Anti-EGFR / MET antibodies and uses thereof

Abstract

The present disclosure relates to antigen binding protein constructs (e.g., bispecific antibodies, antigen binding fragments thereof, or ADCs) wherein the antigen binding protein constructs specifically bind to two different antigens (e.g., EGFR and MET).

Description

[0001] Priority declaration

[0002] This application claims the benefit of PCT Application No. PCT / CN2022 / 102231, filed on June 29, 2022, PCT Application No. PCT / CN2022 / 108838, filed on July 29, 2022, PCT Application No. PCT / CN2022 / 116678, filed on September 2, 2022, and PCT Application No. PCT / CN2022 / 141416, filed on December 23, 2022. The entire contents of the foregoing applications are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to antigen binding protein constructs (eg, bispecific antibodies or antigen binding fragments thereof). Background Art

[0004] Bispecific antibodies are artificial proteins 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 targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has become an important milestone in the development of bispecific antibodies.

[0005] 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 antigen binding protein constructs, wherein the antigen binding protein construct specifically binds to two different antigens (e.g., EGFR and MET). In some embodiments, multispecific antibodies (e.g., bispecific antibodies) have identical light chain variable regions. In some embodiments, multispecific antibodies (e.g., bispecific antibodies) have common light chains. In some embodiments, multispecific antibodies (e.g., bispecific antibodies) form part of an antibody drug conjugate.

[0007] In one aspect, the present disclosure relates to an antigen-binding protein construct comprising: a first antigen-binding domain that specifically binds to EGFR; and a second antigen-binding domain that specifically binds to MET. In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0008] In some embodiments, the first heavy chain variable region (VH1) includes complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region includes 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) includes CDR1, 2, and 3, in some embodiments, the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 The CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, and in some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequence and the selected VL1 CDR1, 2, and 3 amino acid sequence are one of:

[0009] (1) the selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4-6, 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 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;

[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) includes CDR1, 2, and 3, in some embodiments, the VH2 CDR1 region includes an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region includes an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region includes 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) includes CDR1, 2, and 3, in some embodiments, the VL2 CDR1 region includes an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region includes an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region includes an amino acid sequence that is at least 80% identical to a selected VL2 The CDR3 amino acid sequence has an amino acid sequence with at least 80% identity, in some embodiments, the selected VH2 CDR1, 2 and 3 amino acid sequence and the selected VL2 CDR1, 2 and 3 amino acid sequence are one of the following:

[0014] (1) the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively;

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

[0016] (3) the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and

[0017] (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

[0018] In some embodiments, the antigen binding protein constructs as described herein have one of the following features:

[0019] (1) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 4-6, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively;

[0020] (2) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 7-9, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively;

[0021] (3) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 7-9, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively;

[0022] (4) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 16-18, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively;

[0023] (5) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 19-21, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively; and

[0024] (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

[0025] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:28, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32.

[0026] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32.

[0027] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32.

[0028] In some embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of:

[0029] (1) the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO: 32; and

[0030] (2) The selected VH sequence is SEQ ID NO:29, and the selected VL sequence is SEQ ID NO:32.

[0031] 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:

[0032] (1) the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO: 32; and

[0033] (2) The selected VH sequence is SEQ ID NO:29, and the selected VL sequence is SEQ ID NO:32.

[0034] In some embodiments, VH2 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of:

[0035] (1) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 32; and

[0036] (2) The selected VH sequence is SEQ ID NO:31, and the selected VL sequence is SEQ ID NO:32.

[0037] 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 and the selected VL sequence are one of:

[0038] (1) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 32; and

[0039] (2) The selected VH sequence is SEQ ID NO:31, and the selected VL sequence is SEQ ID NO:32.

[0040] In some embodiments, VH1 comprises the sequence of SEQ ID NO:28, and VL1 comprises the sequence of SEQ ID NO:32.

[0041] In some embodiments, VH1 comprises the sequence of SEQ ID NO:30, and VL1 comprises the sequence of SEQ ID NO:32.

[0042] In some embodiments, VH2 comprises the sequence of SEQ ID NO:31 and VL2 comprises the sequence of SEQ ID NO:32.

[0043] In some embodiments, the first antigen binding domain specifically binds human EGFR or monkey EGFR; and / or the second antigen binding domain specifically binds human MET or monkey MET.

[0044] In some embodiments, the first antigen binding domain is human or humanized; and / or the second antigen binding domain is human or humanized.

[0045] In some embodiments, the antigen binding protein construct is a multispecific antibody (e.g., a bispecific antibody). 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. In some embodiments, the first light chain variable region and the second light chain variable region are the same.

[0046] In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding an antigen binding protein construct as described herein.

[0047] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids as described herein.

[0048] In one aspect, the disclosure relates to a cell comprising a vector as described herein. In some embodiments, the cell is a CHO cell.

[0049] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0050] In one aspect, the present disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof or an antigen-binding protein construct, the method comprising: (a) culturing a cell as described herein under conditions sufficient for the cell to produce an antigen-binding protein construct; and (b) collecting the antigen-binding protein construct produced by the cell.

[0051] In one aspect, the present disclosure relates to an antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to an antigen binding protein construct as described herein. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent. In some embodiments, the therapeutic agent is MMAE or MMAF.

[0052] In some embodiments, the therapeutic agent is selected from

[0053]

[0054]

[0055] In some embodiments, the therapeutic agent is connected to the antigen binding protein construct via a linker. In some embodiments, the linker has the following structure:

[0056]

[0057] In some embodiments, the antibody drug conjugate has the following structure:

[0058]

[0059] In some embodiments, n=1, 2, 3, 4, 5, 6, 7, or 8; in some embodiments, "Ab" represents an antigen binding protein construct described herein.

[0060] On the one hand, the present disclosure relates to a method for treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antigen binding protein construct as described herein or an antibody drug conjugate as described herein. In some embodiments, the subject suffers from cancer expressing EGFR and / or MET. In some embodiments, the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma or lung cancer), gastric 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 carcinoma or brain cancer. In some embodiments, the subject is a person. In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject. In some embodiments, the method further comprises administering chemotherapy to the subject.

[0061] In one aspect, the disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antigen binding protein construct as described herein or an antibody drug conjugate as described herein.

[0062] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antigen binding protein construct as described herein or an antibody drug conjugate as described herein.

[0063] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) an antigen binding protein construct as described herein, and / or (b) an antibody drug conjugate as described herein.

[0064] As used herein, the term "antigen-binding protein construct" is a complex of (i) a single polypeptide comprising at least two different antigen-binding domains or (ii) two or more polypeptides (e.g., the same or different polypeptides) that together form at least two different antigen-binding domains. Non-limiting examples and aspects of antigen-binding protein constructs are described herein. Other examples and aspects of antigen-binding protein constructs are known in the art.

[0065] 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 embodiments, 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 embodiments, the antigen binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen). In other embodiments, the antigen binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen).

[0066] 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 specifically 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.

[0067] As used herein, the term "multi-specific antigen-binding protein construct" is an antigen-binding protein construct 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 on different antigens (e.g., different proteins present on the same cell surface or on different cell surfaces). In some aspects, a multi-specific antigen-binding protein construct binds two different epitopes (i.e., "bi-specific antigen-binding protein construct"). In some aspects, a multi-specific antigen-binding protein construct binds three different epitopes (i.e., "tri-specific antigen-binding protein construct"). In some aspects, a multi-specific antigen-binding protein construct binds four different epitopes (i.e., "tetra-specific antigen-binding protein construct"). In some aspects, a multi-specific antigen-binding protein construct binds five different epitopes (i.e., "five-specific antigen-binding protein construct"). Each binding specificity can exist in any suitable valence state. Non-limiting examples of multi-specific antigen-binding protein constructs are described herein.

[0068] 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.

[0069] As used herein, the term "common light chain" refers to a light chain that can interact with two or more different heavy chains to form different antigen binding sites, wherein these different antigen binding sites can specifically bind 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, an antigen binding construct may have a common light chain. In some embodiments, an antigen binding construct may have a common light chain variable region.

[0070] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one 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. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0071] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is a schematic diagram showing a bispecific antibody with a knobs-into-holes structure having a common light chain.

[0073] Figure 2 The cancer cell killing activity of E-6C4-M-2F11 and E-6C4-M-2F11-ADC at 10 μg / mL for 72 hours is shown. ISO-ADC is a human IgG1 isotype control coupled to MMAE. Amivantamab analog is a bispecific antibody control for E-6C4-M-2F11. The culture medium is a blank control.

[0074] Figure 3 Shown are the average tumor volumes of different groups of B-NDG mice injected with NCI-H1975 cells and treated with 10 mg / kg E-6C4-M-2F11-ADC (G2), 3 mg / kg E-6C4-M-2F11-ADC (G3), 10 mg / kg E-6C4-M-2F11 (G4) or 3 mg / kg E-6C4-M-2F11 (G5). PBS was injected as a control (G1).

[0075] Figure 4 Shown are the mean tumor volumes of different groups of B-NDG mice injected with NCI-H292 cells and treated with 10 mg / kg E-6C4-M-2F11-ADC (G2), 3 mg / kg E-6C4-M-2F11-ADC (G3), 1 mg / kg E-6C4-M-2F11-ADC (G4), 10 mg / kg ervantumab analog (G5), 3 mg / kg ervantumab analog (G6), or 1 mg / kg ervantumab analog (G7). PBS was injected as a control (G1).

[0076] Figure 5 Shown are the mean tumor volumes of different groups of B-NDG mice injected with NCI-H292 cells and treated with ISO-ADC (G2), E-6C4-M-2F11-ADC (G3), E-6C4-M-2G10-ADC (G4), E-6C4-ADC (G5), M-2G10-ADC (G6), M-2F11-ADC (G7), CD28-E-6C4-ADC (G8), CD28-M-2F11-ADC (G9) or M-2G10-CD28-ADC (G10) at a dose level of 3 mg / kg. PBS was injected as a control (G1).

[0077] Figure 6 Shown are the mean tumor volumes of different groups of B-NDG mice injected with SNU-5 cells and treated with 10 mg / kg ervantumab analog (G2), 3 mg / kg ervantumab analog (G3), 10 mg / kg E-6C4-M-2F11-ADC (G4), 3 mg / kg E-6C4-M-2F11-ADC (G5), 10 mg / kg E-6C4-M-2G10-ADC (G6), 3 mg / kg E-6C4-M-2G10-ADC (G7), 10 mg / kg E-6C4-M-2F11 (G8), 3 mg / kg E-6C4-M-2F11 (G9), 10 mg / kg E-6C4-M-2G10 (G10), or 3 mg / kg E-6C4-M-2G10 (G11). PBS was injected as a control (G1).

[0078] Figure 7 Shown are the mean tumor volumes of different groups of B-NDG mice injected with A431 cells and treated with ISO-ADC (G2), E-6C4-M-2F11-ADC (G3), E-6C4-M-2F11 (G4) or ervantumab analogs (G5) at a dose level of 3 mg / kg. PBS was injected as a control (G1).

[0079] Figure 8 The heavy chain variable region CDR sequences of the anti-EGFR antibodies E-1G11 and E-6C4 and the anti-MET antibodies M-2F11 and M-2G10 defined according to Kabat numbering are listed.

[0080] Fig. 9 The heavy chain variable region CDR sequences of the anti-EGFR antibodies E-1G11, E-6C4 and the anti-MET antibodies M-2F11, M-2G10 defined according to the Chothia numbering are listed.

[0081] Fig.10 The CDR sequences of the common light chain are listed according to the Kabat and Chothia numbering definitions.

[0082] Fig.11 The antibody heavy and light chain variable region sequences discussed in this disclosure are listed.

[0083] Fig.12 Other amino acid sequences discussed in this disclosure are listed.

[0084] Fig.13 Shown are the mean tumor volumes of different groups of B-NDG mice injected with NCI-H1975 cells and treated with M-2F11-ADC (G2 and G4), E-6C4-ADC (G3 and G5), E-6C4-M-2F11 (G9 and G10), E-6C4-M-2F11-ADC (G11 and G12), or a combination of E-6C4-ADC and M-2F11-ADC (G6, G7, and G8). PBS was injected as a control (G1).

[0085] Fig.14 Shown are the mean tumor volumes of different groups of B-NDG mice implanted with patient-derived gastric tumor fragments and treated with E-6C4-M-2F11-ADC (G2-G4) or evitumomab (G5). PBS was injected as a control (G1).

[0086] Fig.15 Shown are the average tumor volumes of different groups of Balb / c nude mice injected with NCI-H292 cells and treated with E-6C4-M-2F11-ADC (G2-G4), Telisotuzumab analog-ADC (G5), MRG003 analog-ADC (G6) or Evantuzumab (G7). PBS was injected as a control (G1).

[0087] Fig.16 Shown are the mean tumor volumes of different groups of B-NDG mice transplanted with patient-derived ampullary tumor fragments and treated with E-6C4-M-2F11-ADC (G2-G4), E-6C4-ADC (G5), Cetuximab analog-ADC (G6-G7), or Evantumab (G8). PBS was injected as a control (G1). DETAILED DESCRIPTION

[0088] Bispecific antibodies or their antigen-binding fragments are artificial proteins that can simultaneously bind to two different epitopes (e.g., on two different antigens). In some embodiments, a bispecific antibody or its antigen-binding fragment may have two arms. Each arm may 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, a bispecific antibody has a common light chain.

[0089] The present disclosure relates to antigen binding protein constructs (eg, bispecific antibodies or antigen-binding fragments thereof) and antibody drug conjugates that specifically bind to two different antigens (eg, EGFR and MET).

[0090] Anti-EGFR / MET antigen binding protein constructs

[0091] 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, 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 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, thereby initiating a signal transduction cascade that ultimately affects a variety of cellular functions, including cell proliferation and survival. Increased expression or kinase activity of EGFR is associated with a variety of human cancers, making EGFR an attractive target for therapeutic intervention. Increased EGFR gene copy number and protein expression correlate with response to the EGFR tyrosine kinase inhibitor IRESSA in non-small cell lung cancer TM The study was associated with a favorable response to gefitinib.

[0092] MET (also known as c-Met, tyrosine-protein kinase Met, or hepatocyte growth factor receptor (HGFR)) is a protein that is encoded in humans by the MET gene. The protein has tyrosine kinase activity. The primary single-chain precursor protein is post-translationally cleaved to produce an alpha subunit and a beta subunit, which are linked by a disulfide bond to form the mature receptor. Upon activation by its ligand, hepatocyte growth factor (HGF), MET stimulates a wide range of cellular processes, including growth, motility, invasion, metastasis, epithelial-mesenchymal transition, angiogenesis / wound healing, and tissue regeneration. The exact stoichiometry of HGF:MET binding is not known, but it is generally believed that two HGF molecules bind to two MET molecules, resulting in receptor dimerization and autophosphorylation at tyrosines 1230, 1234, and 1235. Ligand-independent MET autophosphorylation also occurs, sometimes due to gene amplification, mutation, or overexpression of the receptor.

