGFR an attractive target for therapeutic intervention. In non-small cell lung cancer, increases in EGFR gene copy number and protein expression have been associated with increased resistance to the EGFR tyrosine kinase inhibitor IRESSA. TM (gefitinib) was associated with a favorable response.

[0106] The binding of ligands such as EGF (epidermal growth factor) to EGFR stimulates receptor dimerization, autophosphorylation, activation of the cytoplasmic tyrosine kinase domain inside the receptor, and the initiation of multiple signal transduction and transactivation pathways involved in regulating DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of EGFR signaling may result in inhibition of one or more EGFRs. In some embodiments, EGFR ligands include EGF, TGFα, heparin-binding EGF (HB-EGF), amphiregulin (AR) and epiregulin (EPI).

[0107] 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.

[0108] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. It has a high structural sequence similarity with the epithelial adhesion molecule Epcam. TROP2 is a protein closely associated with tumors. It mainly promotes tumor cell growth, proliferation and metastasis by regulating calcium ion signaling pathways, cell cycle protein expression and reducing fibronectin adhesion. Studies have found 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, spread and other processes. In addition, it has been found that in breast cancer and other cancers, high expression of TROP2 is closely associated with more aggressive diseases and poor clinical prognosis of tumors.

[0109] TROP2 is an intracellular calcium signaling factor that is 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 has prognostic significance. Several ligands that interact with TROP2 have been proposed. TROP2 signals to cells through different pathways and is transcriptionally regulated by a complex network of several transcription factors. TROP2 expression in cancer cells is associated with drug resistance.

[0110] 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.

[0111] In some embodiments, the bispecific anti-TROP2 / EGFR antibodies described herein can be designed to have an IgG1 subtype structure containing a knob-in-hole (KIH) mutation, which can promote heterodimerization and avoid mispairing 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 control bispecific antibody.

[0112] In some embodiments, the bispecific anti-TROP2 / EGFR antibodies described herein can be conjugated to a therapeutic agent to form an antibody drug conjugate (ADC). In some embodiments, the drug-to-antibody ratio (DAR) of the ADC described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADC described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4 ...4, 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 .7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 ... 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 7.9 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 8.5 to about 8.4, about 8.6 to about 8.4, about 8.6 to about 8.4, about 8.7 to about 8.8, about 8.9 to about 8.9. .7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8.2 to about 8.3, about 7.5 to about 8.2, about 7.6 to about 8.2, about 7.7 to about 8.2, about 7.8 to about 8.2, about 7.9 to about 8.2, about 8.0 to about 8.2, about 8.1 to about 8.2, about 7.5 to about 8.1, about 7.6 to about 8.1, about 7.7 to about 8.1, about 7.8 to about 8.1, about 7.9 to about 8.1, about 8.0 to about 8.1, about 7.5 to about 8. 0, about 7.6 to about 8.0, about 7.7 to about 8.0, about 7.8 to about 8.0, about 7.9 to about 8.0, about 7.5 to about 7.9, about 7.6 to about 7.9, about 7.7 to about 7.9, about 7.8 to about 7.9, about 7.5 to about 7.8, about 7.6 to about 7.8, about 7.7 to about 7.8, about 7.5 to about 7.7, about 7.6 to about 7.7, or about 7.5 to about 7.6.

[0113] In some embodiments, the anti-TROP2 / EGFR ADC described herein can be 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. Effectively inhibit the growth of cancer cells in vitro. In some embodiments, in a xenograft mouse model, the anti-TROP2 / EGFR ADC described herein can be 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. The dosage level inhibits the growth of cancer cells in vivo (e.g., lung cancer, gastric cancer, or skin cancer).

[0114] In some embodiments, the anti-TROP2 / EGFR antibodies described herein have a common light chain. In some embodiments, the anti-TROP2 / EGFR antibodies include 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 embodiments, the anti-TROP2 / EGFR antibodies have a heavy chain variable region targeting EGFR (e.g., any of the VHs targeting EGFR described herein), a heavy chain variable region targeting TROP2 (e.g., any of the VHs targeting TROP2 described herein) and two identical common light chain variable regions.

[0115] As defined by Kabat numbering, the CDR sequences of the 1G11 antigen binding domain include CDRs of the heavy chain variable domain (SEQ ID NOs: 7-9) and CDRs of the light chain variable domain (SEQ ID NOs: 1-3). CDRs may also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown by SEQ ID NOs: 16-18, and the CDR sequences of the light chain variable domain are shown by SEQ ID NOs: 1-3. The human light chain variable region and the human heavy chain variable region of 1G11 are shown in SEQ ID NOs: 22 and 23, respectively.

[0116] As defined by Kabat numbering, the CDR sequences of the 6C4 antigen binding domain include CDRs of the heavy chain variable domain (SEQ ID NOs: 10-12) and 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 shown by SEQ ID NOs: 19-21, and the CDR sequences of the light chain variable domain are shown by SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region of 6C4 are shown in SEQ ID NOs: 22 and 24, respectively.

[0117] 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.

[0118] In some embodiments, 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), the heavy chain variable region comprising complementarity determining regions (CDRs) 1, 2, 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 CDR1, 2, 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 The CDR1 amino acid sequence is at least 80%, 85%, 90% or 95% identical to or consists of an amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VLCDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. Figure 4-6 Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in .

[0119] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO:9 with zero, one or two amino acid insertions, deletions or substitutions.

[0120] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions or substitutions.

[0121] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions or substitutions.

[0122] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions or substitutions.

[0123] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions or substitutions.

[0124] Insertions, deletions and substitutions may be within the CDR sequence, or at one or both termini of the CDR sequence.

[0125] In some embodiments, the anti-TROP2 / EGFR antibody contains a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising or consisting of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH sequence, and the light chain variable region comprising or consisting of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 23 or 24, and the selected VL sequence is SEQ ID NO: 22.

[0126] 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). CDRs can also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown by SEQ ID NOs: 13-15, and the CDR sequences of the light chain variable domain are shown by SEQ ID NOs: 1-3. The human light chain variable region and the human heavy chain variable region of 6F7 are shown as SEQ ID NOs: 22 and 25, respectively.

[0127] In addition, in some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein may also contain one, two or three heavy chain variable region CDRs (selected from SEQ ID NOs: 4-6 and SEQ ID NOs: 13-15) and / or one, two or three light chain variable region CDRs selected from SEQ ID NOs: 1-3.

[0128] In some embodiments, 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), the heavy chain variable region comprising complementarity determining regions (CDRs) 1, 2, 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 CDR1, 2, 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 The CDR1 amino acid sequence is at least 80%, 85%, 90% or 95% identical to or consists of an amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VLCDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. Figure 4 and 6 (Kabat CDR) and Figure 5 and 6 Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in (Chothia CDR).

[0129] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO:6 with zero, one or two amino acid insertions, deletions or substitutions.

[0130] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions or substitutions.

[0131] In some embodiments, the anti-TROP2 / EGFR antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: 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; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions or substitutions.

[0132] Insertions, deletions and substitutions may be within the CDR sequence, or at one or both termini of the CDR sequence.

[0133] In some embodiments, the anti-TROP2 / EGFR antibody contains a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising or consisting of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH sequence, and the light chain variable region comprising or consisting of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22.

[0134] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment may have 3 VH CDRs identical to the CDRs of any VH sequence as described herein. In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment may have 3 VL CDRs identical to the CDRs of any VL sequence as described herein.

[0135] The present disclosure also 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 Figure 4 , Figure 5 or Figure 6 The CDRs shown, or having Figure 7 When the polypeptide is paired with a corresponding polypeptide (eg, a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to TROP2 and / or EGFR.

[0136] Anti-TROP2 / EGFR antibodies can also be anti-TROP2 / EGFR antibody variants (including derivatives and conjugates) or antibody fragments of anti-TROP2 / EGFR antibodies. Additional anti-TROP2 / EGFR antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (multimers, such as bispecific) antibodies, human antibodies, chimeric antibodies (such as human-mouse chimeras), single-chain antibodies, antibodies produced in cells (i.e., intracellular antibodies) and antigen-binding fragments thereof. Anti-TROP2 / EGFR antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, anti-TROP2 / EGFR antibodies or antigen-binding fragments are IgG (e.g., IgG1) antibodies or antigen-binding fragments thereof.

[0137] Fragments of anti-TROP2 / EGFR antibodies are suitable for use in the provided methods as long as these antibodies retain the desired affinity and specificity for both TROP2 and EGFR.Thus, fragments of anti-TROP2 / EGFR antibodies will retain the ability to bind to TROP2 and EGFR.

[0138] Antibodies and their antigen-binding fragments

[0139] In some embodiments, multispecific anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies) include 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 can have various forms.

[0140] Typically, antibodies (also known as immunoglobulins) can be composed of two types of polypeptide chains (light chains and heavy chains). The non-limiting anti-TROP2 / EGFR antibodies disclosed herein can be complete four immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the anti-TROP2 / EGFR antibody can be any isotype, including IgM, IgG, IgE, IgA or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

[0141] The hypervariable regions, called complementarity determining regions (CDRs), form loops that comprise the major antigen binding surface of the antibody. The four framework regions adopt primarily a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are brought into close proximity by the framework regions and, together with the CDRs from the other chain, form an antigen binding domain.

[0142] Methods for identifying the CDR regions of an antibody by analyzing its amino acid sequence are well known, and a number of definitions of CDRs are in common use. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

[0143] CDR is important for recognizing antigen epitopes. As used herein, "epitope" is the smallest part of a target molecule that can be specifically bound by the antigen binding domain of an antibody. The minimum size of an epitope may be about three, four, five, six or seven amino acids, but these amino acids are not necessarily located in a continuous linear sequence of the primary structure of the antigen, because the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0144] In some embodiments, the anti-TROP2 / EGFR antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3 and IgG4) are highly conserved, except for their constant regions, especially their hinge and CH2 upper domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0145] Anti-TROP2 / EGFR antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camelids). Antigen binding domains or antigen binding fragments are antibody portions that retain the specific binding activity of intact antibodies, i.e., any portion of antibodies that can specifically bind to epitopes on target molecules of intact antibodies. It includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, anti-TROP2 / EGFR antibodies or antigen binding fragments thereof may include, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide including a binding domain as an antibody binding domain or a binding domain homologous thereto. Non-limiting examples of antigen binding domains include, for example, heavy and / or light chain CDRs of intact antibodies, heavy and / or light chain variable regions of intact antibodies, full-length heavy or light chains of intact antibodies, or separate CDRs of heavy or light chains from intact antibodies.

[0146] 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 the corresponding target antigen with different affinities.

[0147] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof may comprise a Figure 4-5 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 Figure 6 One, two or three light chain variable region CDRs.

[0148] In some embodiments, the anti-TROP2 / EGFR antibodies described herein can be conjugated to a therapeutic agent. The anti-TROP2 / EGFR antibody drug conjugates comprising an antibody or an antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids (such as DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker (e.g., a VC linker). Details of linkers for 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.

[0149] In certain embodiments, anti-TROP2 / EGFR antibody is a bispecific antibody. Bispecific antibodies can be prepared by maximizing the percentage of heterodimers recovered from recombinant cell culture by the interface engineering between a pair of antibody molecules. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (for example, tyrosine or tryptophan). Compensation "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule by replacing the large amino acid side chain with a smaller amino acid side chain (for example, alanine or threonine). This provides a mechanism for increasing the output of heterodimers relative to other unwanted end products such as homodimers. This method is described in, for example, WO 96 / 27011, which is incorporated by reference in its entirety.

[0150] Any anti-TROP2 / EGFR antibody or antigen-binding fragment thereof described herein may 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 solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). The conjugation of stabilizing molecules can increase the half-life of the anti-TROP2 / EGFR antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body) or prolong its biological activity in vitro or in vivo.

[0151] Anti-TROP2 / EGFR antibodies or their antigen-binding fragments can also have various forms. Many different forms of bispecific antibodies or their antigen-binding fragments are known in the art and are described in, for example, 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.