[0093] MET is frequently amplified, mutated, or overexpressed in many types of cancer, including gastric cancer, lung cancer, colon cancer, breast cancer, bladder cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, and CNS cancer. Missense mutations, usually located in the kinase domain, are often found in hereditary papillary renal cell carcinoma (PRCC) and 13% of sporadic PRCC (Schmidt et al., Oncogene, Vol. 18: pp. 2343-2350, 1999), and MET mutations located in the signaling protein domain or juxtamembrane domain of MET are frequently found in gastric cancer, head and neck cancer, liver cancer, ovarian cancer, NSCLC, and thyroid cancer. MET amplification, which is often associated with disease progression, has been detected in brain cancer, colorectal cancer, gastric cancer, and lung cancer. Up to 4% and 20% of non-small cell lung cancer (NSCLC) and gastric cancer, respectively, show MET amplification. MET overexpression is also frequently observed in lung cancer. Furthermore, in clinical samples, nearly half of lung adenocarcinomas exhibited high levels of MET and HGF, both of which are associated with increased tumor growth rate, metastasis, and poor prognosis.

[0094] Of all tumors that develop resistance to EGFR tyrosine kinase inhibitors, almost 60% of these tumors increase MET expression, amplify MET, or increase MET's only known ligand, HGF, suggesting the existence of an EGFR compensatory pathway through MET. MET amplification was first identified in cultured cells that were resistant to the EGFR kinase inhibitor gefitinib, and statistically showed that cell survival was increased through the Her3 pathway. This was further validated in clinical samples, where nine of 43 patients with acquired resistance to erlotinib or gefitinib exhibited MET amplification.

[0095] Aberrant MET signaling has been implicated in the development / progression of many human cancers. This is due to overexpression of MET, activating mutations in MET, transactivation, autocrine or paracrine signaling, or MET gene amplification. Studies of cancer treatment outcomes have revealed a significant relationship between EGFR signaling and MET signaling. MET is a key factor in the development of resistance to targeted therapies, including those directed against EGFR. Similarly, EGFR and downstream gene mutations, such as KRAS, histological transformation, and activation of alternative pathways, including the MET signaling pathway, have been identified as mechanisms of resistance to EGFR-targeted therapies. Therefore, blocking one receptor often upregulates the other, which leads to resistance to monotherapy. Amplification of MET and / or high levels of HGF ligand expression have been observed in NSCLC patients with intrinsic or acquired resistance to EGFR tyrosine kinase inhibitors, including erlotinib and gefitinib. Conversely, MET-amplified lung cancer cells that are chronically exposed to MET inhibitors develop resistance through the EGFR pathway. Because of the signaling crosstalk between EGFR and Met, combined inhibition of both receptors may result in improved treatment outcomes for patients with MET- and EGFR-driven cancers. In addition, simultaneous inhibition may overcome or delay treatment resistance compared to blocking only one pathway.

[0096] After the ligand (such as EGF) binds to EGFR, it stimulates receptor dimerization, autophosphorylation, activation of the cytoplasmic tyrosine kinase domain inside the receptor, and the initiation of multiple signal transduction and transactivation pathways, which are involved in the regulation of DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of EGFR signaling can cause inhibition of one or more EGFRs. In some embodiments, EGFR ligands include EGF, TGFα, heparin-binding EGF (HB-EGF), amphiregulin (AR) and epiregulin (EPI).

[0097] After HGF binds to MET, it stimulates receptor dimerization, autophosphorylation, activation of the cytoplasmic tyrosine kinase domain within the receptor, and the initiation of multiple signal transduction and transactivation pathways, which are involved in the regulation of DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of MET signaling will lead to the inhibition of one or more MET downstream signaling pathways. Therefore, neutralization of MET can have multiple effects, including inhibition of cell proliferation and differentiation, angiogenesis, cell motility and metastasis.

[0098] The role of EGFR and MET in cancer is described in, for example, WO2014081954A1, WO2008 / 127710, WO2009 / 111691, WO2009 / 126834, WO2010 / 039248, WO2010 / 115551, and US2009 / 0042906; Engelman et al., "MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.", science 316.5827 (2007): 1039-1043; Bean et al., "MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.", Proceedings of the National Academy of Sciences 104.52 (2007): pp. 20932-20937, the entire text of which is incorporated herein by reference.

[0099] The anti-EGFR antibodies (e.g., E-1G11 ("1G11") and E-6C4 ("6C4")) and anti-MET antibodies (e.g., M-2F11 ("2F11") and M-2G10 ("2G10")) of the present disclosure are Human antibodies produced in mice. Since these antibodies have the same fully humanized common light chain, an anti-EGFR / MET bispecific antibody with a heavy chain variable region targeting EGFR (e.g., any of the VHs targeting EGFR described herein), a heavy chain variable region targeting MET (e.g., any of the VHs targeting MET described herein) and two identical common light chain variable regions is produced. In some embodiments, the anti-EGFR antigen binding domain includes the CDRs of anti-EGFR antibodies 1G11 or 6C4. In some embodiments, the anti-MET antigen binding domain includes the CDRs of anti-MET antibodies 2F11 or 2G10. In some embodiments, the anti-EGFR antigen binding domain includes the VH and VL of anti-EGFR antibodies 1G11 or 6C4. In some embodiments, the anti-MET antigen binding domain includes the VH and VL of anti-MET antibodies 2F11 or 2G10. For example, E-1G11-M-2F11 refers to a bispecific anti-EGFR / MET antibody that contains an anti-EGFR antigen binding domain derived from E-1G11 and an anti-MET antigen binding domain derived from M-2F11. In some embodiments, the anti-EGFR antigen binding domain includes the CDRs of E-1G11. In some embodiments, the anti-MET antigen binding domain includes the CDRs of M-2F11. In some embodiments, the anti-EGFR antigen binding domain includes the VH and VL of E-1G11. In some embodiments, the anti-MET antigen binding domain includes the VH and VL of M-2F11.

[0100] The bispecific antibodies described herein can be designed as an IgG1 subtype structure with a knob-in-hole (KIH) mutation, which can promote heterodimerization and avoid mispairing between the two heavy chains.

[0101] In some embodiments, the bispecific antibody has a higher endocytosis rate than a corresponding monoclonal antibody or a control bispecific antibody.

[0102] In some embodiments, the bispecific antibodies described herein can be conjugated with a therapeutic agent to form an antibody drug conjugate (ADC). In some embodiments, the 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, about 4.7, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 to about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4.4, about 3.7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 to about 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.2, about 3.8 to about 4.2, about 3.9 to about 4.2, about 4.0 to about 4.2, about 4.1 to about 4.2, about 3.5 to about 4.1, about 3.6 to about 4.1, about 3.7 to about 4.1, about 3.8 to about 4.1, about 3.9 to about 4.1, about 4.0 to about 4.1, about 3.5 to about 4. .0, about 3.6 to about 4.0, about 3.7 to about 4.0, about 3.8 to about 4.0, about 3.9 to about 4.0, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7 or about 3.5 to about 3.6.

[0103] In some embodiments, the DAR of the ADCs described herein is about 7.5 to about 8.5, about 7.6 to about 8.5, about 7.7 to about 8.5, about 7.8 to about 8.5, about 7.9 to about 8.5, about 8.0 to about 8.5, about 8.1 to about 8.5, about 8.2 to about 8.5, about 8.3 to about 8.5, about 8.4 to about 8.5, about 7.5 to about 8.4, about 7.6 to about 8.4, about 7.7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8.2 to about 8.3, about 7.5 to about 8.2, about 7.6 to about 8.2, about 7.7 to about 8.2, about 7.8 to about 8.2, about 7.9 to about 8.2, about 8.0 to about 8.2, about 8.1 to about 8.2, about 7.5 to about 8.1, about 7.6 to about 8.1, about 7.7 to about 8.1, about 7.8 to about 8.1, about 7.9 to about 8.1, about 8.0 to about 8.1, about 7.5 to about 8 .0, about 7.6 to about 8.0, about 7.7 to about 8.0, about 7.8 to about 8.0, about 7.9 to about 8.0, about 7.5 to about 7.9, about 7.6 to about 7.9, about 7.7 to about 7.9, about 7.8 to about 7.9, about 7.5 to about 7.8, about 7.6 to about 7.8, about 7.7 to about 7.8, about 7.5 to about 7.7, about 7.6 to about 7.7 or about 7.5 to about 7.6.

[0104] In some embodiments, the anti-EGFR / MET 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 in vitro cancer cell growth. In some embodiments, the anti-EGFR / MET ADC described herein can inhibit in vivo cancer cell growth (e.g., lung cancer, gastric cancer, or skin cancer) in a xenograft mouse model at a dose level of less than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg.

[0105] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof described herein have a common light chain.

[0106] The anti-EGFR / MET antigen-binding protein construct (e.g., a bispecific antibody) may include an anti-EGFR antigen-binding domain (e.g., E-1G11 ("1G11"), E-6C4 ("6C4")) or an anti-MET antigen-binding domain (e.g., M-2F11 ("2F11"), M-2G10 ("2G10")). In some embodiments, the anti-EGFR / MET antigen-binding protein construct has a heavy chain variable region targeting EGFR (e.g., any of the VHs targeting EGFR described herein), a heavy chain variable region targeting MET (e.g., any of the VHs targeting MET described herein), and two identical common light chain variable regions.

[0107] The CDR sequences of 1G11 and 1G11-derived antibodies (e.g., human antibodies) include CDRs of the heavy chain variable domain (SEQ ID NOs: 4-6) and CDRs of the light chain variable domain (SEQ ID NOs: 1-3) defined according to the Kabat numbering. CDRs may also be defined according to the Chothia system. According to the Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 16-18, and the CDR sequences of the light chain variable domain are shown in 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: 32 and 28, respectively.

[0108] The CDR sequences of 6C4 and 6C4-derived antibodies (e.g., human antibodies) 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) defined according to Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 19-21, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 1-3. The human light chain variable region and the human heavy chain variable region of 6C4 are shown in SEQ ID NOs: 32 and 29, respectively.

[0109] The CDR sequences of 2F11 and 2F11-derived antibodies (e.g., human antibodies) include CDRs of heavy chain variable domains (SEQ ID NOs: 10-12) and CDRs of light chain variable domains (SEQ ID NOs: 1-3) defined according to Kabat numbering. CDRs may also be defined according to the Chothia system. According to Chothia numbering, the CDR sequences of heavy chain variable domains are shown in SEQ ID NOs: 22-24, and the CDR sequences of light chain variable domains are shown in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region of 2F11 are shown in SEQ ID NOs: 32 and 30, respectively.

[0110] The CDR sequences of 2G10 and 2G10-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NOs: 13-15) and CDRs of the light chain variable domain (SEQ ID NOs: 1-3) defined according to Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 25-27, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region of 2G10 are shown in SEQ ID NOs: 32 and 31, respectively.

[0111] In addition, in some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may also contain one, two or three heavy chain variable region CDRs selected from the following items: SEQ ID NO: 4-6, SEQ ID NO: 7-9, SEQ ID NO: 10-12, SEQ ID NO: 13-15, SEQ ID NO: 16-18, SEQ ID NO: 19-21, SEQ ID NO: 22-24 and SEQ ID NO: 25-27; and / or one, two or three light chain variable region CDRs selected from the following items: SEQ ID NO: 1-3.

[0112] In some embodiments, the anti-EGFR / MET antigen binding protein construct may have a heavy chain variable region (VH) 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 a light chain variable region (VL) comprising 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 VL 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 VL The CDR2 amino acid sequence has at least 80%, 85%, 90% or 95% identity or consists of an amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to the selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, 3 amino acid sequences and the selected VL CDR1, 2, 3 amino acid sequences are shown in Figure 8 and Fig.10 (Kabat CDR) and Fig. 9 and Fig.10 (Chothia CDR).

[0113] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 4 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 5 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 6 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0114] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 7 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 8 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 9 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0115] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO: 10 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 11 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 12 having zero, one or two amino acid insertions, deletions or substitutions.

[0116] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 13 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 14 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 15 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0117] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 16 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 17 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 18 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0118] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 19 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 20 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 21 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0119] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 22 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 23 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 24 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0120] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO:25 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:26 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:27 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0121] In some embodiments, the anti-EGFR / MET antigen binding protein constructs described herein may include a light chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 1 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 2 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 3 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0122] Insertions, deletions and substitutions may be within the CDR sequence or at one or both ends of the CDR sequence.

[0123] In some embodiments, the anti-EGFR / MET antigen binding protein construct contains: a heavy chain variable region (VH) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90% or 95% identity with a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90% or 95% identity with a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 28, 29, 30 or 31, and the selected VL sequence is SEQ ID NO: 32.

[0124] In some embodiments, the anti-EGFR / MET antigen binding protein construct may have 3 VH CDRs identical to the CDRs of any VH sequence as described herein. In some embodiments, the anti-EGFR / MET antigen binding protein construct may have 3 VL CDRs identical to the CDRs of any VL sequence as described herein.

[0125] The present disclosure also provides nucleic acids comprising polynucleotides encoding anti-EGFR / MET antigen binding protein constructs, the constructs comprising immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chains or immunoglobulin light chains include, for example, Figure 8 , Fig. 9 or Fig.10 The CDRs shown, or having Fig.11 sequence shown.

[0126] The anti-EGFR / MET antigen-binding protein construct can also be an antibody variant (including derivatives and conjugates) of the anti-EGFR / MET antigen-binding protein construct. Other antibodies provided herein are polyclonal antibodies, multispecific (multimers, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies prepared in cells (i.e., intrabodies), and antigen-binding fragments thereof. The anti-EGFR / MET antigen-binding protein construct 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, the anti-EGFR / MET antigen-binding protein construct is an IgG (e.g., IgG1) antibody or an antigen-binding fragment thereof.

[0127] Fragments of the anti-EGFR / MET antigen binding protein constructs are suitable for use in the provided methods as long as they retain the desired affinity and specificity for both EGFR and MET. Thus, fragments of the anti-EGFR / MET antigen binding protein constructs will retain the ability to bind to EGFR and MET.

[0128] Antigen binding protein constructs

[0129] The present disclosure provides antigen-binding protein constructs (e.g., bispecific antibodies). Antigen-binding protein constructs (e.g., bispecific antibodies) may include anti-EGFR antibodies or antigen-binding fragments thereof, and anti-MET antibodies or antigen-binding fragments thereof. These antigen-binding protein constructs (e.g., bispecific antibodies) may have various forms.

[0130] Typically, antibodies (also referred to as immunoglobulins) can be composed of two types of polypeptide chains, i.e., light chains and heavy chains. The non-limiting antigen-binding protein constructs disclosed herein can be complete tetra-immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype (including IgM, IgG, IgE, IgA or IgD) or 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.

[0131] These hypervariable regions, called complementarity determining regions (CDRs), form loops that comprise the major antigen-binding surface of the antibody. The four framework regions largely adopt 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 held tightly together by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0132] 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 commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains", Antibody engineering, Springer Berlin Heidelberg, 2001, pp. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecular immunology 45.14 (2008): pp. 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med., Vol. 132: pp. 211-250; Martin et al., Methods Enzymol., Vol. 203: pp. 121-153 (1991); Morea et al., Biophys Chem., Vol. 68, No. 1-3: pp. 9-16 (October 1997); Morea et al., J. Chem., Vol. 68, No. 1-3: pp. 9-16 (October 1997); Morea et al., J. Mol Biol., Vol. 275, No. 2: pp. 269-294 (January 1998); Chothia et al., Nature, Vol. 342, No. 6252: pp. 877-883 (December 1989); Ponomarenko and Bourne, BMC Structural Biology, Vol. 7, No. 64 (2007), each of which is incorporated herein by reference in its entirety.

[0133] CDR is very important for identifying the epitope of an antigen. As used herein, an "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 can be about three, four, five, six or seven amino acids, but these amino acids do not have to be in a continuous linear sequence of the primary structure of the antigen, because the epitope can depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0134] In some embodiments, the anti-EGFR / MET antigen binding protein construct 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, but their constant regions differ, particularly in the hinge and upper CH2 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 these documents is incorporated herein by reference in its entirety.

[0135] The anti-EGFR / MET antigen binding protein construct can also be an immunoglobulin molecule derived from any species (e.g., humans, rodents, mice, rats, camels). The anti-EGFR / MET antigen binding protein construct disclosed herein also includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. An antigen binding domain or antigen binding fragment is a portion of an antibody that retains the specific binding activity of a complete antibody, that is, any portion of an antibody that is capable of specifically binding to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, an anti-EGFR / MET antigen binding protein construct or an antigen binding fragment thereof can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising an antibody binding domain or a binding domain homologous thereto. Non-limiting examples of antigen binding domains include, e.g., heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a single CDR from a heavy or light chain of an intact antibody.

[0136] In some embodiments, the scFv in the anti-EGFR / MET antigen binding protein construct has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFv has two antigen binding regions (antigen binding regions A and B), and the two antigen binding regions can bind to the corresponding target antigens with different affinities.

[0137] In some embodiments, anti-EGFR / MET antigen binding protein constructs or antigen binding fragments may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) fused to a CD3-ζ transmembrane domain and an intracellular domain as described herein. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase effectiveness. Therefore, on the one hand, the present disclosure also provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0138] In some embodiments, an antigen binding protein construct (eg, a bispecific antibody) can bind to two different antigens or two different epitopes.

[0139] In some embodiments, the antigen binding protein construct (eg, bispecific antibody) may include one, two, or three selected from Figure 8 and Fig. 9 In some embodiments, the antigen binding protein construct (eg, a bispecific antibody) may include one, two, or three selected from Fig.10 The light chain variable region CDRs.

[0140] The multimerization of antibodies can be achieved by natural aggregation of antibodies or by chemical or recombinant connection techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0141] In some embodiments, the antigen-binding protein construct is a bispecific antibody. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules so that the percentage of the heterodimer recovered from the recombinant cell culture is maximized. For example, the interface can include at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (for example, tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), a compensation "cavity" identical or similar to the larger side chain size is produced on the interface of the second antibody molecule. This provides a mechanism that can make the yield of heterodimers higher than other unwanted end products (such as homodimers). The method is described in, for example, WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0142] Any antigen binding protein construct described herein (e.g., a bispecific antibody) can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of stabilizing molecules can increase the half-life of the antibody or antigen binding fragment in vitro (e.g., in tissue culture or when stored in a pharmaceutical composition) or in vivo (e.g., in the human body) or extend its biological activity.

[0143] Antigen binding protein constructs (e.g., bispecific antibodies) can also have various forms. Many different forms of antigen binding constructs 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.

[0144] In some embodiments, the antigen binding protein construct is a BiTe, (scFv)2, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HSA, or a tandem scFv. In some embodiments, the antigen binding protein construct is VHH-scAb, VHH-Fab, dual 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-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, diabody-CH3, triple antibody (triple body), miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body or scFv1-PEG-scFv2.

[0145] In some embodiments, the antigen binding protein construct can be a TrioMab. In a TrioMab, the two heavy chains are from different species, with different sequences restricting heavy chain-light chain pairing.

[0146] In some embodiments, the antigen binding protein construct has two different heavy chains and one common light chain. Heterodimerization of the heavy chains can be based on knobs-in-holes or some other heavy chain pairing technology.

[0147] In some embodiments, bispecific antibodies can be produced using CrossMAb technology. Correct light chain binding can be achieved in bispecific heterodimer IgG antibodies using CrossMAb technology, which allows the production of various bispecific antibody formats, 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 that can achieve correct light chain binding. The method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-andmultispecific antibodies.", MAbs. Vol. 8, No. 6, Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are exchanged.

[0148] The antigen binding protein construct can be a Duobody. The naturally occurring Fab exchange mechanism in IgG4 antibodies is similar to the controlled substance in IgG1 antibodies, which is called controlled Fab exchange. This format ensures specific pairing between heavy chain and light chain.

[0149] 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 corresponding N-termini alone, rather than scFv binding to each heavy chain N-terminus.

[0150] In scFv-IgG, two scFvs are linked to the C-terminus (CH3) of the heavy chain. 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.

[0151] In some embodiments, the antigen binding protein construct may have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-termini of the heavy chains.

[0152] The antigen binding protein construct may 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. This format ensures efficient preparation and purification.

[0153] In some embodiments, the antigen binding protein construct can be TF. Three Fab fragments are linked by disulfide bridges. Two fragments target tumor associated antigens (TAA) and one fragment targets a hapten. The TF form does not have an Fc region.

[0154] ADAPTIR has two scFvs bound to either side of the Fc region. This format abandons the full IgG as the basis of its construction, but retains the Fc region to extend half-life and facilitate purification.

[0155] Bispecific T cell engagers ("BiTEs") consist of two scFvs, VLA VHA and VHB VLB, on one peptide chain. They only have binding domains and no Fc region.

[0156] In BiTE-Fc, the Fc region is fused to the BiTE construct. The addition of the Fc region increases the half-life, thereby extending the effective concentration and avoiding continuous intravenous (IV) administration.

[0157] Dual-affinity retargeting (DART) antibodies have two peptide chains connecting opposite fragments, namely VLA and VHB and VLB and VHA, and have a sulfide bond at their C-termini fusing them together. In DART, the sulfide bond can improve its stability compared to BiTE.

[0158] In DART-Fc, the Fc region is attached to the DART structure. It can be produced by assembling three chains, two of which are linked by disulfide bonds, like DART. One chain contains half of the Fc region, which will dimerize with the third chain that only expresses the Fc region. The addition of the Fc region increases the half-life, thereby extending the effective concentration and avoiding continuous intravenous (IV) administration.

[0159] In a tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules with half an Fc region are generated and will dimerize. This format is a divalent binding to both targets, making it a tetravalent molecule.

[0160] Tandem diabodies (TandAb) include two diabodies. Each diabody consists of a VHA and a VLB fragment, and the VHA and VLB fragments are covalently bound. The two diabodies are connected to a peptide chain. Compared with a diabody consisting of two scFvs, its stability is improved. It has two bivalent binding sites.

[0161] ScFv-scFv-toxins include a toxin and two scFvs with a stabilizing linker. They can be used to specifically deliver payloads.

[0162] In the module scFv-scFv-scFv, one scFv targeting a TAA is tagged with a short recognizable peptide and assembled onto a bsAb consisting of two scFvs, one targeting CD3 and one targeting the recognizable peptide.

[0163] In ImmTAC, a stable and soluble T cell receptor is fused to a scFv that recognizes CD3. By using the TCR, ImmTAC is suitable for targeting processed proteins, such as intracellular proteins.

[0164] Trispecific nanobodies have two single variable domains (nanobodies), and another module for extending half-life. Additional modules are added to increase half-life.

[0165] In the trispecific killer adapter (TriKE), two scFvs are connected by a polypeptide linker incorporating human IL- 15. The linker connecting IL-15 is added to increase the survival and proliferation of NK.

[0166] In some embodiments, the antigen binding protein construct is a bispecific antibody. In some embodiments, the bispecific antibody of the present disclosure is designed as 1+1 (each target is monovalent) and has an IgG1 subtype structure. This can reduce the affinity for cells with low expression levels of EGFR and MET, and increase the affinity for cells co-expressing EGFR and MET to achieve enhanced targeting function.

[0167] In some embodiments, an anti-EGFR / MET antigen binding protein construct (e.g., an antibody, a bispecific antibody, or an antibody fragment thereof) comprises a KIH mutation. In some embodiments, an antigen binding protein construct comprises a first antigen binding domain that specifically binds to EGFR and a second antigen binding domain that specifically binds to MET. 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-EGFR / MET antigen binding protein construct comprises a knob heavy chain having 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: 33. In some embodiments, the anti-EGFR / MET antigen binding protein construct comprises a hole heavy chain having 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: 34.

[0168] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof described herein have a common light chain.

[0169] Antibody Drug Conjugate (ADC)

[0170] In some embodiments, the antigen binding protein constructs described herein (e.g., bispecific antibodies) can be optionally conjugated to a therapeutic agent with a linker to form an antibody drug conjugate. Antibody drug conjugates comprising antibodies or antigen binding fragments thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, camptothecin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoid alkaloids (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.

[0171] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of the present invention, chemical elements are identified according to the CAS version of the periodic table of the elements in the inner cover of the 75th edition of "Handbook of Chemistry and Physics", and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry and specific functional parts and reactivity are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, "March's Advanced Organic Chemistry", 5th edition, John Wiley & Sons, New York, 2001; Larock, "Comprehensive Organic Transformations", VCH Publishers, New York, 1989; Carruthers, "Some Modem Methods of Organic Synthesis", 3rd edition, Cambridge University Press, Cambridge, 1987.

[0172] Unless explicitly stated to the contrary, all ranges cited herein are inclusive. When a numerical range is listed, it is intended to include every numerical value and sub-range within that range. For example, "C 1-6 "Aims to cover C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 .

[0173] Compounds or any formula depicting and describing compounds of the present disclosure may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of compounds or any formula depicting and describing compounds of the present disclosure. Asymmetric centers present in compounds or any formula depicting and describing compounds of the present disclosure may all have (R) or (S) configurations independently of one another. When the bonds to chiral carbons in a structural formula are depicted as straight lines, or when the compound name is recited without the (R) or (S) chiral designation of the chiral carbon, it is understood that both the (R) and (S) configurations of each such chiral carbon, as well as each enantiomer or diastereomer and mixtures thereof, are included in the formula or name.

[0174] The disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, such as mixtures of enantiomers and / or diastereomers (various ratios). Therefore, enantiomers are the subject of the disclosure, which exist in the form of pure enantiomers, including both left-handed enantiomers and right-handed enantiomers, and in the form of racemates and mixtures of two enantiomers (various ratios). In the case of cis / trans isomerism, the disclosure includes both cis-forms and trans-forms and mixtures of these forms (various ratios). If necessary, the mixture can be separated by conventional methods, such as by chromatography or crystallization, by using stereochemically uniform synthetic raw materials or by stereoselective synthesis to prepare a single stereoisomer. Optionally, derivatization can be carried out before separating stereoisomers. The separation of stereoisomer mixtures can be carried out in an intermediate step during compound synthesis, or can be carried out on final racemic product. If necessary, the absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates derivatized with reagents containing stereocenters of known configuration. Alternatively, the absolute stereochemistry can be determined by vibrational circular dichroism (VCD) analysis.

[0175] 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 prevalent in nature. Such compounds are termed "isotopic variants." The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of a compound or any formula depicting and describing a compound of the invention. Examples of suitable isotopes 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; isotopes of carbon, such as 11 C. 13 C and 14C; isotopes of chlorine, such as 36 Cl; isotopes of fluorine, such as 18 F; isotopes of iodine, such as 123 I and 125 I; isotopes of nitrogen, such as 13 N and 15 N; isotopes of oxygen, such as 15 O. 17 O and 18 O; isotopes of phosphorus, such as 32 P; and isotopes of sulfur, such as 35 Certain isotopic variations of the compounds or any formula depicting and describing the disclosed compounds, such as those containing radioactive isotopes, may be useful in drug and / or substrate tissue distribution studies. In particular, compounds having the described structures differ only in the substitution of a heavier isotope, such as deuterium ( 2 H or D) instead of hydrogen, such compounds have certain therapeutic advantages, such as greater metabolic stability, increased in vivo half-life or reduced dosage requirements, and therefore may be used in some specific situations. Isotopic variations of the compounds or any formula depicting and describing the compounds of the present disclosure 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 appropriate isotopically labeled reagents in place of the unlabeled reagents previously used for synthesis.

[0176] The compounds provided herein are described with reference to both general formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a variety of different forms or derivatives, all of which are within the scope of the present disclosure. For example, these forms or derivatives include pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, solvated forms, different crystal forms or polymorphs and active metabolites, etc.

[0177] Unless otherwise indicated, the term "pharmaceutically acceptable salt" as used herein 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 of the present disclosure contain one or more acidic or basic groups, the present disclosure also includes their corresponding pharmaceutically acceptable salts. Therefore, the compounds of the present invention containing acidic groups (such as carboxyl) can exist in the form of salts, and can be used, for example, in the form of alkali metal salts, alkaline earth metal salts, aluminum salts or ammonium salts according to the present invention. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts formed with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine or amino acids. 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), these salts can be easily obtained. Other basic salts of the disclosed compounds include, but are not limited to, copper (I) salts, copper (II) salts, iron (II) salts, iron (III) salts, manganese (II) salts, and zinc salts. The disclosed compounds containing one or more basic groups (e.g., groups that can be protonated) can exist in the form of salts and can be used in accordance with the present disclosure in the form of addition salts thereof with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, 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, as well as others known to those skilled in the art. The salts formed are particularly hydrochlorides, chlorides, hydrobromides, bromides, iodides, sulfates, phosphates, mesylates (methanesulfonates), toluenesulfonates, carbonates, bicarbonates, formates, acetates, sulfoacetates, trifluoromethanesulfonates, oxalates, malonates, maleates, succinates, tartrates, malates, pamoates, mandelates, fumarates, lactates, citrates, glutarates, stearates, aspartates and glutamates. In addition, the stoichiometry of the salts formed by the compounds of the present disclosure can be an integer multiple or a non-integer multiple of one.

[0178] The compounds of the present disclosure containing basic nitrogen-containing groups can be quaternized using agents such as C 1-4 Alkyl halides, such as methyl, ethyl, isopropyl and tert-butyl chlorides, bromides and iodides; di-C 1-4Alkyl sulfates, such as dimethyl, diethyl and dipentyl sulfates; C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aryl C 1-4 Alkyl halides such as benzyl chloride and phenethyl bromide.

[0179] 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. The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in 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 drugs due to their low physiological compatibility, but which can be used as, for example, intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002).

[0180] Compounds or any formulas depicting and describing compounds of the present disclosure 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.

[0181] Pharmaceutically acceptable solvates according to the present disclosure may include those solvents wherein the solvent of crystallization may be isotopically substituted, for example D2O, d6-acetone, d6-DMSO.

[0182] Linker compounds

[0183] 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 be linked together by, for example, a coupling reaction with a set of ligand compounds and therapeutic agent compounds, respectively, to form a ligand-drug conjugate, such as an antigen-binding protein construct described herein (e.g., a bispecific antibody) and a therapeutic agent (e.g., any therapeutic agent described herein).

[0184] In some embodiments, a linker described herein is a compound having the formula:

[0185] Q-L

[0186] Formula (I),

[0187] or a pharmaceutically acceptable salt, solvate, stereoisomer or isotopic variant thereof, wherein Q represents a linker moiety capable of coupling to a ligand through a bond selected from a carbonyl, a thioether, an amide, a disulfide and a hydrazone bond; and L represents a linker moiety capable of connecting Q to a therapeutic agent.

[0188] In some embodiments, the linking moiety (Q in Formula (I)) has the following structure:

[0189]

[0190] In some embodiments, the linker moiety (L in formula (I)) has the following formula:

[0191]

[0192] Wherein L1 is a polypeptide residue consisting of three to eight amino acid residues, the polypeptide residue including 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;

[0193] L2 does not exist or is a monodentate, bidentate or tridentate hydrophilic group connected to the side chain carboxyl group of the 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 , 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

[0194] represents the N-terminal side of the polypeptide residue covalently linked to the linking moiety Q.