[0152] 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-HSA, or tandem-scFv. In some embodiments, the anti-TROP2 / EGFR antibody is VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, 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 antibody, TriBi small antibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, intracellular antibody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0153] In some embodiments, the anti-TROP2 / EGFR antibody can be a TrioMab. In TrioMab, the two heavy chains are from different species, wherein different sequences restrict the pairing of the heavy chain and the light chain.

[0154] In some embodiments, the anti-TROP2 / EGFR antibody has two different heavy chains and one common light chain. Heterodimerization of the heavy chains can be based on a knob-in-hole structure or some other heavy chain pairing technology.

[0155] In some embodiments, CrossMAb technology can be used to produce bispecific anti-TROP2 / EGFR antibodies. CrossMAb technology can be used to enforce correct light chain binding in bispecific heterodimer IgG antibodies, and this technology allows the production of various bispecific antibody forms, including bi(1+1), tri(2+1) and tetra(2+2) valence bispecific antibodies, and antibodies based on non-Fc tandem antigen binding fragments (Fab). These forms can be derived from any existing antibody pair using domain crossovers, without the need to identify common light chains, post-translational processing / in vitro chemical assembly or introduce a set of mutations to enforce correct light chain binding. The method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multi-specific antibodies." MAbs [monoclonal antibodies]. Vol. 8. No. 6. Taylor & Francis [Taylor Francis Group], 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are interchanged.

[0156] The anti-TROP2 / EGFR antibody can be a Duobody. In the IgG1 antibody, the Fab exchange mechanism naturally present in the IgG4 antibody is simulated with a controlled substance, and this mechanism is called controlled Fab exchange. This form can ensure the specific pairing between the heavy chain and the light chain.

[0157] In dual variable domain antibodies (DVD-Ig), additional VH and variable light (VL) domains are added to each N-terminus for bispecific targeting. This format is similar to IgG-scFv, but the added binding domains bind to their respective N-termini alone, rather than scFv binding to each heavy chain N-terminus.

[0158] In scFv-IgG, two scFvs are attached to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is therefore also called tetravalent. There is no heavy chain and light chain pairing problem in scFv-IgG.

[0159] In some embodiments, the anti-TROP2 / EGFR antibody may have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-termini of the heavy chains.

[0160] Anti-TROP2 / EGFR antibodies can also have a Fab-scFv-Fc format. In the Fab-scFv-Fc format, a light chain, a heavy chain, and a third chain containing an Fc region and scFv are assembled. It can ensure efficient manufacturing and purification.

[0161] In some embodiments, the anti-TROP2 / EGFR antibody can be TF. Three Fab fragments are linked by disulfide bonds. Two fragments target tumor-associated antigens (TAA), and one fragment targets a hapten. The TF form has no Fc region.

[0162] ADAPTIR has two scFvs bound to each side of the Fc region. It abandons the full IgG as the basis of its construction, but retains the Fc region to extend half-life and facilitate purification.

[0163] Dual-affinity retargeting proteins (DARTs) have two peptide chains connecting opposing fragments (so VLA to VHB and VLB to VHA), and a sulfide bond fusing them together at their C-termini. In DARTs, the sulfide bond improves stability, making it superior to BiTEs.

[0164] In DART-Fc, the Fc region is attached to the DART structure. It can be produced by assembling three chains, two of which are assembled via disulfide bonds, just like DART. One chain contains half of the Fc region, which will dimerize with the third chain, expressing only the Fc region. The addition of the Fc region enhances the half-life, resulting in longer effective concentrations, avoiding continuous IV.

[0165] In a tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created from half of the Fc region and will dimerize. This form binds to two targets bivalently, so it is a tetravalent molecule.

[0166] Tandem diabodies (TandAb) contain two diabodies. Each diabody consists of covalently bound VHA and VLB fragments and VHA and VLB fragments. The two diabodies are connected by a peptide chain. It can improve stability and make it better than a diabody composed of two scFvs. It has two bivalent binding sites.

[0167] scFv-scFv-toxin includes a toxin and two scFvs with a stabilizing linker. It can be used for specific delivery of payloads.

[0168] In some embodiments, the anti-TROP2 / EGFR antibody is a bispecific antibody. In some embodiments, the bispecific antibody in the present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce the affinity for cells with low expression levels of EGFR and TROP2, and increase the affinity for cells co-expressing EGFR and TROP2 to achieve enhanced targeting function.

[0169] 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 NO:27 and 28.

[0170] 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 comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27. In some embodiments, the anti-TROP2 / EGFR antibody comprises a mortar heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0171] Antibody and ADC characteristics

[0172] The anti-TROP2 / EGFR antibody may include an anti-EGFR antigen binding domain as described herein and any anti-TROP2 antigen binding domain.

[0173] The present disclosure provides anti-TROP2 / EGFR antibodies and antigen-binding fragments thereof that can specifically bind to EGFR. These anti-TROP2 / EGFR antibodies can 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 the binding between EGFR and TGFα. By blocking the binding between EGFR and EGF and / or the binding between EGFR and TGFα, anti-TROP2 / EGFR antibodies can inhibit EGFR-related signal transduction pathways, thereby treating cancer (e.g., NSCLC). In some embodiments, anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof can initiate CMC or ADCC.

[0174] Common techniques that can be used to measure the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be 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 can be used in a reaction of less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 The dissociation rate (koff) of the EGFR is greater than 0.01 s. -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0176] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×106 / Ms or less than 1×10 7 / Ms.

[0177] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof can be used at a concentration of less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 - 8 M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0178] Anti-TROP2 / EGFR antibodies or their antigen-binding fragments can also include an antigen-binding domain that can specifically bind to TROP2. Anti-TROP2 / EGFR antibodies or their antigen-binding fragments 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, anti-TROP2 / EGFR antibodies can also inhibit TROP2-related signal transduction pathways, thereby inhibiting cell proliferation, differentiation, and / or metastasis. Therefore, in some embodiments, anti-TROP2 / EGFR antibodies as described herein are TROP2 agonists. In some embodiments, anti-TROP2 / EGFR antibodies are TROP2 antagonists.

[0179] In some embodiments, the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof can be used in a time interval of less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 The dissociation rate (koff) of the antibody binds to TROP2 (eg, human TROP2, monkey TROP2, mouse TROP2, and / or chimeric TROP2). In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1, greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0180] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0181] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0182] Since anti-TROP2 / EGFR antibodies (e.g., bispecific antibodies) bind to both TROP2 and EGFR, for cells expressing both TROP2 and EGFR, the antibodies have a higher binding affinity to these cells. Avidity can be used to measure the binding affinity of antibodies to these cells. Avidity is the cumulative strength of multiple affinities of a single non-covalent binding interaction.

[0183] In some embodiments, 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.

[0184] Thermal stability can also be determined. The Tm of anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof as described herein can 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. Since IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, the first denaturation temperature Tm D1 and the second denaturation temperature Tm D2. The presence of these two peaks usually indicates the denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When there are two peaks, Tm is generally referred to as TmD2. Therefore, in some embodiments, the Tm D1 of the anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as 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 an anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as 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, Tm, TmD1, Tm D2 is 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.

[0185] In some embodiments, the anti-TROP2 / EGFR antibody or its antigen binding fragment can bind to human EGFR or monkey EGFR. In some embodiments, the anti-TROP2 / EGFR antibody or its antigen binding fragment cannot bind to human EGFR or monkey EGFR. In some embodiments, the anti-TROP2 / EGFR antibody or its antigen binding fragment can bind to human TROP2 or monkey TROP2. In some embodiments, the anti-TROP2 / EGFR antibody or its antigen binding fragment cannot bind to human TROP2 or monkey TROP2.

[0186] 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%, for example, 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%, for example, as measured by HPLC.

[0187] In some embodiments, the tumor growth inhibition rate or percentage (TGI%) of the anti-TROP2 / EGFR antibody, antigen-binding fragment or ADC is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the tumor growth inhibition percentage of the anti-TROP2 / EGFR antibody, antigen-binding fragment or ADC is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150%. TGI (%) can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 days after the start of treatment. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:

[0188] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0189] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day zero. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day zero.

[0190] In some embodiments, the anti-TROP2 / EGFR antibody, antigen binding fragment or ADC has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4.

[0191] 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 its antigen binding fragment is a Fab, Fab', F(ab')2 and Fv fragment. In some embodiments, the anti-TROP2 / EGFR antibody or its antigen binding fragment as described herein has an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has a LALA mutation (EU numbered L234A and L235A mutations), or a LALA-PG mutation (EU encoded L234A, L235A, P329G mutations).

[0192] Some other modifications may be made to the Fc region. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The resulting homodimeric fusion protein may have any increased in vitro and / or in vivo half-life.

[0193] In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0194] 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 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 within the Asn297 sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 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., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region may be further engineered to replace asparagine at position 297 with alanine (N297A).

[0195] In some embodiments, the major peak by HPLC-SEC after purification by Protein A-based affinity chromatography and / or size exclusion chromatography accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% of the protein complex described herein.

[0196] In some embodiments, the anti-TROP2 / EGFR ADC described herein has an IC50 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 1 μg / mL, less than 2 μg / mL, less than 1 μg / mL, less than 2 μg / mL, less than 1 μg / mL, less than 2 μg / mL, less than 3.5 μg / mL, less than 3 μg / mL, less than 3.5 μg / mL, less than 3 μg / mL, less than 3.5 μg / mL, less than 3.5 μg / mL, less than 3.5 μg / mL, less than 3.5 μg / mL, less than 3.5 μg / mL, less than 3.5 μg / mL, less than 3.5 μg / mL, less than 2.5 μg / mL, less than 2.5 μg / mL, less than 2.5 μg / mL, less than 2.5 μg / mL, less than 1 ...2.5 μg / mL, less than 2.5 μg / mL, less than 2.5 μg / mL, less than 2.5 μg / mL, less than 2.5 μg / mL, less 0.9 μg / mL, less than 0.8 μg / mL, less than 0.7 μg / mL, less than 0.6 μg / mL, less than 0.5 μg / mL, less than 0.4 μg / mL, less than 0.3 μg / mL, less than 0.2 μg / mL, less than 0.1 μg / mL, less than 0.05 μg / mL, less than 0.025 μg / mL, less than 0.0125 μg / mL, less than 0.005 μg / mL or less than 0.0025 μg / mL. In some embodiments, the IC50 of the anti-TROP2 / EGFR ADC described herein for killing cancer cells (e.g., HCC827 cells, NCI-H292 cells, A431 cells, or Panc 02.03 cells) in vitro is 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.

[0197] 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 cetuximab analogs. In some embodiments, the anti-TROP2 / EGFR antibodies or ADCs described herein have a higher endocytosis rate than DS-1062 analogs and / or gosatuzumab analogs. In some embodiments, the bispecific anti-TROP2 / EGFR antibodies or ADCs described herein have a higher endocytosis rate than ervantuzumab analogs. In some embodiments, the anti-TROP2 / EGFR ADCs described herein have a higher endocytosis rate than isotype control ADCs (e.g., ISO-CPT2).

[0198] In some embodiments, the half-life of an anti-TROP2 / EGFR ADC described herein is 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 1-20 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 the 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., ISO-ADC). In some embodiments, the clearance of the 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 embodiments, the PK profile of an anti-TROP2 / EGFR ADC described herein is determined based on the serum concentration of the administered anti-TROP2 / EGFR ADC or total antibodies derived from the anti-TROP2 / EGFR ADC.

[0199] In some embodiments, the ratio of free therapeutic agent (e.g., MMAE or CPTx) derived from the 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% after adding the anti-TROP2 / EGFR ADC to plasma (e.g., human, monkey, or rat plasma) for at least 1 day, 2 days, 6 days, 8 days, 11 days, 14 days, 18 days, or 21 days. In some embodiments, the final concentration of the ADC is about 10-500 μg / mL (e.g., 100 μg / mL).

[0200] Antibody Drug Conjugate (ADC)

[0201] The anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent (drug). The therapeutic agent can covalently or non-covalently bind 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 has a common light chain.