[0195] In some embodiments, polypeptide residue L1 is NH -Glu-Val-Ala- COOHIn some embodiments, the hydrophilic group L2 has the following structure:

[0196]

[0197] 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 .

[0198] In some embodiments, a linker described herein is a compound having the following structure:

[0199]

[0200] In some embodiments, the linker is a VC linker. Details of linkers for ADCs can be found in, for example, Su, Z. et al., "Antibody-drug conjugates: Recent advances in linker chemistry.", ActaPharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0201] Therapeutic agents

[0202] In some embodiments, therapeutic agents conjugated to the antigen binding protein constructs (eg, bispecific antibodies) described herein are discussed below.

[0203] 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:

[0204]

[0205] wherein X is selected from -CH2-, O and S; and Y is selected from H, D and F.

[0206] 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:

[0207]

[0208] 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:

[0209]

[0210] In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below:

[0211]

[0212] 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:

[0213]

[0214] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and 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, 5,530,0 97, 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.

[0215] 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. There are many different assays known in the art that can be used to determine whether an auristatin or a resulting antibody drug conjugate exerts a cytostatic or cytotoxic effect on the desired cells.

[0216] 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, metodopa and uredopa); ethyleneimines and methylmelamines (including hexamethylmelamine, triethylphosphamide, triethylthiophosphamide and trihydroxymethylmelamine); nitrogen mustards (such as chlorambucil, chlornaphazoline, cholephosphamide, estramustine, ifosfamide, bischloroethyl, mechlorethamine hydrochloride) oxide hydrochloride), melphalan, novembichin, phenylephrine, prednimustine, trofosamide, uracil mustard); nitrosoureas (such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); antibiotics (such as aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogamycin, olive mycin, peplomycin, potofiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, tuberculocin, ubenimex, fenstatin, doxycycline); antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (such as folate, methotrexate, pteropterin, trimetrexate); purine analogs (such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine); pyrimidines analogs (such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU); androgens (such as calosterone, drostanolone propionate, cyclothiodine, melastane, testolactone); antiadrenal agents (such as aminoglutethimide, mitotane, trilostane); folic acid supplements (such as folinic acid); aceglucuronolide; aldophosphamide glycosides (such as leucovorin); glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diazocine; eflornithine; elliptonium acetate; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; diamine nitracrine; pentostatin; phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizoran; spirogermanium;Alternaria tenuisporic acid; triazoquinone; 2',2',2'-trichlorotriethylamine; ethyl carbamate; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes (e.g., paclitaxel (; Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antoni, France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs (such as cisplatin and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; mitoxantrone (Novantrone); teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. The definition also includes anti-hormonal agents that regulate or inhibit the effect of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazoles, 4-hydroxytamoxifen, trioxifene, raloxifene, LY117018, onapristone and toremifene (Falutone); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. A detailed description of chemotherapeutic agents can be found in, for example, US20180193477A1, which is incorporated herein by reference in its entirety.

[0217] Linker-Therapeutic Agent Compounds

[0218] In some embodiments, a linker (eg, any linker described herein) and a therapeutic agent (eg, any therapeutic agent described herein) can be linked to form a "linker-therapeutic agent" compound.

[0219] In some embodiments, the linker-therapeutic agent compound has the structure:

[0220]

[0221] In some embodiments, the linker-therapeutic agent compound has the structure:

[0222]

[0223] In some embodiments, an antibody ("Ab"), such as any antigen binding protein construct described herein (e.g., a bispecific antibody), 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:

[0224]

[0225] wherein n=1, 2, 3, 4, 5, 6, 7 or 8.

[0226] Antibody and ADC characterization

[0227] The anti-EGFR / MET antigen binding protein construct (eg, an antibody, a bispecific antibody, or an antibody fragment thereof) may include an antigen binding region derived from any anti-EGFR antibody or any antigen binding fragment thereof as described herein.

[0228] The present disclosure provides anti-EGFR / MET antigen binding protein constructs that specifically bind to EGFR. These antigen binding protein constructs can be agonists or antagonists. The antigen binding protein constructs 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, the antigen binding protein construct can inhibit the EGFR-related signal transduction pathways, thereby treating cancer (e.g., NSCLC). In some embodiments, the antigen binding protein construct can trigger CDC or ADCC.

[0229] The affinity of an antibody for an antigen can be measured using common techniques, including, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be derived from the quotient of kinetic rate constants (KD = koff / kon). In some implementations, an antigen binding protein construct (e.g., a bispecific antibody) can be 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 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 .

[0230] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0231] In some embodiments, the antigen binding protein construct (eg, bispecific antibody) may be less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M or greater than 1×10 -10 M.

[0232] Anti-EGFR / MET antigen binding protein constructs (e.g., bispecific antibodies) may also include an antigen binding region derived from any anti-MET antibody or antigen binding fragment thereof as described herein. The anti-MET antibody or antigen binding fragment thereof described herein can block the binding between MET and HGF. In some embodiments, by binding to MET, the antigen binding protein construct may also inhibit MET-related signaling pathways, thereby inhibiting cell proliferation, differentiation and / or metastasis. Therefore, in some embodiments, the antigen binding protein construct as described herein is a MET agonist. In some embodiments, the antigen binding protein construct is a MET antagonist.

[0233] In some embodiments, the antigen binding protein construct (eg, bispecific antibody) may be less than 0.1 s -1 , less than 0.01s -1 , less than 0.001s -1, less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0234] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0235] 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.

[0236] Since the antigen binding protein construct (e.g., bispecific antibody) binds both MET and EGFR, the antigen binding protein construct has a higher binding affinity to cells expressing both MET and EGFR. Avidity can be used to measure the binding affinity of the antigen binding protein construct to these cells. Avidity is the cumulative strength of multiple affinities of various types of non-covalent binding interactions.

[0237] Thermal stability can also be determined. Antigen binding protein constructs (e.g., bispecific antibodies) as described herein can have a Tm 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, a melting curve is sometimes shown to have two transitions, showing a first denaturation temperature Tm D1 and a second denaturation temperature Tm D2. The presence of these two peaks usually indicates the denaturation of the Fc domain (Tm D1) and the denaturation of the Fab domain (Tm D2), respectively. When there are two peaks, Tm is generally referred to as Tm D2. Thus, in some embodiments, the antibodies or antigen-binding fragments as described herein have a Tm D1 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 antibodies or antigen-binding fragments as described herein have a Tm D2 of 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, Tm D1, 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.

[0238] In some embodiments, the antigen binding protein construct (e.g., bispecific antibody) can bind to human EGFR or monkey EGFR. In some embodiments, the antigen binding protein construct (e.g., bispecific antibody) cannot bind to human EGFR or monkey EGFR. In some embodiments, the antigen binding protein construct (e.g., bispecific antibody) can bind to human MET or monkey MET. In some embodiments, the antigen binding protein construct (e.g., bispecific antibody) cannot bind to human MET or monkey MET.

[0239] In some embodiments, the antigen binding protein constructs (e.g., bispecific antibodies) have a purity greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 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.

[0240] In some embodiments, the antigen binding protein constructs (e.g., bispecific antibodies) have a tumor growth inhibition rate or percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the antibodies have a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150%. TGI (%) can be determined, for example, at 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days or 24 days after the start of treatment. As used herein, tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:

[0241] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0242] Ti is the mean tumor volume in the treatment group at day i. T0 is the mean tumor volume in the treatment group at day zero. Vi is the mean tumor volume in the control group at day i. V0 is the mean tumor volume in the control group at day zero.

[0243] In some embodiments, the antigen binding protein construct (e.g., bispecific antibody) 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.

[0244] In some embodiments, antigen binding protein constructs (e.g., bispecific antibodies) do not have a functional Fc region. For example, protein constructs are Fab, Fab', F(ab')2, and Fv fragments. In some embodiments, protein constructs as described herein have an Fc region without effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has a LALA mutation (L234A and L235A mutations of EU numbering), or a LALA-PG mutation (L234A, L235A, P329G mutations of EU numbering).

[0245] 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 homodimeric fusion protein thus produced may have any increased in vitro and / or in vivo half-life.

[0246] In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0247] In some embodiments, an Fc region is provided, which has a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such Fc region components can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297, as measured by MALDI-TOF mass spectrometry (e.g., as described in WO2008 / 077546). 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 the Fc region sequence, Asn297 may also be located about ±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 the asparagine at position 297 with alanine (N297A).

[0248] 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 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the protein complex described herein.

[0249] In some embodiments, the ADC described herein has an IC50 for killing cancer cells (e.g., lung cancer cell line NCI-H1975) in vitro of less than 2 μg / mL, less than 1.5 μg / mL, less than 1 μg / mL, less than 0.9 μg / mL, less than 0.8 μg / mL, less than 0.7 μg / mL, less than 0.6 μg / mL, less than 0.5 μg / mL, less than 0.4 μg / mL, less than 0.3 μg / mL, less than 0.2 μg / mL, or less than 0.1 μg / mL.

[0250] In some embodiments, the bispecific antibodies described herein have a higher endocytosis rate than the corresponding monoclonal antibodies and / or control bispecific antibodies described herein. In some embodiments, the anti-EGFR antibodies described herein have a higher endocytosis rate than cetuximab analogs. In some embodiments, the anti-MET antibodies described herein have a higher endocytosis rate than teratuzumab analogs. In some embodiments, the bispecific antibodies described herein have a higher endocytosis rate than ervantuzumab analogs.

[0251] Methods for preparing antigen binding protein constructs

[0252] Can use separated human protein fragment as immunogen, use the standard technology for polyclonal and monoclonal antibody preparation to produce antibody.Polyclonal antibody can be produced in animal by multiple injection (for example, subcutaneous or intraperitoneal injection) antigen peptide or protein.In some embodiments, antigen peptide or protein is injected together with at least one adjuvant.In some embodiments, antigen peptide or protein can be conjugated to the medicament with immunogenicity in the species to be immunized.Animal can be injected with more than one (for example twice, three times or four times) antigen peptide or protein.

[0253] Full-length polypeptides or proteins, or alternatively antigenic peptide fragments thereof, can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein, and encompasses the epitope of the protein, such that antibodies generated against the peptide form specific immune complexes with the protein.

[0254] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed or chemically synthesized polypeptides. The preparations may also include an adjuvant (such as Freund's complete or incomplete adjuvant) or a similar immunostimulant.

[0255] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide or its antigenic peptide (e.g., a portion of a protein) as an immunogen. Antibody titers in immunized subjects can be monitored over time using immobilized polypeptides or peptides by standard techniques, such as enzyme-linked immunosorbent assay (ELISA). If necessary, antibody molecules can be isolated from mammals (e.g., from blood) and further purified by well-known techniques (such as protein A or protein G chromatography) to obtain IgG fractions. At an appropriate time after immunization, for example, when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature, Vol. 256, pp. 495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today, Vol. 4, p. 72, 1983), EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., pp. 77-96, 1985) or trioma technique. The techniques for producing hybridomas are well known (see generally "Current Protocols in Immunology", 1994, edited by Coligan et al., John Wiley & Sons, New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest (eg, using a standard ELISA assay).

[0256] Variants of antigen-binding protein constructs described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding people, humanized or chimeric antibodies or their antigen-binding fragments described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence constituting the antigen-binding site of an antibody or antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions and / or substitutions can be performed to obtain antibodies or their antigen-binding fragments with increased binding affinity for the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell), or introducing new glycosylation sites.

[0257] The antibodies disclosed herein can be derived from animals of any species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits, including transgenic rodents genetically engineered to produce human antibodies).

[0258] Phage display (panning) can be used to optimize antibody sequences with desired binding affinity. In this technology, genes encoding single-chain Fv (including VH or VL) can be inserted into phage coat protein genes, so that the phage "displays" scFv on its outside, and contains the gene of the protein inside, thereby generating a connection between genotype and phenotype. These displayed phages can then be screened for target antigens to detect the interaction between the displayed antigen binding site and the target antigen. Therefore, a large number of protein libraries can be screened and amplified in a process called in vitro selection, and antibody sequences with desired binding affinity can be obtained.

[0259] Human and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having the same amino acid sequence as antibodies derived from such human germline immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), such as in the CDRs.

[0260] Humanized antibodies generally have a human framework (FR) grafted with non-human CDRs. Therefore, humanized antibodies have one or more amino acid sequences introduced therein from non-human sources. Therefore, "humanized" antibodies are chimeric antibodies in which a small portion of a complete human V domain is replaced by a corresponding sequence from a non-human species. In fact, humanized antibodies are generally mouse antibodies in which some CDR residues and some FR residues are replaced by residues from similar sites in human antibodies.

[0261] More importantly, antibody retains high specificity and affinity to antigen and other favorable biological properties while humanization.In order to achieve this goal, humanized antibodies can be prepared by using the three-dimensional model analysis of parental sequence and humanized sequence and the method for various conceptual humanized products.Three-dimensional immunoglobulin model is that those skilled in the art are usually available and familiar.The computer program of the possible three-dimensional conformation structure of the candidate immunoglobulin sequence of explanation and display selection can be obtained.The inspection of these displays allows the possible effect of analysis residues in the function of candidate immunoglobulin sequence, i.e., analyzes the residues that affect the ability of candidate immunoglobulin to combine its antigen.By this method, can select and combine FR residues from receptor and import sequence, so as to obtain desired antibody characteristics, such as the affinity to target antigen increased.

[0262] In some embodiments, a mouse (e.g., RenMab) having a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used. TM Mouse) produces antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains antibody heavy chain genes. The locus may include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes. The κ chain immunoglobulin locus is a region on the chromosome that contains genes encoding antibody light chains (κ chains). The κ chain immunoglobulin locus may include, for example, human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. About RenMab TM For a detailed description of mice, see PCT / CN2020 / 075698 or US20200390073A1, the entire text of which is incorporated herein by reference.

[0263] In some embodiments, mice (e.g., RenLite TM Mouse) produces antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains antibody heavy chain genes. The locus may include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes. The κ chain immunoglobulin locus is a region on the chromosome that contains genes encoding common light chains. The κ chain immunoglobulin locus may include, for example, human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. About RenLite TM A detailed description of mice can be found in PCT / CN2021 / 097652, the entire text of which is incorporated herein by reference.

[0264] Identity or homology relative to a native sequence is typically the percentage of amino acid residues present in a candidate sequence that are identical to the sequence present in a human, humanized or chimeric antibody or fragment, after aligning the candidate sequence with the native sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0265] In some embodiments, antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) may be covalently modified. These covalent modifications may be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with the selected side chain or N-terminal or C-terminal residues.

[0266] In some embodiments, antibody variants are provided, which have a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297, as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). 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 at 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 antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0267] In some embodiments, to promote production efficiency by avoiding Fab arm exchange, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). For a detailed description of the S228 mutation, see, for example, Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.", Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated herein by reference in its entirety.

[0268] In some embodiments, the methods described herein are designed to prepare bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules so that the percentage of heterodimers recovered from recombinant cell culture is maximized. For example, the interface may include at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (for example, tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), a compensation "cavity" identical or similar to the larger side chain size is produced on the interface of the second antibody molecule. This provides a mechanism that can make the yield of heterodimers higher than other unwanted end products (such as homodimers). The method is described in, for example, WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0269] In some embodiments, the knob-into-hole (KIH) technology can be used, which involves engineering the CH3 domain to produce a "knob" or "hole" in each heavy chain to promote heterodimerization. The KIH technology is described in, for example, 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 herein by reference in its entirety. In some embodiments, one heavy chain has T366W and / or S354C (knob) substitutions (EU numbering), while the other heavy chain has Y349C, T366S, L368A and / or Y407V (hole) substitutions (EU numbering). In some embodiments, one heavy chain has one or more of the following substitutions: Y349C and T366W (EU numbering). The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A and Y407V (EU numbering). In addition, substitutions may be introduced in the hinge region of both substituted IgGs (-ppcpScp-->-ppcpPcp-).

[0270] Bispecific antibodies may also include, for example, cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate may be coupled to avidin and the other antibody may be coupled to biotin. Heteroconjugate antibodies may also be prepared using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

[0271] Methods for producing bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science, Vol. 229: p. 81, 1985) described a method in which an intact antibody is proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then converted into a Fab'thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'TNB derivative to form a bispecific antibody.

[0272] Recombinant vector

[0273] 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 these recombinant vectors are introduced (i.e., such that these host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant technology.

[0274] 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 the host cell. "Expression vector" can deliver one or more polynucleotides of interest in a host cell into which the expression vector has been introduced and express them as encoded polypeptides. Therefore, in an expression vector, by being operably connected to a regulatory element (such as a promoter, enhancer and / or poly-A tail) at or near or on both sides of the integration site of the polynucleotide of interest in the vector or in the genome of the host cell, the polynucleotide of interest is positioned to be expressed in the vector so that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

[0275] 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). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0276] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation typically occurs only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA, Vol. 86: 317-321; Flexner et al., 1989, Ann. NY Acad Sci., Vol. 569: 86-103; Flexner et al., 1990, Vaccine, Vol. 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, Vol. 6: pp. 616-627, 1988; Rosenfeld et al., 1991, Science, Vol. 252: pp. 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, Vol. 91: pp. 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 11498-11502; Guzman et al., 1993, Circulation, Vol. 88: pp. 2838-2848; and Guzman et al., 1993, Cir. Res., Vol. 73: pp. 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, Vol. 259: pp. 1745-1749, and Cohen, 1993, Science, Vol. 259: pp. 1691-1692. The uptake of naked DNA may be increased by coating the DNA on biodegradable beads that are efficiently transported into cells.

[0277] For expression, the DNA insert comprising the polynucleotide encoding the antibody or polypeptide disclosed herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, E. coli (E. coli) lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, etc. Other suitable promoters are known to those skilled in the art. The expression construct may also contain sites for transcription initiation and termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon at the start of the polypeptide to be translated and a termination codon (UAA, UGA or UAG) at an appropriate position at the end of the polypeptide to be translated.

[0278] As described above, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes 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, Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as fruit fly S2 and fall armyworm Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0279] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0280] Suitable non-limiting bacterial promoters include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoters of retroviral LTR (such as the promoter of Rous sarcoma virus (RSV)) and metallothionein promoters (such as mouse metallothionein-I promoter).

[0281] In Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters are available, such as alpha factor, alcohol oxidase, and PGH. For reviews, see Ausubel et al. (1989), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, NY, and Grant et al., "Methods Enzymol.", Vol. 153: 516-544 (1997).

[0282] The construct is introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., "Basic Methods In Molecular Biology" (1986), which is incorporated herein by reference in its entirety.

[0283] The transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 bp to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the late side of the replication origin between base pairs 100 and 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

[0284] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space of the cell or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. These signals can be endogenous signals of the polypeptide or heterologous signals.

[0285] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., a GST fusion protein) or with a histidine tag, and can include not only a secretion signal, but also additional heterologous functional regions. For example, a region of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the polypeptide to improve stability and durability in a host cell, during purification, or during subsequent processing and storage. In addition, peptide moieties can be added to the polypeptide to facilitate purification. These regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to a polypeptide to cause secretion or excretion, improve stability, and facilitate purification, etc., is a conventional technique well known in the art.

[0286] The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any of the nucleotide sequences described herein. The present invention also provides the present invention relates to any amino acid sequence as described herein, and amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any amino acid sequence as described herein.

[0287] The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the nucleotide sequences described herein. The present invention also provides the present invention relates to amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any amino acid sequence as described herein.

[0288] In some embodiments, the disclosure relates to a nucleotide sequence encoding any peptide described herein, or 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.

[0289] 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.

[0290] 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.

[0291] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences are compared for the purpose of optimal comparison (e.g., a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal comparison, and non-homologous sequences can be ignored for comparison purposes). 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 same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). Taking into account the number of gaps that need to be introduced to achieve the optimal comparison of the two sequences and the length of each gap, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For example, the comparison of sequences and the determination of the percent identity between the two sequences can be completed using the Blossum62 scoring matrix, wherein the gap penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5.

[0292] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine the percentage of sequence homology is known in the art. In some embodiments, conservative amino acid residues (homology percentage) with similar physicochemical properties, such as leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology is higher than the percent identity.

[0293] The present disclosure provides one or more nucleic acids encoding any one of the polypeptides as described herein. In some embodiments, nucleic acids (e.g., cDNA) 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.

[0294] In some embodiments, the vector may have two of the nucleic acids as described herein, wherein the vector encodes a VL region and a VH region that jointly bind to EGFR. In some embodiments, a pair of vectors is provided, wherein each vector includes one of the nucleic acids as described herein, wherein the pair of vectors jointly encodes a VL region and a VH region that jointly bind to EGFR.

[0295] In some embodiments, the vector comprises two of the nucleic acids as described herein, wherein the vector encodes a VL region and a VH region that jointly bind MET. 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 jointly encodes a VL region and a VH region that jointly bind MET.

[0296] The vector can also be constructed to express specific antibodies or polypeptides. In some embodiments, the vector can be constructed to co-express anti-EGFR antibody light chain (EGFR-K) and heavy chain (EGFR-H). In some embodiments, the vector may contain sequences of cytomegalovirus promoter (CMV), EGFR-K, polyadenylation (PolyA), CMV, EGFR-H, PolyA, simian vacuolating virus 40 terminator (SV40), and glutamine synthetase marker (GS) from the 5' end to the 3' end. In some embodiments, the vector can be constructed to co-express anti-MET antibody light chain (MET-K) and anti-MET antibody heavy chain (MET-H). In some embodiments, the vector may contain sequences of CMV, MET-K, PolyA, CMV, MET-H, SV40, and GS from the 5' end to the 3' end. In some embodiments, the vector can be constructed to express anti-MET antibody scFv polypeptide chain. In some embodiments, a first vector expressing an antibody heavy chain (e.g., any heavy chain described herein) and a second vector expressing an antibody light chain (e.g., any light chain described herein) are used to co-transfect cells (e.g., CHO cells) to produce a monoclonal antibody or antigen-binding fragment thereof described herein. In some embodiments, a first vector expressing an anti-EGFR antibody heavy chain (e.g., any anti-EGFR antibody heavy chain described herein), a second vector expressing an anti-MET antibody heavy chain (e.g., any anti-MET antibody heavy chain described herein) and a third vector expressing a common light chain (e.g., any common light chain described herein) are used to co-transfect cells (e.g., CHO cells) to produce an antigen-binding protein construct described herein (e.g., any anti-EGFR / MET bispecific antibody described herein).

[0297] Treatment

[0298] The methods described herein include methods for treating a disorder associated with cancer. Typically, these methods include administering a therapeutically effective amount of an antigen-binding protein construct (e.g., a bispecific antibody) as described herein to a subject in need or determined to be in need of such treatment.

[0299] As used herein, "treating" means improving at least one symptom of a condition associated with cancer. Typically, cancer leads to death; therefore, treatment can increase life expectancy (e.g., at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years). Administration of a therapeutically effective amount of an agent described herein to treat a condition associated with cancer will reduce the number of cancer cells and / or alleviate symptoms.

[0300] As used herein, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid 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 their histopathological type or invasion stage. As used herein, the term "tumor" refers to a cancer cell, such as a cancer cell mass. Cancers that can be treated or diagnosed using the methods described herein include malignancies of various organ systems, such as malignancies affecting the lungs, breasts, thyroid, lymph, gastrointestinal tract, and urogenital tracts; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and esophageal cancers. In some embodiments, the agents described herein are designed to treat or diagnose a subject's carcinoma. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory carcinoma, gastrointestinal carcinoma, genitourinary carcinoma, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine carcinoma, and melanoma. In some embodiments, the cancer is a renal carcinoma or a melanoma. Exemplary carcinomas include carcinomas formed by tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, for example, it includes a malignant tumor composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or 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.

[0301] In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0302] In one aspect, the present disclosure also provides a method for treating cancer in a subject, a method for reducing the rate of increase of tumor volume over time in a subject, a method for reducing the risk of forming metastases, or a method for reducing the risk of forming additional metastases in a subject. In some embodiments, treatment may terminate, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment may reduce the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0303] In one aspect, the disclosure features a method comprising administering a therapeutically effective amount of an antigen binding protein construct (e.g., a bispecific antibody) or an antibody drug conjugate disclosed herein to a subject in need thereof (e.g., a subject having, identified or diagnosed as having, a cancer, such as a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung carcinoma), gastric cancer (e.g., gastric carcinoma), skin cancer (e.g., skin carcinoma), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal carcinoma, ampullary carcinoma, or brain cancer.

[0304] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe an animal (human or non-human) for which treatment is provided according to the method of the present invention. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adults or adolescents (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, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic animals, farm animals, and zoo animals.

[0305] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer.Cancer patients can be identified using various methods known in the art.

[0306] As used herein, "effective amount" means an amount or dosage sufficient to achieve beneficial or desired results, including stopping, slowing, delaying or inhibiting the progression of a disease (e.g., cancer). The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody drug conjugate, polynucleotide encoding the 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 therefore administration can be determined on an individual basis.

[0307] The effective amount can be administered once or multiple times.For example, the effective amount of an antibody, an Fab or an antibody drug conjugate is enough to improve, terminate, stabilize, reverse, inhibit, slow down and / or delay the progress of the patient's autoimmune disease or cancer, or is enough to improve, terminate, stabilize, reverse, slow down and / or delay the amount of cells (e.g., biopsy cells, any one of the cancer cells described herein or cell line (e.g., cancer cell line)) in vitro propagation.As understood in the art, the effective amount of an antibody, an Fab or an antibody drug conjugate can vary according to, especially the patient's medical history and other factors (such as the type (and / or dosage) of the antibody used).

[0308] The effective amount and administration regimen of the antigen-binding protein constructs, polynucleotides encoding antibodies, antibody drug conjugates and / or compositions disclosed herein can be determined empirically, and making such determinations is within the technical scope of the art. It will be understood by those skilled in the art that the dosage that must be administered will vary according to, for example, the mammal to which the antibodies, polynucleotides encoding antibodies, antibody drug conjugates and / or compositions disclosed herein will be received, the route of administration, the specific type of antigen-binding protein constructs, polynucleotides encoding antibodies, antigen-binding fragments, antibody drug conjugates and / or compositions disclosed herein used, and other drugs administered to mammals. Guidance for selecting the appropriate dosage of antibodies or antigen-binding fragments can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, such as "Handbook of Monoclonal Antibodies", edited by Ferrone et al., Noges Publications, Park Ridge, NJ, 1985, Chapter 22, pages 303-357; Smith et al., "Antibodies in Human Diagnosis and Therapy", edited by Haber et al., Raven Press, New York, 1977, pages 365-389.

[0309] The typical daily dose of an effective amount of an antibody, an antigen-binding fragment thereof, or an antigen-binding protein construct (e.g., a bispecific antibody) 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 dose is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0310] In any of the methods described herein, at least one antigen-binding protein construct (e.g., bispecific antibody), antibody drug conjugate or pharmaceutical composition (e.g., any of the protein constructs, antigen-binding fragments, antibody drug conjugates or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered with the same composition (e.g., liquid composition). In some embodiments, at least one protein construct, its antigen-binding fragment, antigen-binding protein construct (e.g., bispecific antibody) or antibody drug conjugate and at least one additional therapeutic agent are administered with the same composition (e.g., liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered with two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered in the form of a pill, tablet or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0311] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is overlap in the biologically active period of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) in the subject.

[0312] In some embodiments, at least one protein construct, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the protein constructs, antigen-binding antibody fragments, or pharmaceutical compositions described herein) may be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to observe 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 antibody or antigen-binding antibody fragment, antibody drug conjugate (and / or one or more additional therapeutic agents) administered to a subject, and can also adjust (e.g., increase or decrease) the dosage or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0313] In some embodiments, one or more additional therapeutic agents may be administered to a subject. The additional therapeutic agent may include one or more inhibitors, and the one or more inhibitors are selected from B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0314] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors that activate the hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0315] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, enib), Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0316] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapeutic agents targeting CX3CL1, therapeutic agents targeting CXCL9, therapeutic agents targeting CXCL10, therapeutic agents targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0317] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0318] 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.

[0319] Pharmaceutical compositions and routes of administration

[0320] Also provided herein are pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antigen-binding protein constructs, antibodies (e.g., bispecific antibodies), antigen-binding fragments, or antibody drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody drug conjugates described herein may be present in any combination in the pharmaceutical composition. The pharmaceutical composition may be formulated in any manner known in the art.

[0321] The pharmaceutical composition is formulated to be compatible with their expected route of administration (for example, intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (for example, sterile water or saline), fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetate, citrate or phosphate) and isotonic agents (such as sugar (for example, glucose), polyols (for example, mannitol or sorbitol) or salt (for example, sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent number 4,522,811). The preparation of the composition may be formulated and encapsulated in an ampoule, a disposable syringe or a multi-dose bottle. When necessary (as, for example, in injectable formulations), suitable fluidity can be maintained by, for example, using a coating (such as lecithin) or a surfactant. Absorption of the antibody or its antigen-binding fragment 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 and Nova Pharmaceuticals).

[0322] Compositions containing one or more of any of the antigen binding protein constructs, antibodies, antigen binding fragments, 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 predetermined quantities of active compound to facilitate administration and uniformity of dosage).

[0323] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical methods in cell culture or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. When an agent exhibits undesirable side effects, care should be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical methods.

[0324] The data obtained from cell culture assays and animal studies can be used to formulate any given agent for a subject (e.g., a person) of an appropriate dose. The therapeutically effective amount of one or more (e.g., one, two, three, or four) antigen-binding protein constructs, antibodies, or antigen-binding fragments thereof (e.g., any one of the antibodies or antibody fragments described herein) will be a treatment subject (e.g., a human subject identified as having cancer) or a subject identified as having a risk of illness (e.g., a subject previously suffering from cancer but now cured) disease (e.g., killing cancer cells), reducing the severity, frequency, and / or duration of one or more symptoms of the subject's (e.g., a person) disease. The effectiveness and dosage of any one of the antigen-binding protein constructs, antibodies, or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more symptoms of the subject's (e.g., a person) disease. Certain factors can affect the dosage and timing required for the effective treatment of a subject (e.g., the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and the presence of other diseases).

[0325] Exemplary dosages include milligram or microgram amounts of any of the antigen-binding protein constructs, antibodies or antigen-binding fragments or antibody drug conjugates 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). Although these dosages cover a wide range, it will be understood by those of ordinary skill in the art that the efficacy of therapeutic agents (including antigen-binding protein constructs, antibodies and antigen-binding fragments thereof) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, after which the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the development phase) can gradually increase the dose until an appropriate response is obtained. Furthermore, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion and the half-life of the antibody or antibody fragment in vivo.