[0202] 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, dihydroxy anthracin, maytansines (such as DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like). Useful classes of cytotoxic, cytostatic or immunomodulatory agents include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors and alkylating agents.

[0203] In some embodiments, the therapeutic agent may include, but is not limited to, a cytotoxic agent (such as a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antimicrobial agent. In some embodiments, the therapeutic agent to be conjugated may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0204] Definitions of specific functional groups and chemical terms are described in more detail below.For purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed., inside cover, and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987.

[0205] Unless expressly stated otherwise, all ranges cited herein are inclusive. When a numerical range is listed, it is intended to include every value and sub-range within that range. For example, "C 1–6 " is intended to cover C1, C2, C3, C4, C5, C6, C 1–6 , C 1–5 , C 1–4 , C 1–3 , C 1–2 , C 2–6 , C 2–5 , C 2–4 , C 2–3 , C 3–6 , C 3–5 , C 3–4 , C 4–6 , C 4–5 and C 5–6 .

[0206] The compounds of the present disclosure or any formula depicting and describing the compounds may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the compounds of the present invention or any formula depicting and describing the compounds. Asymmetric centers present in the compounds of the present invention or any formula depicting and describing the compounds may have the (R) or (S) configuration independently of one another. When the bond to a chiral carbon is depicted as a straight line in a structural formula, or when there is no (R) or (S) chiral designation of a chiral carbon in the name of the compound, it is understood that both the (R) and (S) configurations of each such chiral carbon, and therefore each enantiomer or diastereomer and mixtures thereof, are included in the formula or name.

[0207] This disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, such as mixtures of enantiomers and / or diastereomers of all ratios. Therefore, enantiomers are the subject of this disclosure, which are enantiomer-pure forms (both left-handed and right-handed enantiomers), racemate forms, and mixtures of all ratios of two enantiomers. In the case of cis / trans isomerism, this disclosure includes mixtures of all ratios of cis-form and trans-form and these forms. If necessary, the mixture can be separated by conventional methods (e.g., by chromatography or crystallization), by synthesizing using stereochemically uniform starting materials or by stereoselective synthesis to prepare a single stereoisomer. Optionally, derivatization can be performed before separating stereoisomers. The separation of stereoisomer mixtures can be performed in an intermediate step during compound synthesis, or can be performed on the final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates, and if necessary, these crystalline products or crystalline intermediates are derivatized with reagents containing stereocenters of known configurations. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0208] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. Such compounds are termed "isotopic variants". The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds of the invention or any formula depicting and describing the compounds. Examples of isotopes suitable for inclusion in the compounds of the invention include, but are not limited to, isotopes of hydrogen, such as 2 H (i.e. D) and 3 H; carbon, such as 11 C. 13 C and 14 C; chlorine, such as 36 Cl; fluorine, such as18 F; iodine, such as 123 I and 125 I; Nitrogen, such as 13 N and 15 N; oxygen, such as 15 O. 17 O and 18 O; phosphorus, such as 32 P; and sulfur, such as 35 S. Certain isotopic variations of the compounds of the present disclosure or any formula depicting and describing the compounds, such as those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. In particular, compounds having the depicted structures (only when replaced by heavier isotopes, such as by deuterium ( 2 H or D) instead of hydrogen) may provide certain therapeutic advantages (e.g., greater metabolic stability, increased in vivo half-life or reduced dosage requirements) and may therefore be used in some specific situations. Isotopic variations of the compounds of the present disclosure or any formula depicting and describing the compounds can generally be prepared by techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and by using an appropriate isotopically labeled reagent in place of the unlabeled reagent previously used.

[0209] Compounds as provided herein are described with reference to both general formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a variety of different forms or derivatives, all of which are within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites, etc.

[0210] As used herein, unless otherwise indicated, the term "pharmaceutically acceptable salt" includes salts that retain the biological effectiveness of the free acid / base form of a particular compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids. In the case where the compounds disclosed herein contain one or more acidic or basic groups, the disclosure also includes its corresponding pharmaceutically acceptable salts. Therefore, the compounds of the present invention containing acidic groups (such as carboxyl groups) may exist in salt form and may be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts or ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts or salts with ammonia or organic amines (such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine or amino acids). These salts are easily obtained, for example, by reacting a compound with an acidic group with a suitable base (such as lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide or barium hydroxide). Other alkali salts of the compound disclosed herein include but are not limited to copper (I), copper (II), iron (II), iron (III), manganese (II) and zinc salts. The compound disclosed herein containing one or more basic groups (for example, a protonated group) can exist in salt form, and can be used in the form of an addition salt with an inorganic acid or an organic acid according to the disclosure. The example of suitable acid includes hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, 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 well known to persons skilled in the art. The salts formed are, among others, hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (methanesulfonate / mesylate), toluenesulfonate, carbonate, bicarbonate, formates, acetate, sulfoacetate, trifluoromethanesulfonate, oxalate, malonate, maleate, succinate, tartrate, malate, pamoate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate and glutamate. In addition, the stoichiometry of the salt formed by the compounds of the present disclosure can be an integer multiple or a non-integer multiple of 1.

[0211] Compounds of the present disclosure containing basic nitrogen-containing groups may be quaternized with agents such as C 1-4Alkyl halides, such as methyl, ethyl, isopropyl and tert-butyl chlorides, bromides and iodides; di-C 1-4 Alkyl sulfates, such as dimethyl, diethyl and diamyl sulfate; C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aryl C 1-4 Alkyl halides such as benzyl chloride and phenethyl bromide.

[0212] If the compounds of the present disclosure contain both acidic and basic groups in the molecule, the present disclosure also includes inner salts or betaines (zwitterions) in addition to the salt forms mentioned. The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example by contacting these with organic or inorganic acids or bases in solvents or dispersants, or by anion exchange or cation exchange with other salts. The present disclosure also includes all salts of the compounds of the present disclosure, which are not directly suitable for use in pharmaceuticals due to low physiological compatibility, but can be used as intermediates for chemical reactions or preparation of pharmaceutically acceptable salts, for example. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0213] The compounds disclosed herein or any formulas depicting and describing the compounds and pharmaceutically acceptable salts thereof may exist in unsolvated and solvated forms. As used herein, the term "solvate" refers to a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules. For example, when the solvent is water, the term "hydrate" is used.

[0214] Pharmaceutically acceptable solvates according to the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, for example D2O, d6-acetone, d6-DMSO.

[0215] Linker compounds

[0216] In some embodiments, 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 set of ligand compounds and therapeutic agent compounds, respectively, by, for example, coupling reactions, to link a ligand (e.g., an antibody or antigen-binding fragment thereof described herein) and a therapeutic agent (e.g., any therapeutic agent described herein) together to form a ligand-drug conjugate.

[0217] In some embodiments, the linker described herein is a compound having the formula:

[0218] QL

[0219] Formula (I),

[0220] or a pharmaceutically acceptable salt, solvate, stereoisomer or isotopic variant thereof, wherein Q represents a linking portion capable of being coupled 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 represents a linker portion capable of connecting Q to a therapeutic agent.

[0221] In some embodiments, the linking moiety (Q in formula (I)) has the following structure:

[0222]

[0223] In some embodiments, the linker moiety (L in formula (I)) has the formula:

[0224]

[0225] Wherein L1 is a polypeptide residue consisting of three to eight 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, wherein "-COOH" represents the carboxyl group of the amino acid residue at the C-terminus of the polypeptide residue;

[0226] L2 is absent or is attached to a monodentate, bidentate or tridentate hydrophilic group of a side chain carboxyl group of an amino acid residue of polypeptide residue L1, and L2 has -NHC(R L2a )(R L2b )(R L2c ) structure, where R L2a , R L2b and R L2c Each independently selected from H, -(CH2O)(CH2CH2O) m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O) m (CH2) p C(O)NHR L2d The group composed of R L2d is H or C optionally substituted by 1 to 6 hydroxyl groups 1-6 Alkyl, each m is independently an integer from 0 to 10, preferably from 0 to 4, such as 0, 1, 2, 3 or 4, particularly preferably m is 0, and each p is independently an integer from 1 to 4, such as 1, 2, 3 or 4; and

[0227] represents the N-terminal side of the polypeptide residue covalently attached to the linking moiety Q.

[0228] In some embodiments, polypeptide residue L1 is NH -Glu-Val-Ala- COOH In some embodiments, the hydrophilic group L2 has the following structure:

[0229]

[0230] Wherein "*" indicates the site of covalent attachment to polypeptide residue L1, e.g. NH -Glu-Val-Ala- COOH The side chain of the Glu residue in .

[0231] In some embodiments, the linker described herein is a compound having the structure:

[0232]

[0233] In some embodiments, the linker is a VC linker. Details of linkers for 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.

[0234] Therapeutic agents

[0235] In some embodiments, the therapeutic agent conjugated to the antibody or antigen-binding fragment thereof described herein is as described below.

[0236] In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, an analog or derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the following structure:

[0237]

[0238] wherein X is selected from the group consisting of -CH2-, O and S; and Y is selected from the group consisting of H, D and F.

[0239] 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:

[0240]

[0241] 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:

[0242]

[0243] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below:

[0244]

[0245] 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:

[0246]

[0247] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. For example, the auristatin can be an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 02 / 088172 and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530 ,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated herein by reference in its entirety and for all purposes.

[0248] Auristatins have been shown to interfere with microtubule dynamics and nuclear and cell division and have anticancer activity. Auristatins bind to tubulin and can exert cytotoxic or cytostatic effects on cancer cells. A number of different assays known in the art can be used to determine whether an auristatin or a resulting antibody drug conjugate exerts a cytostatic or cytotoxic effect on the desired cells.

[0249] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, trothamide, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine; nitrogen mustards, such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, mechlorethamineoxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas (such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); antibiotics (such as aclacinomycins, actinomycins, authramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carminomycin, chromomycin, actinomycin D, daunorubicin, detoximum, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin, Mycophenolic acid, noramycin, olivomycin, piroxicam, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, zinastatin, levorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folinic acid, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, Thiamiprine, thioguanine; pyrimidine analogs, such as cyclocytidine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as caprotestosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone; antiadreners, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; acetoglucuronolide;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidarol; nitroamine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK7; aproximate; sizoran; spirogermanium; tenuazonic acid acid); triaminoquinone; 2',2',2'-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannitol nitrogen mustard; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel (; Bristol-Myers Squibb Oncology (Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer (Antoine, France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novarutamide; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof. This definition also includes anti-hormonal agents for regulating or inhibiting the effect of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, 4 (5) -imidazoles, 4-hydroxytamoxifen, travoxifene, naloxifene (keoxifene), LY117018, onapristone and toremifene (Fareston) for inhibiting aromatase; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and any of the above-mentioned pharmaceutically acceptable salts, acids or derivatives. A detailed description of chemotherapeutic agents can be found in, for example, US20180193477 A1, which is incorporated by reference in its entirety.

[0250] Linker-Therapeutic Agent Compounds

[0251] In some embodiments, 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.

[0252] In some embodiments, the linker-therapeutic agent compound has the structure:

[0253]

[0254] In some embodiments, the linker-therapeutic agent compound has the structure:

[0255]

[0256] In some embodiments, 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 embodiments, the antibody drug conjugate has the following structure:

[0257]

[0258] Wherein n=1-8. In some embodiments, n=1-8. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8. In some embodiments, n is about 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8. In some embodiments, n is an integer multiple or non-integer multiple of 1.