[0326] The pharmaceutical composition may be contained in a container, pack or dispenser together with instructions for administration. The present disclosure also provides methods for preparing the anti-EGFR / MET antigen binding protein constructs or antibody drug conjugates as described herein for various uses.

[0327] Example

[0328] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0329] Example 1. Preparation and analysis of anti-EGFR / MET bispecific antibodies

[0330] Provided herein are bispecific antigen binding molecules targeting EGFR and MET. These antigen binding molecules are hereinafter referred to as anti-EGFR / MET bispecific antibodies.

[0331] Preparation of anti-EGFR / MET bispecific antibodies

[0332] Anti-EGFR antibodies (E-1G11, VH SEQ ID NO: 28, VL SEQ ID NO: 32 and E-6C4, VH SEQ ID NO: 29, VL SEQ ID NO: 32) and anti-MET antibodies (M-2F11, VH SEQ ID NO: 30, VL SEQ ID NO: 32 and M-2G10, VH SEQ ID NO: 31, VL SEQ ID NO: 32) can be paired to form various bispecific antibodies. Vectors encoding antibody light chains and heavy chains were constructed. CHO-S cells were co-transfected with three vectors, including a first vector encoding an anti-EGFR antibody heavy chain, a second vector encoding an anti-MET antibody 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.

[0333] Various methods can be used to reduce the probability of mispairing between the two heavy chains. For example, a knob-in-hole mutation is introduced into the Fc region of the anti-EGFR arm heavy chain and the anti-MET arm heavy chain. Exemplary bispecific antibodies obtained include: E-1G11-M-2F11, E-6C4-M-2F11, and E-6C4-M-2G10. In order to verify the binding affinity of the bispecific antibodies, anti-EGFR or anti-MET control bispecific antibodies are also generated, wherein one arm of the control bispecific antibody recognizes EGFR or MET and the other arm recognizes CD28. These control bispecific antibodies are generated using similar methods, for example by pairing RenLite TM Mice were immunized to obtain VH sequences. Exemplary control bispecific antibodies were named E-1G11-CD28, E-6C4-CD28, CD28-M-2G10, and CD28-M-2F11.

[0334] Knob-hole mutations were introduced into all bispecific antibodies. For example, in E-1G11-M-2F11, the heavy chain constant region of E-1G11 includes a knob mutation, and the heavy chain constant region of M-2F11 includes a hole mutation. In E-6C4-M-2F11, the heavy chain constant region of E-6C4 includes a knob mutation, and the heavy chain constant region of M-2F11 includes a hole mutation. Figure 1 Exemplary antibody structures are shown in , where target 1 and target 2 can be EGFR and MET, respectively; MET and EGFR, respectively; EGFR and CD28, respectively; or CD28 and MET, respectively.

[0335] 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: 35, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.

[0336] Internalization of antibodies targeting EGFR and MET

[0337] The anti-EGFR / MET bispecific antibody and pHAb-goat anti-human IgG secondary antibody were added to NCI-H1975 cells (ATCC, reference number: CRL-5908), HCC827 cells (ATCC, reference number: CRL-2868) and NCI-H292 cells (ATCC, reference number: CRL-1848), respectively, and incubated for 1 hour. The cells were centrifuged and washed with FACS buffer. MFI was measured using a flow cytometer. The endocytosis rate of the antibody was calculated. For isotype control (ISO), antibodies targeting unrelated target proteins were used. The results are shown in the table below.

[0338] Table 1

[0339]

[0340] The results showed that the bispecific antibody E-6C4-M-2F11 had a higher endocytosis rate than E-6C4-M-2G10. The two bispecific antibodies also showed a higher endocytosis rate than the corresponding monoclonal antibodies M-2F11 or M-2G10.

[0341] Purity analysis of antibodies

[0342] The purified anti-EGFR / MET bispecific antibody was analyzed by non-reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and SEC-UPLC (size exclusion chromatography ultra-performance liquid chromatography).

[0343] Non-reducing SDS-PAGE was performed using 4%-12% acrylamide gel. 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 number: LC2676) and 3.6 μL distilled water. The mixture was then boiled for 2 minutes and centrifuged immediately before loading. 4 μg of each sample was loaded onto the gel.

[0344] In the SEC-UPLC method, antibody samples were diluted to 1 mg / mL with purified water and analyzed 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 μL; sample plate temperature: 6°C; run time: 7 minutes. The results are summarized in the table below.

[0345] Table 2

[0346]

[0347] Binding activity of anti-EGFR / MET bispecific antibodies

[0348] A Biacore was used with a pre-immobilized protein A sensor chip. TM (Biacore, Piscataway, NJ) 8K biosensor, the binding activity of the anti-EGFR / MET bispecific antibody to human EGFR, human MET, monkey EGFR, and monkey MET was verified by surface plasmon resonance (SPR).

[0349] Specifically, hEGFR-His (ACROBiosystems, catalog number: EGR-H5222), hMET-His (ACROBiosystems, catalog number: MET-H5227), fasEGFR-His (ACROBiosystems, catalog number: EGR-C52H5), and fasMET-His (Sino Biological, catalog number: 90304-C08H) were diluted to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 6.25 nM, 3.125 nM, and 1.5626 nM with 1×HBS-EP+ buffer (pH 7.4), and then injected into the Biacore at 10 μL / min. TM 8K biosensor for about 50 seconds to obtain the desired protein density (e.g., about 100 response units (RU)). Then, a purified antibody at a concentration of 1 μg / mL with 1×HBS-EP+ buffer (pH 7.4) was injected at 10 μL / min for 50 seconds. Dissociation was monitored for 400 seconds. After the last injection of each round of titration, the chip was regenerated with glycine solution (pH 1.5) at a rate of 30 μL / min for 30 seconds.

[0350] By using Biacore TM8K 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, Vol. 6, pp. 99-110), thereby simultaneously obtaining the kinetic association rate (kon) and dissociation rate (koff). Affinity is derived from the quotient of the kinetic rate constant (KD = koff / kon).

[0351] As will be appreciated by one of ordinary skill in the art, the same approach with appropriate adjustment of parameters (eg, antibody concentration) was employed for each antibody tested. The results for the antibodies tested are summarized in the table below.

[0352] Table 3

[0353]

[0354]

[0355] Cetuximab is a chimeric monoclonal IgG1 antibody targeting EGFR that was originally developed by ImClone Systems and first marketed in Switzerland in 2003 as Erbitux. TM was launched by Merck KGaA under the name of , as a monotherapy in combination with irinotecan for the treatment of irinotecan-resistant metastatic colorectal cancer, and its heavy and light chain sequences are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively.

[0356] Terituzumab is a humanized IgG1 monoclonal antibody targeting MET that is in early clinical development at AbbVie for the treatment of advanced solid tumors with MET gene amplification, and its heavy chain and light chain sequences are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0357] The results showed that the anti-EGFR / MET bispecific antibodies E-6C4-M-2F11 and E-6C4-M-2G10 could bind to human EGFR, human MET, monkey EGFR and monkey MET.

[0358] The binding activity of the anti-EGFR / MET bispecific antibody E-6C4-M-2F11 to other human EGF family proteins was also confirmed, and the results showed that E-6C4-M-2F11 could not bind to human HER2, HER3 and HER4 (data not shown).

[0359] Stability of anti-EGFR / MET bispecific antibodies

[0360] A pH 6.0 buffer (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL 80) The anti-EGFR / MET bispecific antibodies E-1G11-M-2F11, E-6C4-M-2F11 and E-6C4-M-2G10 were diluted to 5 mg / mL. The diluted antibodies were placed in sealed Eppendorf tubes and stored at 4°C±3°C (hereinafter referred to as 4°C) for 7 days; or stored 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 frozen at -80°C and then thawed at room temperature. The freeze-thaw experiment was repeated 10 times (5 days), and the antibody samples were detected after the last thawing at room temperature. The bispecific antibodies were also incubated under low pH conditions. Specifically, the antibodies were incubated in 1 mol / L acetic acid at pH 3.5 for 0 hours or 6 hours.

[0361] After completing the above treatments, the following analyses were performed: (1) observing the appearance of the solution and the presence of visible insoluble objects; (2) detecting changes in the purity of the antibody by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area to the total area of ​​all peaks (purity, %)); (3) detecting changes 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 changes in the purity of the antibody 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 total area of ​​all peaks (purity, %)); (5) detecting charge variations in the antibody by capillary isoelectric focusing (cIEF) (expressed as the percentage of the main component, acidic component, and basic component).

[0362] In the SEC-UPLC experiment, the antibody samples were diluted to 1 mg / mL with purified water and analyzed using an Agilent 1290 chromatography system (with XBridge TM Protein BEH SEC Column ( The following parameters were used: mobile phase: 100 mmol / L phosphate buffer (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 μL; sample plate temperature: about 6°C; running time: 7 minutes.

[0363] In the HIC-HPLC experiments, an Agilent 1260 chromatography system (with ProPac TMHIC-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: 100% A at 0 min, 100% A at 2 min, 100% B at 32 min, 100% B at 34 min, 100% A at 35 min, and 100% A at 45 min; column temperature: 30° C.; detection wavelength: 280 nm; injection volume: 10 μL; sample plate temperature: approximately 6° C.; run time: 45 min.

[0364] In the cIEF experiment, the Maurice cIEF method development kit (Protein Simple, catalog number: PS-MDK01-C) was used for sample preparation. Specifically, 40 μg of protein sample was mixed with the following reagents in the kit: 1 μL Maurice cIEF pI marker-4.05, 1 μL Maurice cIEF pI marker-9.99, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500mM arginine, 4 μL ampholytes (Pharmalyte pH range 3-10) and water (added to make the final volume 100 μL). On the Maurice analyzer (Protein Simple, Santa Clara, California), the imaging capillary isoelectric focusing spectra were generated using the Maurice cIEF box (PS-MC02-C). The sample was 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 280nm.

[0365] 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). 54 μL sample buffer, 6 μL antibody sample, 2.4 μL 25× internal standard, 3 μL 250nM iodoacetamide (SIGMA, catalog number: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3000 rpm 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 10000 rpm for 3 minutes. The supernatant sample preparation was then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.

[0366] The detailed results of the anti-EGFR / MET bispecific antibodies are shown in the table below. The results showed that E-6C4-M-2F11 and E-6C4-M-2G10 had better stability and physical and chemical properties than the other tested antibodies.

[0367] Table 4

[0368]

[0369]

[0370] Example 2. Antibody Drug Conjugates

[0371] After protein A purification, the bispecific antibodies E-1G11-M-2F11, E-6C4-M-2F11 and E-6C4-M-2G10 were dialyzed and concentrated into PBS buffer by ultrafiltration. The concentration was determined by UV absorption. These antibodies were used for subsequent antibody drug conjugation reactions.

[0372] Conjugation of antibodies to drug molecules

[0373] Each purified antibody was conjugated to MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.

[0374] For the names of antibody drug conjugates, add "ADC" directly after the antibody name. For example, if E-1G11-M-2F11 is conjugated with MMAE, it will be named E-1G11-M-2F11-ADC.

[0375] 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 μL; sample plate temperature: about 6° C.; run time: 45 min.

[0376] For isotype control, human IgG1 isotype control was coupled with MMAE to form isotype-ADC (ISO-ADC). The results of HIC-HPLC detection are shown in the following table. The results show that the drug-antibody ratio (DAR) of ADC is about 4. The average DAR is determined by multiplying the PA% (PA% is the peak area percentage of the area measured under the 280nm peak) by the corresponding drug loading of 0, 2, 4, 6 or 8 and dividing by 100. For example, the average DAR of E-1G11-M-2F11-ADC can be calculated as: [(6.69×0)+(23.45×2)+(41.60×4)+(18.60×6)+(9.67×8)] / 100=4.0.

[0377] Table 5

[0378]

[0379] In vitro killing activity

[0380] Human lung cancer cell line NCI-H1975 (5×10 3 ), and at IncuCyte (Sartorius AG, After incubation for 72 hours in S3), the killing activity was detected. The results are shown in the table below.

[0381] Table 6

[0382]

[0383]

[0384] Evantumab is a fully human bispecific antibody developed by Janssen targeting EGFR and MET. The antibody was approved in the United States and the European Union in 2021 under the name Rybrevant for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations and whose disease has progressed after platinum-based chemotherapy or chemotherapy. The heavy and light chain sequences of Evantumab are shown in SEQ ID NO: 40-43.

[0385] Some data were further analyzed, for example, the cancer cell killing results of the bispecific antibody E-6C4-M-2F11 and the corresponding ADC E-6C4-M-2F11-ADC at a concentration of 10 μg / mL are shown in Figure 2 The detailed killing results of E-6C4-M-2F11-ADC at different concentrations and different times are shown in the table below.

[0386] Table 7

[0387] 10 μg / mL 3.33 μg / mL 1.11 μg / mL 0.37 μg / mL 0.12 μg / mL 0.04 μg / mL 0.01 μg / mL 0 hours 37.36 39.91 37.09 39.32 40.52 42.14 41.63 8 hours 35.54 38.55 37.19 42.39 45.58 49.02 49.97 16 hours 31.49 33.57 34.14 39.14 46.69 56.13 60.43 24 hours 32.63 35.03 35.64 40.68 48.16 63.21 72.08 32 hours 39.29 42.54 43.09 49.66 57.32 71.08 82.00 40 hours 45.83 48.88 49.60 56.70 65.99 79.45 90.99 48 hours 48.38 51.54 51.50 61.07 70.95 86.52 95.97 56 hours 49.51 53.29 54.17 64.16 75.85 91.12 98.27 64 hours 51.34 56.09 55.99 67.57 79.84 95.07 99.25 72 hours 52.86 57.44 57.58 70.57 83.68 97.12 99.71

[0388] Note: Values ​​represent phase object confluence (%).

[0389] The above results show that both E-6C4-M-2F11 and ervantumab analogs were unable to kill NCI-H1975 at the highest concentration of 10 μg / mL. However, E-6C4-M-2F11-ADC can effectively inhibit the growth of tumor cells in a dose-dependent manner at various concentrations.

[0390] Example 3. Antitumor activity in the NCI-H1975 xenograft model

[0391] The effect of the antibodies on in vivo tumor growth was tested in a lung adenocarcinoma model. 6 NCI-H1975 cells were subcutaneously injected into B-NDG mice (Beijing Biocytogen Pharmaceuticals Co., Ltd., catalog number: B-CM-002). When the tumor volume in the mice reached about 300 mm 3 At 1 , mice were randomly divided into different groups according to tumor volume. Then mice were injected with phosphate buffered saline (PBS) or antibody by intravenous (iv) administration. The frequency of administration was once a week (2 administrations in total). The detailed information is shown in the table below.

[0392] The lengths of the long and short axes of the tumor were measured and the value was 0.5 × (long axis) × (short axis). 2 Calculate the volume of the tumor. Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100%. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero. Statistical analysis was performed using the t-test. A TGI higher than 60% indicates that tumor growth is significantly inhibited. P < 0.05 is the threshold for significant differences.