[0259] In some embodiments, the anti-TROP2 / EGFR antibody is coupled 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 coupled to the drug via a non-cleavable linker (e.g., an MCC linker formed using SMCC or sulfo-SMCC). A skilled person can easily select a suitable linker for a given ADC based on the knowledge in the art and taking into account relevant factors such as the attachment site of the anti-TROP2 / EGFR antibody, any structural constraints of the drug, and the hydrophobicity of the drug (e.g., see Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer. [Springer Press] for a review). In certain embodiments, a variety of specific linker-toxin combinations have been described and can be used to prepare ADCs with the anti-TROP2 / EGFR antibodies or antigen-binding fragments thereof described herein. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins (such as MMAE and MMAF), camptothecins (such as SN-38), duocarmycins and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicin and doxorubicin; disulfide-based linkers with maytansines (such as DM1 and DM4), and bismaleimidotrioxyethylene glycol (BMPEO)-based linkers with 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. US2015 / 0374847 and US20180193477 A1; which are incorporated herein by reference in their entirety.

[0260] According to the desired drug and the selected linker, those skilled in the art can choose a suitable method to couple them together. For example, some conventional coupling methods (such as amine coupling methods) can be used to form the desired drug-linker complex, which still contains reactive groups for conjugation with anti-TROP2 / EGFR antibodies or their antigen-binding fragments by covalent bonds. In some embodiments, drug-maleimide complexes (i.e., maleimide-connected drugs) can be used for payloads carrying reactive groups in the present disclosure. In ADC preparation, the most common reactive group that can be combined with a thiol group is maleimide. In addition, organic bromides and iodides are also often used.

[0261] 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 (e.g., see Bioconjugate Techniques (GT Hermanson, 2013, Academic Press). For example, conjugation can be achieved by: (1) reacting a nucleophilic or electrophilic group of an antibody with a divalent linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, which is then reacted with an activated drug moiety D; or (2) reacting a nucleophilic or electrophilic group of a drug moiety with a linker reagent to form a drug-linker intermediate DL via a covalent bond, which is then reacted with a nucleophilic or electrophilic group of an antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the anti-TROP2 / EGFR ADCs described herein. Various prepared linkers, linker components, and toxins are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith and March, 2006, 6th ed., Wiley); Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (GT Hermanson, 2013, Academic Press); US20210379193 A1 and US20180193477 A1, which are incorporated herein by reference in their entirety. In addition, a number of preformed drug-linkers suitable for reacting with a selected anti-TROP2 / EGFR antibody or antigen-binding fragment are also commercially available, for example, linker-toxins (including DM1, DM4, MMAE, MMAF or duocarmycin SA) are available from Creative BioLabs, Inc. (Shirley, NY).

[0262] Several specific examples of methods for preparing anti-TROP2 / EGFR ADCs are known in the art and described in U.S. Pat. No. 8,624,003 (pot method), U.S. Pat. No. 8,163,888 (one-step method), and U.S. Pat. No. 5,208,020 (two-step method) and US20180193477 A1, which are incorporated herein by reference in their entirety. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer.

[0263] The drug loading is represented by the number of drug moieties per antibody in the ADC molecule. For some antibody drug conjugates, the drug loading may be limited by the number of attachment sites on the antibody. For example, in the case where the attachment is a cysteine ​​thiol, as in certain exemplary embodiments described herein, the drug loading may be in the range of 0 to 8 drug moieties per antibody. In certain embodiments, a higher drug loading (e.g., p≥5) may result in aggregation, insolubility, toxicity, or cell permeability loss of certain antibody drug conjugates. In certain embodiments, the average drug loading of the anti-TROP2 / EGFR antibody drug conjugate 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 certain antibody drug conjugates, the optimal ratio of the drug moiety per antibody can be about 4. In some embodiments, the DAR of the anti-TROP2 / EGFR ADC composition is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the anti-TROP2 / EGFR ADC composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0264] In some embodiments, anti-TROP2 / EGFR antibody variants having a carbohydrate structure are provided that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody 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 within the Asn297 sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues, or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-TROP2 / EGFR antibody may be further engineered to replace asparagine at position 297 with alanine (N297A).

[0265] In some embodiments, in order to improve production efficiency by avoiding Fab-arm exchange, the Fc region of the anti-TROP2 / EGFR antibody or its antigen-binding fragment is further engineered to replace the serine (S228P) at position 228 (EU numbering) of IgG4 with proline. A detailed description of the S228 mutation is described in, for example, Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. [As demonstrated by the combination of novel quantitative immunoassays and physiological matrix preparation, the S228P mutation prevents in vivo and in vitro IgG4 Fab arm exchange]" Journal of Biological Chemistry [Journal of Biological Chemistry] 290.9 (2015): 5462-5469, which is incorporated by reference in its entirety.

[0266] In certain embodiments, the methods described herein are designed to prepare bispecific anti-TROP2 / EGFR antibodies. Bispecific anti-TROP2 / EGFR antibodies can be prepared by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). Compensation "cavities" of the same or similar size as large side chains are produced on the interface of the second antibody molecule by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products such as homodimers. This method is described in, for example, WO 96 / 27011, which is incorporated by reference in its entirety.

[0267] In some embodiments, knob-into-hole (KIH) technology can be used, which involves engineering the CH3 domain to create a "knob" or a "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. Vol. 7 No. 1 Taylor & Francis, 2015, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has T366W, and / or S354C (knob) substitutions (EU numbering), and the other heavy chain has Y349C, T366S, L368A, and / or Y407V (hole) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the following substitutions: Y349C and T366W (EU numbering). Another heavy chain may have one or more of the following substitutions: E356C, T366S, L368A, and Y407V (EU numbering). In addition, substitutions (-ppcpScp-->-ppcpPcp-) may also be introduced in the hinge region of the two substituted IgGs.

[0268] Recombinant vector

[0269] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that these host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and the production of anti-TROP2 / EGFR antibody polypeptides or fragments thereof by recombinant technology.

[0270] As used herein, "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into a host cell."expression vector" can deliver one or more polynucleotides of interest in a host cell introduced into an expression vector and express them as encoded polypeptides.Therefore, in an expression vector, the polynucleotide of interest is positioned in a vector for expression by being operably connected to regulatory elements such as promoters, enhancers and / or poly-A tails, and these regulatory elements are located in the vector or in the genome of the host cell, at the integration site of the polynucleotide of interest or near the integration site or on both sides of the integration site, so that the polynucleotide of interest will be translated in the host cell introduced into the expression vector.

[0271] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0272] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation generally occurs only in complementing viral packaging cells. Suitable systems are disclosed in, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked", as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA can be increased by coating the DNA on biodegradable beads, which can be efficiently transported into cells.

[0273] For expression, the DNA insert sequence comprising the polynucleotide encoding the polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λ 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 the promoters of the retroviral LTR (to name a few). Other suitable promoters are known to the skilled person. 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 transcript expressed by the construct may include a translation initiation codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately located at the end of the polypeptide to be translated.

[0274] As noted, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia 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 HK293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0275] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be apparent to the skilled artisan.

[0276] Suitable non-limiting bacterial promoters include E. coli lacI promoter and lacZ promoter, T3 and T7 promoter, gpt promoter, lambda PR and PL promoter, and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, SV40 early and late promoters, promoters of retroviral LTR (such as the promoter of Rous sarcoma virus (RSV)), and metallothionein promoters, such as mouse metallothionein-I promoter.

[0277] In the yeast Saccharomyces cerevisiae, a variety of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For review, 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).

[0278] The construct can be introduced into the host cell 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 [Basic Methods in Molecular Biology] (1986), which is incorporated herein by reference in its entirety.

[0279] The transcription of the DNA encoding the anti-TROP2 / EGFR antibody disclosed herein by higher eukaryotic organisms can be increased by inserting an enhancer sequence into a vector. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer (which is located on the rear side of the replication origin of base pairs 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the rear side of the replication origin, and an adenovirus enhancer.

[0280] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide or a heterologous signal.

[0281] Polypeptides (e.g., anti-TROP2 / EGFR antibodies) can be expressed in a modified form, such as a fusion protein (e.g., GST fusion) or with a histidine tag, and can include not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in host cells during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to the polypeptide to promote purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to the polypeptide to cause secretion or excretion, thereby improving stability and promoting purification, is especially a familiar and conventional technique in the art.

[0282] The present disclosure also provides a nucleic 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 nucleotide sequence as described herein, And provided is 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.

[0283] The present disclosure also provides a nucleic 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 nucleotide sequence as described herein, And provided is 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 as described herein.

[0284] In some embodiments, the disclosure relates to a nucleotide sequence encoding any peptide described herein, or to any amino acid sequence encoded by any nucleotide sequence as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0285] 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.

[0286] 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.

[0287] In order to determine the percentage identity of two amino acid sequences or two nucleotide sequences, for the best comparison purpose, the sequences are compared (for example, a gap can be introduced in one or both of the first and second amino acid or nucleotide sequences for the best comparison, and for the purpose of comparison, non-homologous sequences can be ignored). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the amino acid residue or nucleotide identical to the corresponding position in the second sequence, these molecules are identical at this position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percentage identity between the two sequences is a function of the number of identical positions shared by these sequences, wherein the number of gaps and the length of each gap that need to be introduced to achieve the best comparison of the two sequences are considered. For example, the sequence comparison between the two sequences and the determination of the percentage identity can be realized using the Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0288] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine the percentage of sequence homology is known in the art. In certain embodiments, conservative amino acid residues such as leucine and isoleucine with similar physicochemical properties (homology percentage) can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology is higher than the percent identity.

[0289] The present disclosure provides one or more nucleic acids encoding any polypeptide as described herein. In some embodiments, nucleic acids (e.g., cDNA) include polynucleotides encoding heavy chain polypeptides as described herein. In some embodiments, nucleic acids include polynucleotides encoding light chain polypeptides as described herein. In some embodiments, nucleic acids include polynucleotides encoding scFv polypeptides as described herein.

[0290] In some embodiments, a vector can have two nucleic acids as described herein, wherein the vector encodes a VL region and a VH region that bind together to EGFR. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein the pair of vectors encodes a VL region and a VH region that bind together to EGFR.

[0291] In some embodiments, the vector comprises two nucleic acids as described herein, wherein the vector encodes a VL region and a VH region that bind together to TROP2. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein the pair of vectors encodes a VL region and a VH region that bind together to TROP2.

[0292] Treatment

[0293] The methods described herein include methods for treating disorders associated with cancer. Generally, these methods include administering a therapeutically effective amount of an anti-TROP2 / EGFR antibody or anti-TROP2 / EGFR antibody drug conjugate as described herein to a subject in need or determined to be in need of such treatment.

[0294] As used in this context, "treating" means improving at least one symptom of a disorder associated with cancer. Cancer typically results in death; therefore, 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 disorder associated with cancer will result in a decrease in the number of cancer cells and / or relief of symptoms.

[0295] As used herein, the term "cancer" refers to cells with autonomous growth ability, i.e., abnormal states or conditions characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasion stage. As used herein, the term "tumor" refers to cancer cells, such as cancer cell clusters. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems (such as those affecting the lungs, breasts, thyroids, lymph nodes, gastrointestinal tracts, and genitourinary tracts), and adenocarcinomas (including malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, small intestinal cancers, and esophageal cancers). In some embodiments, the agents described herein are designed to treat or diagnose cancers of subjects. The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancers, gastrointestinal cancers, urogenital cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include those formed by cervical, lung, prostate, breast, head and neck, colon and ovarian tissue. The term also includes carcinosarcoma, for example, these carcinosarcoma include malignant tumors composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or a cancer in which tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a mesenchymal derived malignant tumor.

[0296] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0297] In one aspect, the disclosure also provides methods for treating cancer in a subject, methods for reducing the rate at which a subject's tumor volume increases over time, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0298] In one aspect, the disclosure features a method comprising 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 having, or identified or diagnosed as having, a cancer, e.g., a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung cancer), 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.

[0299] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe a human or non-human animal to which treatment is provided according to the method of the present invention. Veterinary and non-veterinary applications are encompassed by the present invention. Human patients can be adults or minors (e.g., people under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Including, for example, 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), horses, dogs, felines, bovines and other domestic animals, farm animals, and zoo animals.

[0300] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer.A variety of methods known in the art can be used to identify patients suffering from cancer.

[0301] As used herein, "effective amount" means an amount or dosage sufficient to achieve a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease (e.g., cancer). The effective amount will depend on, for example, the age and weight of the subject to whom 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 a composition thereof is to be administered, the severity of the symptoms and the route of administration, and thus administration can be determined on an individual basis.

[0302] The effective amount can be administered once or multiple times. For example, the effective amount of an anti-TROP2 / EGFR antibody, an anti-TROP2 / EGFR antigen-binding fragment, or an anti-TROP2 / EGFR antibody-drug conjugate is enough to improve, terminate, stabilize, reverse, inhibit, slow down and / or delay the patient's autoimmune disease or cancer progression, or is enough to improve, stop, stabilize, reverse, slow down and / or delay the amount of in vitro cell (e.g., biopsy cells, any cancer cell described herein, or cell line (e.g., cancer cell line)) proliferation. As understood in the art, the effective amount of an anti-TROP2 / EGFR antibody, an anti-TROP2 / EGFR antigen-binding fragment, or an anti-TROP2 / EGFR antibody-drug conjugate may be different, particularly depending on the patient's medical history and other factors, such as the type (and / or dosage) of the agent used.

[0303] The effective amount and schedule for administering the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, polynucleotides encoding anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antibody drug conjugates, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the purview of those skilled in the art. Those skilled in the art will appreciate that the dosage that must be administered will vary depending on, for example, the mammal to which the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, polynucleotides encoding anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antibody drug conjugates, and / or compositions disclosed herein will be administered, the route of administration, the specific type of agent or composition disclosed herein used, and other drugs being administered to the mammal.

[0304] The typical daily dosage 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 embodiments, the dosage may be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In some embodiments, the dosage may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dosage is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg or 0.1 mg / kg.

[0305] In any of the methods described herein, 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 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 a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) can be administered to the subject.

[0306] In some embodiments, 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 of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, or anti-TROP2 / EGFR ADC). In some embodiments, one or more additional therapeutic agents and 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 so that there is overlap in the biological activity period of the one or more additional therapeutic agents and at least one anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding fragment, or anti-TROP2 / EGFR ADC in the subject.

[0307] In some embodiments, 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 of an anti-TROP2 / EGFR antibody, anti-TROP2 / EGFR antigen-binding antibody fragment, or anti-TROP2 / EGFR ADC) can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein for diagnosing or following up on the effectiveness of treatment (e.g., observing at least one symptom of cancer). As described herein, a skilled medical professional can also change the type and amount (e.g., increase or decrease) of the anti-TROP2 / EGFR antibody or anti-TROP2 / EGFR antigen-binding antibody fragment, anti-TROP2 / EGFR antibody drug conjugate (and / or one or more additional therapeutic agents) administered to the subject, and can also adjust (e.g., increase or decrease) the dose 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 the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0308] In some embodiments, one or more additional therapeutic agents may be administered to a subject. Additional therapeutic agents may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, TROP2 inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) inhibitors and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, additional therapeutic agents are indoleamine 2,3-dioxygenase-1 (IDO1) inhibitors (e.g., edostat).

[0309] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: a HER3 inhibitor, a LSD1 inhibitor, a MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an activated hedgehog signaling pathway inhibitor, and an agent that selectively degrades the estrogen receptor.

[0310] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: trabectedin, nab-paclitaxel, trebanib, pazopanib, cediranib, palbociclib, irolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, terolimus, axitinib, irolimus, sorafenib, vemurafenib (Votrient), pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate and enzatalen.

[0311] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0312] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0313] 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.

[0314] Pharmaceutical compositions and routes of administration

[0315] Also provided herein is a pharmaceutical composition containing at least one (e.g., one, two, three, or four) of an anti-TROP2 / EGFR antibody (e.g., a bispecific antibody), an anti-TROP2 / EGFR antigen-binding fragment, or an anti-TROP2 / EGFR antibody-drug conjugate as described herein. The pharmaceutical composition can be formulated in any manner known in the art.

[0316] The pharmaceutical composition is formulated to be compatible with its expected route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition can include a sterile diluent (e.g., sterile water or sterile saline), fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate or phosphate) and isotonic agents (e.g., sugars, e.g., dextrose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride) or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition can be prepared and packaged in an ampoule, a disposable syringe or a multi-dose bottle. If necessary (e.g., in the form of an injectable formulation), appropriate fluidity can be maintained, for example, by using a coating such as lecithin or a surfactant. The absorption of anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, or anti-TROP2 / EGFR ADCs can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems (which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0317] A composition containing one or more of any of the anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments, anti-TROP2 / EGFR antibody drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0318] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). The LD50 (lethal dose to 50% of the population) and ED50 (therapeutically effective dose to 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indexes are preferred. When an agent exhibits undesirable side effects, care should be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0319] The data obtained from cell culture assays and animal studies can be used to formulate an appropriate dose of any given agent for use in a subject (e.g., a human). The therapeutically effective amount of an anti-TROP2 / EGFR antibody, an anti-TROP2 / EGFR antigen-binding fragment thereof, or an anti-TROP2 / EGFR ADC will be a subject (e.g., a human subject identified as having cancer) or a subject identified as having a risk of developing a disease (e.g., a subject previously suffering from cancer but now cured) to treat the subject's disease (e.g., kill cancer cells), reduce the severity, frequency, and / or duration of one or more symptoms of the subject (e.g., a human). Methods known in the art can be used, as well as by observing one or more symptoms of the subject (e.g., a human), and the effectiveness and administration of any anti-TROP2 / EGFR antibody, an anti-TROP2 / EGFR antigen-binding fragment thereof, or an anti-TROP2 / EGFR ADC described herein can be determined by a health care professional or a veterinary professional. Certain factors may affect the dosage and time course required for the effective treatment of a subject (e.g., the severity of the disease or disorder, previous treatment, the general health status and / or age of the subject, and other diseases present).

[0320] Exemplary dosages 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 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 between 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 dosage level is about 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg or 30mg / kg. In some embodiments, the dosage levels described herein will not induce severe toxic effects to the subject. Although these dosages cover a wide range, it will be appreciated by those of ordinary skill in the art that therapeutic agents differ in their efficacy and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development phase) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, administration time, administration route, excretion rate and the in vivo half-life of the therapeutic agent.

[0321] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods of making anti-TROP2 / EGFR antibodies, anti-TROP2 / EGFR antigen-binding fragments thereof, or anti-TROP2 / EGFR ADCs for various uses as described herein.

[0322] Examples

[0323] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0324] Example 1. Preparation and analysis of anti-TROP2 / EGFR bispecific antibodies

[0325] Provided herein are bispecific antigen binding molecules targeting TROP2 and EGFR. These antigen binding molecules are hereinafter referred to as anti-TROP2 / EGFR bispecific antibodies.

[0326] Preparation of anti-TROP2 / EGFR bispecific antibody

[0327] The anti-TROP2 / EGFR bispecific antibody can 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 structure (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-transduced with three vectors, including a first vector encoding an anti-TROP2 binding arm heavy chain, a second vector encoding an anti-EGFR binding arm heavy chain, and a third vector encoding a common light chain. After 14 days of culture, the cell supernatant was collected and purified by protein A affinity chromatography.

[0328] Various methods can be used to reduce the chance of mispairing between the two heavy chains. For example, a knob-and-hole structure mutation is introduced into the Fc region 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. In order to verify the binding affinity of the bispecific antibodies, anti-TROP2 or anti-EGFR control bispecific antibodies are also generated, wherein one arm of the control bispecific antibody recognizes TROP2 or EGFR and the other arm recognizes CD28. Similar methods are used to generate these control bispecific antibodies, for example, by immunizing RenLite TM Mouse to obtain VH sequences. Exemplary control bispecific antibodies were named T-6F7-CD28, CD28-T-6F7, CD28-E-1G11, CD28-E-6C4, E-1G11-CD28, and E-6C4-CD28.

[0329] Knob-hole structural mutations were introduced into all bispecific antibodies. For example, in T-6F7-E-1G11, the heavy chain constant region of T-6F7 includes a knob mutation, and the heavy chain constant region of E-1G11 includes a hole mutation. In T-6F7-CD28, the heavy chain constant region of T-6F7 includes a knob mutation, and the heavy chain constant region of CD28 includes a hole mutation. Figure 1 Exemplary antibody structures are shown in , where target 1 and target 2 can be TROP2 and EGFR, respectively; EGFR and TROP2, respectively; TROP2 and CD28, respectively; CD28 and TROP2, respectively; EGFR and CD28, respectively, or CD28 and EGFR.

[0330] The constant region may also include one or more mutations. For example, when the SI mutation (EU numbering: S239D and I332E mutations) was introduced into the Fc region of T-6F7, the resulting antibody was named T-6F7-SI.

[0331] 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.

[0332] Internalization of antibodies targeting TROP2 and / or EGFR

[0333] 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. The mean fluorescence intensity (MFI) was measured using a flow cytometer. The endocytosis rate of the antibody was calculated. For isotype control (ISO), human IgG1 protein (hIgG1) was used. The results are shown in the table below.

[0334] Table 1

[0335]

[0336] Cetuximab is a chimeric monoclonal IgG1 antibody targeting EGFR that was originally developed by ImClone Systems and marketed as Erbitux by Merck KGaA in 2003 as a monotherapy. TM First launched in Switzerland, and used in combination with irinotecan for the treatment of 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.

[0337] Sacilotuzumab-SI analog is a humanized anti-TROP2 monoclonal IgG1 antibody with SI mutation in the constant region. The heavy chain and light chain sequences of Sacilotuzumab-SI analog are shown as SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0338] The results showed that the internalization 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. In addition, the internalization 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.

[0339] Purity analysis of antibodies

[0340] 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).

[0341] Non-reducing SDS-PAGE was performed using 4%-12% acrylamide gels. The protein samples were prepared as follows. First, 2.4 μL of protein sample was mixed with 6 μL Tris-glycine SDS sample buffer (2×) (Invitrogen; Catalog No.: 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.

[0342] In the SEC-HPLC method, antibody samples were diluted to 1 mg / mL with PBS (pH 7.2-7.4, 0.01 M) and chromatographically analyzed using an Agilent 1290 chromatography system (with Xbridge TM Protein BEH SEC column ( Waters Corporation)). The following parameters were used: mobile phase: 25mmol / L phosphate buffer (PB) + 300mmol / L NaCl, pH 6.8; flow rate: 1.8mL / min; column temperature: 25°C; detection wavelength: 280nm; injection volume: 10mL; sample tray temperature: about 4°C; and run time: 7 minutes. The following table summarizes the results.

[0343] Table 2. Non-reducing SDS-PAGE and SEC-HPLC analysis results

[0344]

[0345]

[0346] Binding affinity of anti-TROP2 / EGFR bispecific antibodies

[0347] Using a Biacore with a pre-immobilized Protein A sensor chip TM (Biacore, Inc., Piscataway, NJ) 8K biosensor, binding affinity of anti-TROP2 / EGFR bispecific antibody to human TROP2, human EGFR, monkey TROP2, and monkey EGFR was verified by surface plasmon resonance (SPR).

[0348] Specifically, hTROP2-His (ACRO Biosystems Inc., catalog number: TR2-H5223), hEGFR-His (ACRO Biosystems Inc., catalog number: EGR-H5222), fasTROP2-His (ACRO Biosystems Inc., catalog number: TR2-R52H3) and fasEGFR-His (ACRO Biosystems Inc., catalog number: EGR-C52H1) were diluted to 200 nM with 1×HBS-EP+ buffer (pH 7.4). The purified antibodies were injected into the Biacore TM 8K biosensor for about 50 seconds to reach the desired protein density (e.g., about 350 response units (RU)), and then the diluted antigen protein with a concentration of 200nM was injected at 30mL / min for 180 seconds. Dissociation was monitored for 400 seconds. After the last injection of each titration, the chip was regenerated for 30 seconds with glycine solution (pH 1.5) at 30mL / min.

[0349] By using Biacore TM 8K Evaluation Software 3.0 fits the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) to simultaneously obtain the kinetic association rate (kon) and dissociation rate (koff). Affinity was derived from the quotient of the kinetic rate constants (KD = koff / kon).