[0393] Table 8

[0394] Group Number of mice Antibody dose way frequency Total number of applications G1 5 PBS - iv QW 2 G2 5 ISO-ADC 3mg / kg iv QW 2 G3 5 E-1G11-M-2F11-ADC 3mg / kg iv QW 2 G4 5 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G5 5 E-6C4-M-2G10-ADC 3mg / kg iv QW 2 G6 5 Evantamab analogs 10mg / kg iv QW 2

[0395] The body weight of mice was also measured twice a week. On the day of grouping (day 0), the average body weight of each group was in the range of 22.0g-24.3g. At the end of the experiment (day 21), the average body weight of each group was in the range of 20.9g-24.9g. Therefore, the average body weight of each group varied in the range of 91.1%-103.7%. The results showed that the tested antibodies were well tolerated and had no significant toxicity to mice.

[0396] The following table summarizes the results of the experiment, including the tumor volume on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21); the survival rate of mice; TGI (%); and the statistical difference in tumor volume between the treatment group and the control group (P value).

[0397] Table 9

[0398]

[0399] The tumor volumes in all treatment groups (G3-G6) were smaller than those in the control groups (G1 and G2). The treatment groups had different tumor inhibitory effects. All anti-EGFR / MET bispecific antibody ADCs showed sustained and potent tumor inhibitory effects at a dose level of 3 mg / kg (G3-G5), with a TGI exceeding 100%, especially E-6C4-M-2F11-ADC (G4), which had the highest TGI of 110.5%. The TGI values ​​of all tested ADCs were higher than the TGI value of the positive control entuzumab analog at 10 mg / kg (TGI: 98.4%).

[0400] In another experiment, about 5 × 10 5 NCI-H1975 cells were injected subcutaneously into B-NDG mice to determine the antitumor activity of E-6C4-M-2F11 and E-6C4-M-2F11-ADC, and the tumors were cultured when the tumor volume reached approximately 400 mm. 3 At the same time, mice were randomly divided into a control group and different treatment groups according to tumor size. The detailed grouping and dosage are shown in the table below.

[0401] Table 10

[0402]

[0403]

[0404] During the experiment, little difference was observed between the weight of each group of mice. Tumor volume and body weight were measured twice a week. The following table summarizes the results of the experiment, including the tumor volume on the day of grouping (day 0), 12 days after grouping (day 12), and at the end of the experiment (day 22); the survival rate of mice; TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0405] Table 11

[0406]

[0407] Figure 3 The tumor volumes of mice in different groups treated with antibodies, ADCs or PBS are shown, wherein the anti-EGFR / MET bispecific antibody ADC E-6C4-M-2F11-ADC (G2 and G3) showed better tumor inhibition compared to PBS in the control group (G1) and the corresponding anti-EGFR / MET bispecific antibody E-6C4-M-2F11 in the G4 and G5 groups.

[0408] In another experiment, NCI-H1975 cells were injected subcutaneously into Balb / c nude mice to determine the antitumor activity of ADC, and when the tumor volume reached approximately 400 mm 3 When the mice were randomly divided into a control group and different treatment groups according to the tumor size. Then, the mice were injected with PBS, E-6C4-M-2F11-ADC (1.5 mg / kg, 3 mg / kg or 6 mg / kg, QW, 2 times) or ervantumab (Janssen, reference number: LFS0L03) (10 mg / kg, BIW, 4 times) by intravenous (iv) administration. The results showed that the anti-tumor activity of all three treatment groups of E-6C4-M-2F11-ADC (TGI of 97.5%, 110.3% and 110.8%, respectively) was stronger than that of ervantumab at a dose of 10 mg / kg (TGI of 12.9%).

[0409] Example 4. Antitumor activity in the NCI-H292 xenograft model

[0410] The effect of the antibody on in vivo tumor growth was tested in a lung cancer model. 7 NCI-H292 cells were injected subcutaneously into B-NDG mice. When the tumor volume in the mice reached about 300 mm 3 At the time of the experiment, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS or antibody by intravenous (iv) administration. The frequency of administration was once a week (2 times in total). The detailed information is shown in the table below.

[0411] Table 12

[0412] Group Number of mice Antibody dose way frequency Total number of applications G1 5 PBS - iv QW 2 G2 5 E-6C4-M-2F11-ADC 10mg / kg iv QW 2 G3 5 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 5 E-6C4-M-2F11-ADC 1mg / kg iv QW 2 G5 5 Evantamab analogs 10mg / kg iv QW 2 G6 5 Evantamab analogs 3mg / kg iv QW 2 G7 5 Evantamab analogs 1mg / kg iv QW 2

[0413] During the experiment, little difference was observed between the body weights of mice in each group. The tumor sizes in the antibody-treated groups are shown in Figure 4 The following table summarizes the results of the 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 24); the survival rate of mice; TGI (%); and the statistical difference in tumor volume between the treatment group and the control group (P value).

[0414] Table 13

[0415]

[0416]

[0417] During the experiment, the body weight of each group of mice was maintained. The body weight of mice treated with 10 mg / kg E-6C4-M-2F11-ADC (G2) showed an increasing trend, from 21.1 g on day 0 to 21.8 g on day 24, with an average body weight change of 103.4%. At the same dose level, the anti-EGFR / MET bispecific antibody ADC showed better anti-tumor activity than the positive control ervantumab analog in a dose-dependent manner.

[0418] In another experiment, about 5 × 10 6 NCI-H292 cells were injected subcutaneously into B-NDG mice to determine the antitumor activity of E-6C4-M-2F11 and E-6C4-M-2F11-ADC, and the tumors were cultured when the tumor volume reached approximately 200 mm. 3 At the same time, mice were randomly divided into a control group and different treatment groups according to tumor size. The detailed grouping and dosage are shown in the table below.

[0419] Table 14

[0420] Group Number of mice Antibody dose way frequency Total number of applications G1 5 PBS - iv QW 2 G2 5 ISO-ADC 3mg / kg iv QW 2 G3 5 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 5 E-6C4-M-2G10-ADC 3mg / kg iv QW 2 G5 5 E-6C4-ADC 3mg / kg iv QW 2 G6 5 M-2G10-ADC 3mg / kg iv QW 2 G7 5 M-2F11-ADC 3mg / kg iv QW 2 G8 5 CD28-E-6C4-ADC 3mg / kg iv QW 2 G9 5 CD28-M-2F11-ADC 3mg / kg iv QW 2 G10 5 M-2G10-CD28-ADC 3mg / kg iv QW 2

[0421] During the experiment, little difference was observed between the weight of each group of mice. Tumor volume and body weight were measured twice a week. The following table summarizes the results of the experiment, including the tumor volume on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21); the survival rate of mice; TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0422] Table 15

[0423]

[0424] The tumor sizes in the antibody-treated groups are shown in Figure 5 The treatment groups showed different tumor inhibition effects. In summary, the anti-tumor activity of the bispecific antibody ADC was stronger than that of the monoclonal antibody ADC and also stronger than that of the control bispecific antibody ADC.

[0425] In another experiment, about 2 × 10 6 NCI-H292 cells were injected subcutaneously into Balb / c nude mice to determine the antitumor activity of ADC, and when the tumor volume reached approximately 300 mm 3 When the mice were randomly divided into a control group and different treatment groups according to the tumor size, the mice were then injected with PBS, ADC or envantab (Janssen, reference number: LFS0L03) by intravenous (iv) administration. The detailed grouping and dosage are shown in the table below.

[0426] Table 16

[0427]

[0428]

[0429] MRG003 is an antibody drug conjugate consisting of a fully human IgG1 monoclonal antibody targeting EGFR conjugated to monomethyl auristatin E (MMAE), which is in early clinical development at Shanghai Miracogen for the treatment of solid tumors, and its heavy and light chain sequences are shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.

[0430] Tumor volume and body weight were measured twice a week. The tumor sizes in the ADC-treated groups are shown in Fig.15 The results showed that the anti-tumor activity of E-6C4-M-2F11-ADC (G3) was stronger than that of the positive control group (G5-G6) at a dose of 3 mg / kg, and the anti-tumor activity of E-6C4-M-2F11-ADC (G2) at a dose of 6 mg / kg was stronger than that of 10 mg / kg of ervantumab. In addition, the bispecific antibody ADC E-6C4-M-2F11-ADC showed a dose-dependent tumor inhibition effect.

[0431] Example 5. Antitumor activity in the SNU-5 xenograft model

[0432] The effect of the antibody on in vivo tumor growth was tested in a gastric cancer model. 7SNU-5 cells were injected subcutaneously into B-NDG mice. When the tumor volume in the mice reached about 200 mm 3 At the time of the experiment, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS or antibody by intravenous (iv) administration. The frequency of administration was once a week (2 times in total). The detailed information is shown in the table below.

[0433] Table 17

[0434]

[0435]

[0436] During the experiment, little difference was observed between the body weights of mice in each group. The tumor sizes in the ADC-treated groups are shown in Figure 6 The following table summarizes the results of the experiment, including the tumor volume on the day of grouping (day 0), 10 days after grouping (day 10), and at the end of the experiment (day 24); the survival rate of mice; TGI (%); and the statistical difference in tumor volume between the treatment group and the control group (P value).

[0437] Table 18

[0438]

[0439] In the SNU-5 gastric cancer tumor model, both ADCs, namely E-6C4-M-2F11-ADC and E-6C4-M-2G10-ADC, showed strong antitumor activity at a dose level of 10 mg / kg or 3 mg / kg. Their tumor inhibition effects were greater than those of the corresponding bispecific antibodies E-6C4-M-2F11 and E-6C4-M-2G10, and greater than those of the positive control ervantumab analog.

[0440] Example 6. Antitumor activity in A431 xenograft model

[0441] The effect of the antibody on in vivo tumor growth was tested in a skin carcinoma model. Approximately 5×10 6 A431 cells (ATCC, reference number: CRL-1555) were injected subcutaneously into B-NDG mice. When the tumor volume in the mice reached about 300 mm 3 At the time of the experiment, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS or antibody by intravenous (iv) administration. The frequency of administration was once a week (2 times in total). The detailed information is shown in the table below.

[0442] Table 19

[0443] Group Number of mice Antibody dose way frequency Total number of applications G1 5 PBS 3mg / kg iv QW 2 G2 5 ISO-ADC 3mg / kg iv QW 2 G3 5 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 5 E-6C4-M-2F11 3mg / kg iv QW 2 G5 5 Evantamab analogs 3mg / kg iv QW 2

[0444] During the experiment, little difference was observed in the body weight of each group of mice. The tumor sizes in the antibody-treated groups are shown in Figure 7 Similar to the above experimental results, in the A431 skin cancer tumor model in vivo, E-6C4-M-2F11-ADC showed a stronger tumor inhibitory effect than the corresponding bispecific antibody E-6C4-M-2F11, and both had a stronger tumor inhibitory effect than the positive control ervantumab analog.

[0445] Example 7. In vivo efficacy in a human lung cancer patient-derived xenograft (PDX) model

[0446] The effect of the antibody was tested in two human lung cancer PDX models (PDX001 and PDX002). Patient-derived lung tumor fragments were immunofluorescently stained and images were analyzed by HALO version 3.2. The results showed that EGFR-positive cells and MET-positive cells in PDX001 were 24.28% and 25.71%, respectively. In PDX002, EGFR-positive cells and MET-positive cells were 12.88% and 105.93%, respectively.

[0447] In the PDX001 model, patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mouse reached approximately 250 mm 3 -300mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS, ervantuzumab analogs or E-6C4-M-2F11-ADC by intravenous (iv) administration. The detailed administration schedule is shown in the table below.

[0448] Table 20

[0449] Group Number of mice Antibody Dosage (mg / kg) way frequency Total number of applications G1 6 PBS - iv QW 2 G2 6 E-6C4-M-2F11-ADC 10mg / kg iv QW 2 G3 6 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 6 E-6C4-M-2F11-ADC 1mg / kg iv QW 2 G5 6 Evantamab analogs 10mg / kg iv QW 2 G6 6 Evantamab analogs 3mg / kg iv QW 2 G7 6 Evantamab analogs 1mg / kg iv QW 2

[0450] Tumor volume was measured twice a week and the results are shown in Table 26, which show that treatment with E-6C4-M-2F11-ADC at 10 mg / kg (G2) and 3 mg / kg (G3) produced strong tumor growth inhibition in a human lung cancer PDX model co-expressing EGFR / MET, compared with the control group (G1) and the entuzumab analog treatment group (G5, G6 and G7), with TGI% on day 23 (23 days after grouping) of 109.3% and 38.3%, respectively.

[0451] Table 21

[0452]

[0453] In the PDX002 model, when the tumor volume in mice reaches approximately 250 mm 3 -300mm 3 At the time of the experiment, B-NDG mice were divided into a control group and different treatment groups (6 mice per group) according to the tumor size. The treatment groups were randomly selected to receive E-6C4-M-2F11-ADC at 3 mg / kg (G2) and 1 mg / kg (G3), or 3 mg / kg (G4) and 1 mg / kg (G5) for the treatment of ervantumab analogs. The control group mice were injected with PBS (G1). The frequency of administration was once a week (two administrations in total).

[0454] The tumor size in each group is shown in Table 22. Compared with the control group (G1) and the entuzumab analog treatment groups (G4 and G5), treatment with 3 mg / kg of E-6C4-M-2F11-ADC (G2) showed better tumor inhibition in the human lung cancer PDX model with high MET expression.

[0455] Table 22

[0456]

[0457] Example 8. In vivo efficacy in a human pancreatic PDX model

[0458] The effect of the antibody was tested in two human pancreatic PDX models (PDX003 and PDX004). Immunofluorescence staining was performed on patient-derived pancreatic tumor fragments, and the images were analyzed by HALO version 3.2. The results showed that EGFR-positive cells and MET-positive cells in PDX003 were 7.98% and 32.44%, respectively. In PDX004, EGFR-positive cells and MET-positive cells were 54.65% and 36.63%, respectively.

[0459] In the PDX003 model, patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mouse reached approximately 300 mm 3 -400mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS, ervantuzumab analogs or E-6C4-M-2F11-ADC by intravenous (iv) administration. The detailed administration schedule is shown in the table below.

[0460] Table 23

[0461] Group Number of mice Antibody Dosage (mg / kg) way frequency Total number of applications G1 6 PBS - iv QW 2 G2 6 E-6C4-M-2F11-ADC 10mg / kg iv QW 2 G3 6 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 6 E-6C4-M-2F11-ADC 1mg / kg iv QW 2 G5 6 Evantamab analogs 10mg / kg iv QW 2 G6 6 Evantamab analogs 3mg / kg iv QW 2 G7 6 Evantamab analogs 1mg / kg iv QW 2

[0462] Tumor volume was measured twice a week and the results are shown in Table 24, which show that treatment with E-6C4-M-2F11-ADC at 10 mg / kg, 3 mg / kg and 1 mg / kg produced significant tumor growth inhibition in the human pancreatic PDX model compared to the control group, with TGI% on day 21 of 129.2%, 127.7% and 45.8%, respectively.

[0463] Table 24

[0464]

[0465] In the PDX004 model, when the tumor volume in mice reaches approximately 250 mm 3 -300mm 3 At the same time, the mice were divided into a control group and different treatment groups (6 mice in each group) according to the tumor size. The tested antibodies and the frequency of administration were similar to those in Table 28.

[0466] The results showed that the E-6C4-M-2F11-ADC treatment groups (including 1 mg / kg, 3 mg / kg and 10 mg / kg doses) all produced significant anti-tumor activity in the human pancreatic PDX model, with TGI% on day 35 (35 days after grouping) being 46.9%, 109.4% and 110.9%, respectively, which were higher than the anti-tumor activity of the entuzumab analogue treatment group (approximately -13.3%, -0.6% and 4.1%, respectively).