[0350] The same approach with appropriate adjustments of parameters (eg, antibody concentration) was performed for each antibody tested, as would be understood by one of ordinary skill in the art. The following table summarizes the results for the antibodies tested.

[0351] Table 3. Affinity test results

[0352]

[0353]

[0354] The results showed that the anti-TROP2 / EGFR bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 had good binding affinity to human TROP2, monkey TROP2, human EGFR and monkey EGFR.

[0355] Stability of anti-TROP2 / EGFR bispecific antibodies

[0356] The anti-TROP2 / EGFR bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 were diluted in a pH 6.0 buffer (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween TM 80) was diluted to 5 mg / mL. The diluted antibodies were stored in sealed Eppendorf tubes 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, and their thermal stability was evaluated. Alternatively, the bispecific antibodies were also incubated under low pH conditions. In particular, the antibodies were incubated in 1 mol / L acetic acid at pH 3.5 for 0 hours or 6 hours to determine their stability under acidic conditions.

[0357] 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 the change in antibody purity by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area to the sum of all peak areas (purity, %)); (3) detecting the change in the apparent hydrophobicity of the antibody by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (expressed as the retention time of the main peak (HIC, min)); (4) detecting the change 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 to the sum of all peak areas (purity, %)); (5) detecting the charge variants in the antibody by capillary isoelectric focusing (Cief) (expressed as the percentage of the main component, acidic component and basic component).

[0358] In the SEC-UPLC experiment, the antibody sample was diluted to 1 mg / mL with purified water and chromatographed using an Agilent 1290 chromatography system (with Xbridge TM Protein BEH SEC column ( Waters Corporation). 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: about 6°C; and run time: 7 minutes.

[0359] In the HIC-HPLC experiments, an Agilent 1260 chromatography system (with ProPac TM HIC-10 column (4.6×250 mm, Thermo Scientific) was connected), and the sample was diluted to 0.5 mg / mL using 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: about 6° C.; and run time: 45 minutes.

[0360] In the Cief experiment, the Maurice Cief method development kit (Protein Simple, catalog number: PS-MDK01-C) was used for sample preparation. In particular, 40 μg of protein sample was mixed with the following reagents in the kit: 1 mL Maurice Cief Pi marker-4.05, 1 mL Maurice Cief Pi marker-9.99, 35 mL 1% methylcellulose solution, 2 mL Maurice Cief 500 mM arginine, 4 mL ampholytes (Pharmalyte pH range of 3-10) and water (added to make the final volume 100 mL). On the Maurice analyzer (Protein Simple, Santa Clara, California), Maurice Cief Cartridges (PS-MC02-C) were used to generate imaging capillary isoelectric focusing spectra. The samples were focused for a total of 10 minutes. The analysis software installed on the instrument was used to integrate the absorbance of the focused protein at 280 nm.

[0361] In the CE-SDS(NR) experiment, Maurice (Protein simple, Maurice TM) and Maurice CE-SDS size application kit (Protein simple, catalog number: PS-MAK02-S). 54mL sample buffer, 6mL antibody sample, 2.4mL 25× internal standard, 3mL 250nM iodoacetamide (Sigma, catalog number: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3000rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The sample was then cooled to room temperature and then centrifuged at 10000rpm for 3 minutes. The supernatant sample preparation was then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage: 4.6kV; injection time: 20 seconds; separation voltage: 5.75kV; and separation time: 40 minutes.

[0362] The 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 physical and chemical properties than other tested antibodies.

[0363] Table 4

[0364]

[0365] Example 2. Antibody Drug Conjugates

[0366] After protein A purification, the bispecific antibodies T-6F7-E-6C4 and T-6F7-E-1G11 were dialyzed and concentrated in PBS buffer by ultrafiltration. The concentration was determined by UV absorption. These antibodies were used for subsequent antibody drug conjugation reactions.

[0367] Conjugation of antibodies to drug molecules

[0368] Purified antibodies were conjugated to MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0369] For the names of antibody drug conjugates, when the antibody is coupled to MMAE, "ADC" is added directly after the antibody name. For example, if T-6F7-E-6C4 with an IgG1 constant region is coupled to MMAE, it is named T-6F7-E-6C4-ADC. Similarly, if a cetuximab analog with an IgG1 constant region is coupled to MMAE, it is named cetuximab analog-ADC. If an antibody portion analog of MRG003 with 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, resulting in names such as saccharotuzumab-SI analog-ADC, DS-1062-SI analog (DXd), and DS-1062-SI analog (MMAE).

[0370] HIC-HPLC is used to detect the conjugation of antibodies and drug molecules. In the HIC-HPLC experiment, an Agilent 1260 chromatography system (with ProPac TM HIC-10 column (4.6×250 mm, Thermo Scientific) was connected), and the sample was diluted to 0.5 mg / mL using mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile pH 6.5; mobile phase B: 0.1 M PB, 10% acetonitrile pH 6.5; flow rate: 0.8 mL / min; gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30° C.; detection wavelength: 280 nm; injection volume: 10 mL; sample tray temperature: about 6° C.; and run time: 45 minutes.

[0371] For isotype control, human IgG1 was coupled to MMAE to form isotype-ADC (ISO-ADC). HIC-HPLC detection results showed that the drug-antibody ratio (DAR) of ADC was about 4.

[0372] In vitro killing activity

[0373] Human epidermoid carcinoma cell line A431 (ATCC, catalog number: CRL-1555), human breast cancer cell line MCF-7, human lung cancer cell line NCI-H226 or NCI-H292 (5 × 103 ), and at IncuCyte (Sartorius AG, S3) and then incubated for 3 days to detect killing activity. These results are shown in the table below.

[0374] DS-1062 (Datopotamab deruxtecan) is an antibody-drug conjugate targeting TROP2 containing the drug deruxtecan (DXd). SI mutations (EU number: S239D and I332E mutations) were introduced into the constant region of DS-1062 to produce the DS-1062-SI analog (DXd). We further replaced the drug DXd with MMAE to obtain the DS-1062-SI analog (MMAE). The heavy chain 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.

[0375] Table 5

[0376]

[0377] (“NA” means no in vitro killing activity; “—” means not tested)

[0378] The above results showed that T-6F7-E-1G11-ADC and T-6F7-E-6C4-ADC had good killing activity in vitro.

[0379] In another experiment, pancreatic cancer Pan.02.03 cells (TROP2) cultured in cell culture plates were treated with different concentrations of antibodies or ADCs (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). + EGFR + ), human pancreatic adenocarcinoma BxPC-3 cells (TROP2 + EGFR + ), human lung cancer NCI-H292 cells (TROP2 + EGFR + ), and use PrestoBlue TM The cell viability reagent was used to detect killing activity after 72 hours of incubation. These results are shown in the table below.

[0380] Gosartuzumab (from Immunomedics, Inc) is a humanized anti-TROP2 monoclonal antibody-drug conjugate.

[0381] Table 6

[0382]

[0383] (“NA” means no in vitro killing activity; “—” means not tested)

[0384] The results showed that T-6F7-E-6C4-ADC (G5) displayed tumor killing efficacy against several cell lines, comparable to its parental TROP2 or EGFR ADC (G3, G4).

[0385] In another experiment, 0.1 μg / mL T-6F7-E-6C4-ADC was added to three groups of cells: Group 1: BxPC-3 cells + NCI-H520 cells; Group 2: BxPC-3 cells; Group 3: NCI-H520 cells (TROP2 - EGFR - ) to test the tumor killing efficacy. The control group did not add T-6F7-E-6C4-ADC. After incubation at 37°C, 5% CO2 for 72 hours, the cell pellet was stained with dead dye and live dye (eBioscience TM Fixable Viability Dye eFluor TM 780, eBioscience, catalog number: 65-0865-14) for flow cytometry analysis. The results are shown in Fig.9A In the results of the study, it was shown that T-6F7-E-6C4-ADC exhibited strong tumor killing activity against TROP2-EGFR double-positive BxPC-3 cells, but not against TROP2-EGFR double-negative NCI-H520 cells. However, in Group 1, when BxPC-3 cells and NCI-H520 cells were co-cultured ( Figures 9A-9B ), T-6F7-E-6C4-ADC exhibited strong tumor killing activity against NCI-H520 cells, indicating that T-6F7-E-6C4-ADC displayed significant bystander killing effect in vitro.

[0386] Example 3. Antitumor activity in A431 xenograft model

[0387] The effect of the antibodies or ADCs on in vivo tumor growth was tested in an epidermoid carcinoma model. Specifically, approximately 5 × 10 6 A431 cells were subcutaneously injected into B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., catalog number: B-CM-002). When the tumor volume of the mice reached about 300 mm3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Mice were then injected with phosphate buffered saline (PBS), ADC or antibody. These details are shown in the table below.

[0388] The lengths of the long and short axes of the tumor were measured, and the volume of the tumor was calculated as 0.5 × (long axis) × (short axis) 2 . Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100%. Ti is the average tumor volume of the treatment group on day i. T0 is the average tumor volume of the treatment group on day zero. Vi is the average tumor volume of the control group on day i. V0 is the average tumor volume of the control group on day zero. T-test was performed for statistical analysis. A TGI higher than 60% indicates that tumor growth is significantly inhibited. P < 0.05 is the threshold value indicating a significant difference.

[0389] Table 7

[0390]

[0391] The mice were also weighed twice a week. On the day of grouping (day 0), the average body weight of each group ranged from 20.7 g to 22.1 g. At the end of the experiment (day 21), the average body weight of each group ranged from 20.4 g to 22.9 g. Therefore, the average body weight of each group varied from 98.5% to 107.9%. The results showed that the antibodies tested were well tolerated and had no significant toxicity to mice.

[0392] The following table summarizes the results of this experiment, including the tumor volume 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 the statistical differences (P values) in body weight and tumor volume between the treatment group and the control group.

[0393] Table 8

[0394]

[0395] Figure 2 Tumor volumes of different groups of mice treated with antibodies, ADC or PBS are shown. Compared with the control group (G1-G2) treated with PBS or ISO-ADC, the treated groups (G3-G9) showed better tumor inhibition.

[0396] In addition, anti-TROP2 / EGFR bispecific antibody ADC (G5-G6) and anti-TROP2 / EGFR bispecific antibody (G8-G9) showed better tumor inhibition effect compared with saccharotuzumab-SI analog-ADC, cetuximab analog-ADC or DS-1062-SI analog (DXd).

[0397] Example 4. Antitumor activity of Panc 02.03 in xenograft model

[0398] The effects of the ADC on in vivo tumor growth were tested in a xenograft model of pancreatic adenocarcinoma. 6 Pancreatic adenocarcinoma epithelial Panc 02.03 (ATCC, catalog number: CRL-2553) cells were subcutaneously injected into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 1:10, mice were randomly divided into different groups according to tumor volume. Then PBS or ADC was injected into mice by intravenous (iv) administration. The frequency of administration was once a week (1 administration in total). These details are shown in the table below.

[0399] Table 9

[0400]

[0401]

[0402] During the experiment, there was little difference in the body weight of mice in each group.

[0403] The following table summarizes the results of this experiment, including the tumor volume 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 the statistical differences (P values) in body weight and tumor volume between the treatment group and the control group.

[0404] Table 10

[0405]

[0406] Figure 3The tumor size in the group treated with ADC is shown. The treatment group shows different tumor inhibition effects. In general, at a dosage of 10 mg / kg, compared with controls (ISO-ADC, cetuximab analogs-ADC and DS-1062-SI analogs (MMAE)), anti-TROP2 / EGFR bispecific antibody ADC (T-6F7-E-1G11-ADC and T-6F7-E-6C4-ADC) shows better anti-tumor activity. Anti-TROP2 / EGFR bispecific antibody ADC (T-6F7-E-1G11-ADC and T-6F7-E-6C4-ADC) shows dose-dependent anti-tumor activity.

[0407] Example 5. Antitumor activity in a pancreatic adenocarcinoma PDX model

[0408] The effect of the ADC on in vivo tumor growth was tested in a xenograft model of pancreatic adenocarcinoma. Specifically, tumor fragments derived from pancreatic adenocarcinoma patients were subcutaneously inoculated in B-NDG mice. When the tumor volume of the mice reached approximately 250-300mm 3 At 4 ℃, mice were randomly divided into different groups according to tumor volume. Then PBS or ADC was injected into mice by intravenous (iv) administration. These details are shown in the table below.

[0409] Table 11

[0410]

[0411] During the experiment, there was little difference in the body weight of the mice in the various groups.

[0412] The following table summarizes the results of this experiment, including the tumor volume 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 the statistical difference in tumor volume between the treatment group and the control group (P value).

[0413] Table 12

[0414]

[0415] The tumor volumes of all treatment groups (G3-G7) were smaller than those of the control groups (G1 and G2). The treatment groups had different tumor inhibition effects. TROP2 / EGFR bispecific antibody ADC (G5-G6) at a dose level of 3 mg / kg showed sustained and effective tumor inhibition. 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 analogs-ADC or gosartumomab).

[0416] Example 6. Antitumor Activity in Patient-Derived Lung Cancer Xenograft Model

[0417] The effect of ADCs on in vivo tumor growth was tested in a xenograft model of lung cancer. Specifically, tumor fragments derived from lung cancer patients were subcutaneously inoculated in B-NDG mice. When the tumor volume of the mice reached approximately 250-300 mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then PBS, ADC or antibody were injected into mice by intravenous (iv) administration. The frequency of administration was once a week (a total of 2 administrations). These details are shown in the table below.

[0418] Table 13

[0419]

[0420] During the experiment, there was little difference in the body weight of the mice in the various groups.

[0421] The following table summarizes the results of this experiment, including the tumor volume 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 the statistical difference in tumor volume between the treatment group and the control group (P value).

[0422] Table 14

[0423]

[0424] The treatment groups had different tumor inhibitory effects. The TGI value of the TROP2 / EGFR bispecific antibody ADC at a dose level of 3 mg / kg (G5) was higher than that of the positive control ISO-ADC, cetuximab analog ADC, and gosartan.

[0425] Example 7. Antitumor activity in the NCI-H292 xenograft model

[0426] The effects of the antibodies or ADCs on in vivo tumor growth were tested in a xenograft model of lung cancer. 6 NCI-H292 cells were injected subcutaneously into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then mice were injected with PBS, antibody or ADC by intravenous (iv) administration. These details are shown in the table below.

[0427] Cetuximab is a chimeric monoclonal IgG1 antibody targeting EGFR from Merck.

[0428] Table 15

[0429]

[0430] MRG003 is an antibody-drug conjugate consisting of a fully human IgG1 monoclonal antibody targeting EGFR conjugated to 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 chain and light chain sequences of the antibody of MRG003 are shown in SEQ ID NO:35 and SEQ ID NO:36, respectively.

[0431] The following table summarizes the results of this experiment, including the tumor volume on the day of grouping (day 0), 18 days after grouping (day 18), and at the end of the experiment (day 35); mouse survival rate; TGI (%); and the statistical difference in tumor volume between the treatment group and the control group (P value).

[0432] Table 16

[0433]

[0434] Fig.10 Tumor size in the group treated with antibody or ADC is shown. Anti-TROP2 / EGFR bispecific antibody ADCT-6F7-E-6C4-ADC (G5-G7) showed better anti-tumor activity (at a dose level of 10 mg / kg) compared with control (cetuximab and MRG003-ADC), and obtained better tumor inhibition (at a dose level of 3 mg / kg) than the corresponding parental ADC (T-6F7-SI-ADC and E-6C4-ADC). In addition, T-6F7-E-6C4-ADC showed dose-dependent anti-tumor activity. In particular, T-6F7-E-6C4-ADC (G7) showed sustained anti-tumor activity in all 6 mice, and these mice were tumor-free on the 35th day after grouping.

[0435] Example 8. Antitumor activity of NUGC-4 in a xenograft model

[0436] The effects of the antibodies or ADCs on in vivo tumor growth were tested in a xenograft model of gastric cancer. 6 NUGC-4 cells were injected subcutaneously into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then mice were injected with PBS, antibody or ADC by intravenous (iv) administration. These details are shown in the table below.

[0437] Table 17

[0438]

[0439]

[0440] The following table summarizes the results of this experiment, including tumor volume on the day of grouping (day 0), 13 days after grouping (day 13), and at the end of the experiment (day 27); TGI (%); and the statistical difference in tumor volume between the treatment group and the control group (P value).

[0441] Table 18

[0442]

[0443] Fig.11 Tumor sizes in groups treated with antibodies or ADCs are shown. ADC T-6F7-E-6C4-ADC (G7, G8, G9) showed better anti-tumor activity compared to control cetuximab (G4), gosartuzumab (G3) and MRG003-ADC (G5, G6). In addition, T-6F7-E-6C4-ADC showed dose-dependent anti-tumor activity.

[0444] Example 9. Pharmacokinetic characteristics and plasma stability

[0445] The pharmacokinetic clearance of the anti-EGFR / TROP2 bispecific antibody ADC was determined in B-hFcRn mice (Beijing Biocytogen Pharmaceuticals Co., Ltd., catalog number: 110001). In particular, the mice were divided into four groups (6 mice per group) and administered with 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 before and 15 minutes, 1 day, 3 days, 7 days, 10 days, 14 days, and 21 days after administration.

[0446] Serum levels of total antibodies and ADC were determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., catalog number: 109-005-088) or anti-MMAEmIgG (Bipsys, catalog number: MME-M5252) was diluted to a final concentration of 2000 ng / mL, added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 2°C-8°C. After incubation, the plate was washed with PBS-T buffer (supplemented with Tween TM20% PBS) was washed 4 times. The antibody unbound area was blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. Then, the plate was washed 4 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 using a plate washer, 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 at 100 μL / well and incubated at 37°C for 1 hour to determine the serum concentration of total antibodies. Alternatively, Gh-IgGκL-HRP (Abcam, catalog number: ab202549) was added to determine the serum concentration of ADC. After washing the plate, tetramethylbenzidine (TMB) solution was added to a 96-well plate at 100 μL / well as a substrate. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. The luminescent signal of the plate was measured at 450 nm and 630 nm to calculate the concentration. The absorbance values ​​and corresponding concentrations of the calibration samples prepared from each test product were used to create a calibration curve with four parameters (i.e., T 1 / 2 , C max , AUC 0-21天 The total antibody or ADC concentration of each serum sample was calculated using the standard curve. The drug concentration-time curve was created using the sample concentration calculated at each time point. TM WinNolin 8.3 was used to calculate the pharmacokinetic parameters.

[0447] The results are shown in the following table and Figures 12A-12B , which showed that T-6F7-E-6C4-ADC exhibited a similar half-life to the isotype control.

[0448] Table 19

[0449]

[0450]

[0451] 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. In particular, T-6F7-E-6C4-ADC was added to the plasma of humans, cynomolgus monkeys and SD rats, respectively, to a final concentration of 100 μg / mL. In the control group, PBS containing 0.5% BSA was used instead of plasma. The content of free MMAE and ADC was determined at 0 days, 1 day, 2 days, 6 days, 8 days, 11 days and 14 days after the addition of T-6F7-E-6C4-ADC, and the ratio of free MMAE to ADC was calculated. The results are shown in Fig.13 middle.

[0452] The results showed that after 14 days, the percentage of free MMAE in total MMAE in human plasma, monkey plasma and rat plasma was less than 2%, indicating that T-6F7-E-6C4-ADC was relatively stable in human, monkey and rat plasma.

[0453] Example 10. Antibody Drug Conjugates

[0454] Conjugation of antibodies to drug molecules

[0455] The purified antibody is coupled to CPT-1, CPT-2, CPT-3 or CPT-4 via a CPT-L linker. For the name of the antibody drug conjugate, CPTx (x = 1, 2, 3 or 4) is added directly after the antibody name. For example, when T-6F7-E-6C4 is coupled to CPT-1, it is named T-6F7-E-6C4-CPT1. As another example, when T-6F7-E-6C4 is coupled to CPT-2, it is named T-6F7-E-6C4-CPT2. Exemplary ADCs obtained by this method include: T-6F7-E-6C4-CPT1 and T-6F7-E-6C4-CPT2.

[0456] MS (mass spectrometry) is used to detect the coupling of antibodies to drug molecules. Human IgG1 molecules are coupled to CPT-2 to form isotype-CPT2 (ISO-CPT2), which serves as an isotype control. The results of MS detection showed that the drug-antibody ratio (DAR) of the ADC was about 4 or 8. Regarding the ADC name, if the DAR of T-6F7-E-6C4-CPT2 is about 4, the ADC is named T-6F7-E-6C4-CPT2 (DAR4). If the DAR of T-6F7-E-6C4-CPT2 is about 8, the ADC is named T-6F7-E-6C4-CPT2 (DAR8).

[0457] In vitro killing activity

[0458] HCC827 cells, NCI-H292 cells, A431 cells or Panc 02.03 cells cultured in cell culture plates were treated with purified antibodies or ADCs at different concentrations, and the killing activity was detected after 7 days of incubation using CellCounting-Lite 2.0 kit luminescent cell viability assay (Nanjing Vazyme Biotech Co., Ltd., catalog number: DD1101-02). These results are shown in the table below.

[0459] Table 20

[0460]

[0461] (“NA” means no in vitro killing activity; “—” means not tested)

[0462] The above results showed that T-6F7-E-6C4-CPT2 (DAR8) had good in vitro killing activity against HCC827 cells, NCI-H292 cells, A431 cells and Panc 02.03 cells.

[0463] Internalization of anti-TROP2 / EGFR bispecific antibodies and ADCs

[0464] A431 cells or NCI-H292 cells cultured in cell culture plates were treated with anti-TROP2 / EGFR bispecific antibodies and ADCs (as shown in the table below) and stained with IncuCyte (Sartorius, S3) The internalization activity was monitored over a 24-hour period after incubation, with images taken every hour. The results are shown in Figures 14A-14B In the results, it was shown 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 gosartumomab and cetuximab.

[0465] Table 21

[0466] Group Antibodies / ADCs G1 T-6F7-E-6C4 G2 T-6F7-E-6C4-CPT2(DAR4) G3 T-6F7-E-6C4-CPT2(DAR8) G4 ISO-CPT2(DAR8) G5 Gosartumomab G6 Cetuximab

[0467] Binding activity of anti-TROP2 / EGFR bispecific antibodies and ADCs

[0468] The purpose of this experiment was to test the binding activity of anti-TROP2 / EGFR bispecific antibodies and ADCs to tumor cell lines.

[0469] Specifically, A431 cells or human lung cancer HCC827 cells (ATCC, catalog number: CRL-2868) were cultured at 2×10 5Serial dilutions of anti-TROP2 / EGFR bispecific antibodies or ADCs (highest concentration: 130 nM, 9 gradients of 2-fold serial dilutions) were added to the 96-well plates and incubated at 4°C for 25-30 minutes. The cells were then incubated with secondary antibodies Alexa Fluor® before flow cytometry analysis. 647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) was incubated at 4°C in the dark for 25-30 minutes. The results shown in the table below show that 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.

[0470] Table 22

[0471]

[0472] Example 11. Antitumor activity in a patient-derived breast cancer xenograft model

[0473] Breast cancer patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) were implanted into the right side of B-NDG mice. Patient-derived breast tumor fragments were immunofluorescently stained, and images were analyzed by HALO version 3.2. The results showed that 96.92% of EGFR-positive cells and 49.87% of TROP2-positive cells in the tumor fragments were positive, respectively. When the tumor volume of the mice reached approximately 200-300 mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Mice were then injected with PBS or ADC by iv administration. Details of the dosing schedule, route and frequency are shown in the table below.

[0474] Table 23

[0475]

[0476]

[0477] The following table summarizes the results of this experiment, including the tumor volume 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 the statistical difference in tumor volume between the treatment group and the control group (P value).

[0478] Table 24

[0479]

[0480] Fig.15 The tumor size in the group treated with PBS or ADC is shown. The results show that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 both exhibited good tumor inhibition in a dose-dependent manner. In addition, T-6F7-E-6C4-CPT2 with DAR4 exhibited better tumor inhibition than T-6F7-E-6C4-ADC at a dose of 3 mg / kg.

[0481] Example 12. Antitumor Activity in Patient-Derived Pancreatic Cancer Xenograft Model

[0482] Pancreatic cancer patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) were implanted into the right side of B-NDG mice. Immunofluorescence staining results showed that the EGFR-positive cells and TROP2-positive cells in the pancreatic tumor fragments were 71.08% and 89.09%, respectively. When the tumor volume of the mice reached approximately 200-300 mm 3 At 4 ℃, mice were randomly divided into different groups according to tumor volume. Then PBS or ADC was injected into mice by iv administration. These details are shown in the table below.

[0483] Table 25

[0484]

[0485] The following table summarizes the results of this experiment, including tumor volume on the day of grouping (Day 0), 14 days after grouping (Day 14), and 32 days after grouping (Day 32); TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0486] Table 26

[0487]

[0488] The results showed that T-6F7-E-6C4-CPT2 with both DAR4 and DAR8 exhibited tumor inhibitory effects in a dose-dependent manner.

[0489] Example 13. Antitumor activity in the SKOV-3 xenograft model

[0490] The effects of the ADC on in vivo tumor growth were tested in a xenograft model of ovarian adenocarcinoma. 6 SKOV-3 cells (ATCC, catalog number: HTB-77) were subcutaneously injected into B-NDG mice. When the tumor volume of the mice reached about 300 mm 3At 4 ℃ and 7 ℃, mice were randomly divided into different groups according to tumor volume. Then PBS or ADC was injected into mice by intravenous (iv) administration. These details are shown in the table below.

[0491] Table 27

[0492]

[0493] The following table summarizes the results of this experiment, including tumor volume on the day of grouping (Day 0), 17 days after grouping (Day 17), and 35 days after grouping (Day 35); TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0494] Table 28

[0495]

[0496] Fig.16 Tumor sizes in the groups treated with PBS or ADC are shown. The results showed that both T-6F7-E-6C4-CPT2 with DAR4 and DAR8 exhibited tumor inhibition in the ovarian adenocarcinoma model in a dose-dependent manner, and T-6F7-E-6C4-CPT2 (DAR8) exhibited better tumor inhibition than T-6F7-E-6C4-CPT2 (DAR4).

[0497] Example 14. Antitumor activity in A431 xenograft model

[0498] The effects of the ADC on in vivo tumor growth were tested in a xenograft model of epidermoid carcinoma. 6 A431 cells were injected subcutaneously into B-NDG mice. When the tumor volume of the mice reached about 200 mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then mice were injected with PBS, antibody or ADC by intravenous (iv) administration. These details are shown in the table below.

[0499] Table 29

[0500]

[0501] Body weight was measured twice a week. During the experiment, the mice in all groups gained weight, and there was no significant difference in body weight between groups, indicating that the tested ADC was well tolerated and had no obvious toxicity to mice.

[0502] The following table summarizes the results of this experiment, including tumor volume on the day of grouping (Day 0), 17 days after grouping (Day 17), and 31 days after grouping (Day 31); TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0503] Table 30

[0504]

[0505]

[0506] Fig.17 The tumor size in the group treated with PBS, antibody or ADC is shown. The results show that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 all exhibited a better tumor inhibition effect than gosartuzumab or cetuximab in a dose-dependent manner. In addition, the experiment continued to 49 days after grouping (day 49), and 6mg / kg or 10mg / kg of T-6F7-E-6C4-CPT2 with DAR4 and DAR8 still showed a tumor inhibition effect.

[0507] Example 15. Antitumor activity in the NCI-H292 xenograft model

[0508] The effects of the antibodies or ADCs on in vivo tumor growth were tested in a xenograft model of lung cancer. 6 NCI-H292 cells were injected subcutaneously into B-NDG mice. When the tumor volume of the mice reached about 300 mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then mice were injected with PBS, antibody or ADC by intravenous (iv) administration. These details are shown in the table below.

[0509] Table 31

[0510]

[0511] The following table summarizes the results of this experiment, including the tumor volume 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 the statistical difference in tumor volume between the treatment group and the control group (P value).

[0512] Table 32

[0513]

[0514] The results showed that T-6F7-E-6C4-CPT2 with DAR4 and DAR8 showed good tumor inhibition in lung cancer models in a dose-dependent manner. In addition, T-6F7-E-6C4-CPT2 with DAR4 and DAR8 showed good tumor inhibition at a dose level of 10 mg / kg, and its TGI was higher than that of gosartuzumab or cetuximab.

[0515] Example 16. Antitumor Activity in Patient-Derived Xenograft Models

[0516] The effect of T-6F7-E-6C4-CPT2 (DAR8) on tumor growth was tested in head and neck squamous cell carcinoma models, esophageal cancer models, colorectal cancer models, or gastric cancer models. Specifically, BALB / c nude mice were implanted with patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm). When the tumor volume of the mice reached approximately 100-200 mm 3 At 4 pm, mice were randomly divided into different groups (3 mice per group) according to tumor volume. Mice were then injected with saline (G1, control) or 6 mg / kg T-6F7-E-6C4-CPT2(DAR8) (G2) (1 injection in total).

[0517] Immunohistochemistry (IHC) staining was performed on tumor tissues derived from different patients, and the following table shows the histochemical scores (H scores) of EGFR or TROP2 expression levels in patient-derived tumor tissues. Table 33 below also summarizes the TGI (%) of xenograft models derived from different patients.

[0518] Table 33

[0519]

[0520] Figures 18A-18F Tumor sizes in the groups treated with saline or T-6F7-E-6C4-CPT2(DAR8) are shown, which demonstrates that T-6F7-E-6C4-CPT2(DAR8) exhibits good tumor growth inhibition in head and neck squamous cell carcinoma, esophageal cancer, colorectal cancer, and gastric cancer.

[0521] Example 17. Pharmacokinetic characteristics and plasma stability

[0522] The pharmacokinetic clearance of the anti-EGFR / TROP2 bispecific ADC was determined in B-NDG mice. 6 A431 cells were injected subcutaneously into B-NDG mice. When the tumor volume of the mice reached about 300 mm 3At 14:1, mice were randomly divided into different groups (3 mice per group) according to tumor volume, and then 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) were administered by intravenous injection (administered once in total). The G1 group was used as a blank control. Blood samples and tumor tissue samples of mice in the G3-G10 and G11-G18 groups were collected 15 minutes, 2 hours, 6 hours, 1 day, 3 days, 5 days, 7 days and 14 days after administration. Blood samples and tumor tissue samples of mice in the G1 group were collected 1 hour before administration, while these samples of mice in the G2 group were collected 14 days after administration. These collected samples were used to detect the total antibody levels in serum and tumor tissues by sandwich ELISA, and to detect free payload by MS (mass spectrometry).

[0523] The level of total antibody was determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch, catalog number: 109-005-088) was diluted to a final concentration of 2000 ng / mL, added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 2°C-8°C. After incubation, the plate was washed with PBS-T buffer (supplemented with Tween TM 20% PBS) was washed 4 times. The antibody unbound area was blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. Then, the plate was washed 4 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 using a plate washer, 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 at 100 μL / well and incubated at 37°C for 1 hour to determine the concentration of total antibody and payload CPT2. After washing the plate, tetramethylbenzidine (TMB) solution was added to a 96-well plate at 100 μL / well as a substrate. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. The luminescence signal of the plate was measured at 450 nm and 630 nm to calculate the concentration. The absorbance values ​​and corresponding concentrations of the calibration samples prepared from each test product were used to create a calibration curve with four parameters (i.e., T 1 / 2 , C max , AUC 0-21天 The standard curve was used to calculate the antibody or ADC concentration of each serum sample. The drug concentration-time curve was created using the sample concentration calculated at each time point.TM WinNolin 8.3 was used to calculate the pharmacokinetic parameters.

[0524] The results are shown in the following table and Figures 19A-19D , which showed that T-6F7-E-6C4-CPT2 (DAR4) and T-6F7-E-6C4-CPT2 (DAR8) exhibited the expected PK behavior.

[0525] Table 34

[0526]

[0527] In another experiment, the plasma stability of T-6F7-E-6C4-CPT2 (DAR4) and T-6F7-E-6C4-CPT2 (DAR8) in human plasma, monkey (crab-eating macaque) plasma, and rat (SD rat) plasma was determined. In particular, 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, PBS containing 0.5% BSA was used instead of plasma. The content of free payload CPT2 and ADC was determined at 0 days, 1 day, 2 days, 6 days, 8 days, 11 days and 14 days after the addition of ADC, and the ratio of free CPT2 to total ADC was calculated. The results are shown in Figures 20A-20B It was shown that T-6F7-E-6C4-CPT2 (DAR4) and T-6F7-E-6C4-CPT2 (DAR8) were relatively stable in human, monkey and rat plasma, and the release rate of free CPT2 did not exceed 2.0% at most.

[0528] Example 18. Toxicology Evaluation

[0529] In preliminary experiments, in order to study safety and toxicokinetics (TK) characteristics, T-6F7-E-6C4-CPT2 (DAR8) was administered to cynomolgus monkeys three times by iv injection, 3 weeks apart (day 1, day 22 and day 43). The dosage formulation is shown in the table below. Then, the animals were sacrificed on day 50 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 the main TK parameters of payload, total antibody and ADC, such as Tmax, Cmax and AUC (0-t), were calculated. It was found that T-6F7-E-6C4-CPT2 (DAR8) had a good safety profile.

[0530] Table 35

[0531]

[0532] Other embodiments

[0533] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

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

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 anti-TROP2 / EGFR antibody or antigen-binding fragment thereof according to claim 2, wherein the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) 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 the selected VH1 CDR1, 2 and 3 amino acid sequences, the selected VL1 CDR1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7-9, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (2) the selected VH1 CDR1, 2, 3 amino acid sequences are represented by SEQ ID NOs: 10-12, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are represented by SEQ ID NOs: 1-3, respectively; (3) the selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16-18, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21, respectively, and the 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 or 3, wherein 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 the selected VH2 CDR1, 2 and 3 amino acid sequences and the selected VL2 CDR1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4-6, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and (2) The selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

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

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

22.

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

22.

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

22.

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

22.

10. An anti-TROP2 / EGFR antibody or antigen-binding fragment thereof as described in any one of claims 2-9, 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. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2-10, wherein the VH2 comprises VH CDR1, VH CDR2 and VH CDR3 identical to those of a selected VH sequence; and the VL2 comprises VL CDR1, VL CDR2 and VL CDR3 identical to those 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. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2-11, wherein the VH1 comprises the sequence of SEQ ID NO: 23, and the VL1 comprises the sequence of SEQ ID NO:

22.

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

22.

14. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 2-13, 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 as described in any one of claims 1-14, 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. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1-15, 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 of any one of claims 1-16, wherein the antibody is a multispecific antibody (eg, a bispecific antibody).

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

19. The anti-TROP2 / EGFR antibody or antigen-binding fragment thereof of any one of claims 1-18, 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, which cross-competes with the anti-TROP2 / EGFR antibody or an 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 according to 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. The cell of claim 23, wherein the cell is a CHO cell.

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

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

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

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

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

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

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

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

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

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

36. The method of claim 34 or claim 35, wherein the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung cancer), stomach cancer (e.g., gastric cancer), skin cancer (e.g., 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.

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

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

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

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

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

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

43. An anti-TROP2 / EGFR antibody drug conjugate (ADC), comprising a therapeutic agent covalently bound to a bispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody or the 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. The anti-TROP2 / EGFR ADC of any one of claims 27-33 and 43, wherein the drug to antibody ratio (DAR) is about 4 or 8.

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