[0467] Example 9. Pharmacokinetic curves

[0468] The pharmacokinetic clearance rate of anti-EGFR / MET bispecific antibody ADC was determined in C57BL / 6 mice. Specifically, mice were divided into four groups (5 mice per group) and administered E-6C4-M-2F11-ADC (G1, 3 mg / kg; G2, 10 mg / kg) or E-6C4-M-2F11 (G3, 3 mg / kg; G4, 10 mg / kg) by intravenous injection. Blood samples were collected 3 days before administration and 15 minutes, 6 hours, 1 day, 2 days, 5 days, 10 days, 14 days and 21 days after administration.

[0469] Serum levels of antibodies and ADCs were determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch, catalog number: 109-005-088) or anti-MMAE mIgG (ACRO Biosystems, 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 cells were washed with PBS-T buffer (supplemented with TweenTM The plate was washed 4 times with PBS (20% PBS). The unbound area of ​​the antibody was blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. The plate was then 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 specific fragment (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. 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 establish a calibration curve with four parameters (i.e., T 1 / 2 , C max , AUC 0天-21天 The standard curve was used to calculate the antibody or ADC concentration of each serum sample. The drug concentration-time curve was established using the sample concentration calculated at each time point. TM WinNolin 8.3 was used to calculate the pharmacokinetic parameters.

[0470] The results are shown in the table below, which show that conjugation of drug molecules does not affect the in vivo pharmacokinetic clearance rate of the bispecific antibody.

[0471] Table 25

[0472]

[0473] Example 10. In vivo efficacy of ADC combinations

[0474] In another experiment, about 5 × 10 5 NCI-H1975 cells were injected subcutaneously into B-NDG mice to determine the in vivo efficacy of the combination of E-6C4-ADC and M-2F11-ADC. 3 At the same time, mice were randomly divided into a control group and different treatment groups according to tumor size. The detailed grouping and dosage are shown in the table below.

[0475] Table 26

[0476]

[0477]

[0478] During the experiment, little difference was observed between the weight of each group of mice. Tumor volume and body weight were measured twice a week. The following table summarizes the results of the experiment, including the tumor volume on the day of grouping (day 0), 14 days after grouping (day 14), 21 days after grouping (day 21), and 39 days after grouping (day 39, if applicable); the survival rate of mice; TGI (%); and the tumor volume P value between the treatment group and the control group on day 21.

[0479] Table 27

[0480]

[0481] The tumor volumes of mice in different groups treated with ADC or PBS are shown in Fig.13 Compared with the PBS group (G1), the monoclonal antibody ADC groups (E-6C4-ADC (G3 and G5) and M-2F11-ADC (G2 and G4)), the bispecific antibody ADC group (G11 and G12), and the combination group of monoclonal antibody ADC (G6, G7, and G8) all showed significant antitumor activity on day 21.

[0482] In particular, the TGI% of 1.5 mg / kg of M-2F11-ADC (G4) and E-6C4-ADC (G5) was comparable to the TGI% of the combination of 0.75 mg / kg of M-2F11-ADC and E-6C4-ADC (G8), but was lower than the TGI% of 1.5 mg / kg of E-6C4-M-2F11-ADC (G11).

[0483] Similar to the above results, the TGI% of 3 mg / kg of M-2F11-ADC (G2) and E-6C4-ADC (G3) was similar to the TGI% of the combination of 1.5 mg / kg of M-2F11-ADC and E-6C4-ADC (G7), but lower than the TGI% of 3 mg / kg of E-6C4-M-2F11-ADC (G12).

[0484] The tumor volume and survival rate of mice continued to be monitored after day 21. At the end of the experiment on day 39, all mice in the G1, G4, G5, G8, G9 and G10 groups died, while all mice in the G2, G3, G6, G7, G11 and G12 groups survived. The tumor volume results on day 39 showed that at a dose level of 3 mg / kg, E-6C4-M-2F11-ADC (G12) treatment had a better anti-tumor effect than M-2F11-ADC (G2), E-6C4-ADC (G3) or their combination (G7).

[0485] The results also showed that anti-EGFR / MET bispecific antibodies had a synergistic effect on tumor inhibition.

[0486] Example 11. In vivo efficacy in a human ampullary carcinoma PDX model

[0487] The effect of ADC was tested in a human ampullary carcinoma PDX model. Patient-derived ampullary tumor fragments were immunofluorescently stained and images were analyzed by HALO version 3.2. The results showed that EGFR-positive cells and MET-positive cells were 94.03% and 0.25%, respectively.

[0488] Patient-derived ampullary tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 250 mm 3 -300mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS, ADC or envantab (Janssen, catalog number: LFS0L03) by intravenous (iv) administration. The detailed administration scheme is shown in the table below.

[0489] Table 28

[0490] Group Number of mice Antibody Dosage (mg / kg) way frequency Total number of applications G1 6 PBS - iv QW 2 G2 6 E-6C4-M-2F11-ADC 6mg / kg iv QW 2 G3 6 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 6 E-6C4-M-2F11-ADC 1.5 mg / kg iv QW 2 G5 6 E-6C4-ADC 3mg / kg iv QW 2 G6 6 Cetuximab analogs - ADC 6mg / kg iv QW 2 G7 6 Cetuximab analogs - ADC 3mg / kg iv QW 2 G8 6 Evantamab 10mg / kg iv BIW 4

[0491] The following table summarizes the results of the experiment, including the tumor volume on the day of grouping (Day 0), 14 days after grouping (Day 14), and 21 days after grouping (Day 21); TGI (%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0492] Table 29

[0493]

[0494] The tumor volumes of mice in different groups are shown in Fig.16In. The results showed that the treatment groups showed different tumor inhibition effects compared with the control group G1. In the treatment groups, at the same dose, E-6C4-M-2F11-ADC (G2 and G3) showed the best tumor inhibition effect, followed by the corresponding anti-EGFR antibody ADC E-6C4-ADC, and finally the positive control cetuximab analog-ADC. E-6C4-M-2F11-ADC showed a dose-dependent tumor inhibition effect. In addition, the experiment continued until 39 days after grouping (day 39), and E-6C4-M-2F11-ADC (G2-G3) still showed better tumor inhibition, with TGIs of 108.6% and 83.7%, respectively, while the TGI of E-6C4-ADC (G5) was 71.0%, and the TGIs of cetuximab analog-ADC (G6-G7) were 78.7% and 57.5%, respectively.

[0495] Example 12. In vivo efficacy in human gastric cancer PDX model

[0496] Patient-derived gastric tumor fragments were immunofluorescently stained, and the images were analyzed by HALO version 3.2. The results showed that EGFR-positive cells and MET-positive cells in the gastric PDX model were 80.53% and 3.10%, respectively.

[0497] Patient-derived gastric tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mice reached approximately 250 mm 3 -300mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS, ADC or envantab (Janssen, reference number: LFS0L03) by intravenous (iv) administration. The detailed administration scheme is shown in the table below.

[0498] Table 30

[0499] Group Number of mice Antibody Dosage (mg / kg) way frequency Total number of applications G1 5 PBS - iv QW 2 G2 5 E-6C4-M-2F11-ADC 6mg / kg iv QW 2 G3 5 E-6C4-M-2F11-ADC 3mg / kg iv QW 2 G4 5 E-6C4-M-2F11-ADC 1.5 mg / kg iv QW 2 G5 5 Evantamab 10mg / kg iv BIW 6

[0500] Tumor volume was measured twice a week and the results are shown in the following table and Fig.14 These results showed that the E-6C4-M-2F11-ADC (G2-G4) treatment group produced sustained and strong tumor growth inhibition in the human gastric PDX model compared with the control group (G1), and 6 mg / kg of E-6C4-M-2F11-ADC showed better tumor inhibition than 10 mg / kg of ervantumab. Tumor volume continued to be monitored until 77 days after grouping (day 77), and E-6C4-M-2F11-ADC (G2-G4) still showed better tumor inhibition compared with ervantumab (10 mg / kg).

[0501] Table 31

[0502]

[0503] Example 13. Antibody Drug Conjugate

[0504] The purified antibody is coupled to CPT1, CPT2, CPT3 or CPT4 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, if E-6C4-M-2F11 is coupled to CPT1, it is named E-6C4-M-2F11-CPT1. For another example, if E-6C4-M-2F11 is coupled to CPT2, it is named E-6C4-M-2F11-CPT2.

[0505] HIC-HPLC is used to detect the coupling of antibodies to drug molecules. Human IgG1 molecules are coupled to CPT2 to form isotype-CPT2 (ISO-CPT2), which serves as an isotype control. The results of HIC-HPLC detection show that the drug-antibody ratio (DAR) of the ADC is about 4 or 8. Regarding the name of the ADC, if the DAR of E-6C4-M-2F11-CPT1 is 4, the ADC is named E-6C4-M-2F11-CPT1 (DAR4). If the DAR of E-6C4-M-2F11-CPT1 is 8, the ADC is named E-6C4-M-2F11-CPT1 (DAR8).

[0506] Example 14. Antitumor activity of ADC in lung cancer model

[0507] The inhibitory effect of ADC on tumor growth in vivo was tested in a lung cancer model. 6 NCI-H292 cells were injected subcutaneously into Balb / c nude mice. When the tumor volume in the mice reached about 300 mm 3 When, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS, ADC or ervantumab by intravenous (iv) administration. The frequency of administration of ADC was once a week (1 administration in total). The detailed dosing regimen, route and frequency are shown in the table below.

[0508] Table 32

[0509]

[0510]

[0511] The results showed that E-6C4-M-2F11-CPT2 of both DAR4 and DAR8 exhibited good tumor inhibitory effects.

[0512] In a similar experiment, about 2×10 6 NCI-H1975 cells were injected subcutaneously into Balb / c nude mice. When the tumor volume in 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, ADC or ivantuzumab by intravenous (iv) administration. The detailed dosing schedule, route and frequency are shown in the table below.

[0513] Table 33

[0514]

[0515]

[0516] Similar to the above results, E-6C4-M-2F11-CPT2 of DAR4 and DAR8 both achieved good tumor growth inhibition effects.

[0517] Example 15. Antitumor activity of ADC in pancreatic cancer model

[0518] Pancreatic cancer patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. When the tumor volume in the mouse reached approximately 200 mm 3 -300mm 3 At 4 pm, mice were randomly divided into different groups according to tumor volume. Then, mice were injected with PBS, ADC or ivantuzumab by intravenous (iv) administration. The detailed dosing schedule, route and frequency are shown in the table below.

[0519] Table 34

[0520] Group Number of mice Antibody dose way frequency Total number of applications G1 5 PBS - iv QW 1 G2 5 Terituzumab analogs-ADC 3mg / kg iv QW 1 G3 5 MRG003 analogs-ADC 3mg / kg iv QW 1 G4 5 ISO-CPT2(DAR4) 6mg / kg iv QW 1 G5 5 E-6C4-M-2F11-CPT2(DAR4) 1mg / kg iv QW 1 G6 5 E-6C4-M-2F11-CPT2(DAR4) 3mg / kg iv QW 1 G7 5 E-6C4-M-2F11-CPT2(DAR4) 6mg / kg iv QW 1 G8 5 ISO-CPT2(DAR8) 6mg / kg iv QW 1 G9 5 E-6C4-M-2F11-CPT2(DAR8) 1mg / kg iv QW 1 G10 5 E-6C4-M-2F11-CPT2(DAR8) 3mg / kg iv QW 1 G11 5 E-6C4-M-2F11-CPT2(DAR8) 6mg / kg iv QW 1 G12 5 Evantamab 10mg / kg iv BIW 4

[0521] The results showed that E-6C4-M-2F11-CPT2 of both DAR4 and DAR8 showed excellent efficacy in treating pancreatic cancer.

[0522] Other Implementations

[0523] It should be understood that although the invention has been described in conjunction with the specific embodiments of the invention, 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 antigen-binding protein construct, comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to MET.

2. The antigen-binding protein construct 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 antigen-binding protein construct 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 sequence, the selected VL1 CDR1, 2 and 3 amino acid sequence is one of: (1) the selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4-6, 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 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; (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 antigen-binding protein construct 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 sequence and the selected VL2 CDR1, 2 and 3 amino acid sequence are one of: (1) the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; (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; (3) the selected VH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively; and (4) The selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

5. The antigen-binding protein construct according to any one of claims 2 to 4, wherein (1) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 4-6, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively; (2) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 7-9, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively; (3) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 7-9, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 13-15, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively; (4) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 16-18, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively; (5) the selected VH1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 19-21, respectively, the selected VL1 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 22-24, respectively, and the selected VL2 CDR1, 2, 3 amino acid sequences are as shown in SEQ ID NOs: 1-3, respectively; and (6) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19-21, respectively, the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25-27, respectively, and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1-3, respectively.

6. An antigen-binding protein construct according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:28, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:

32.

7. An antigen-binding protein construct according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:30, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:

32.

8. An antigen-binding protein construct according to any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:31, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:

32.

9. The antigen binding protein construct of any one of claims 2-8, wherein the VH1 comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of: (1) the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO:29, and the selected VL sequence is SEQ ID NO:

32.

10. The antigen-binding protein construct of any one of claims 2-9, 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: (1) the selected VH sequence is SEQ ID NO: 28, and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO:29, and the selected VL sequence is SEQ ID NO:

32.

11. The antigen binding protein construct of any one of claims 2-10, 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 and the selected VL sequence are one of: (1) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO:31, and the selected VL sequence is SEQ ID NO:

32.

12. The antigen-binding protein construct of any one of claims 2-11, 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 and the selected VL sequence are one of: (1) the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 32; and (2) The selected VH sequence is SEQ ID NO:31, and the selected VL sequence is SEQ ID NO:

32.

13. The antigen binding protein construct according to any one of claims 2-12, wherein the VH1 comprises the sequence of SEQ ID NO: 28, and the VL1 comprises the sequence of SEQ ID NO:

32.

14. The antigen binding protein construct according to any one of claims 2-13, wherein the VH1 comprises the sequence of SEQ ID NO: 29, and the VL1 comprises the sequence of SEQ ID NO:

32.

15. The antigen binding protein construct of any one of claims 2-14, wherein the VH2 comprises the sequence of the SEQ ID NO:30, and the VL2 comprises the sequence of the SEQ ID NO:

32.

16. The antigen-binding protein construct of any one of claims 2-15, wherein the VH2 comprises the sequence of the SEQ ID NO:31, and the VL2 comprises the sequence of the SEQ ID NO:

32.

17. The antigen-binding protein construct according to any one of claims 1-16, wherein the first antigen-binding domain specifically binds to human EGFR or monkey EGFR; and / or the second antigen-binding domain specifically binds to human MET or monkey MET.

18. The antigen binding protein construct according to any one of claims 1 to 17, wherein the first antigen binding domain is human or humanized; and / or the second antigen binding domain is human or humanized.

19. The antigen binding protein construct according to any one of claims 1 to 18, wherein the antigen binding protein construct is a multispecific antibody (eg, a bispecific antibody).

20. The antigen binding protein construct according to any one of claims 1-19, wherein the first antigen binding domain is a single chain variable fragment (scFV); and / or the second antigen binding domain is a scFv.

21. The antigen binding protein construct of any one of claims 1-20, wherein the first light chain variable region and the second light chain variable region are identical.

22. A nucleic acid comprising a polynucleotide encoding the antigen binding protein construct according to any one of claims 1 to 21.

23. A vector comprising one or more of the nucleic acids according to claim 22.

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

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

26. A cell comprising one or more of the nucleic acids of claim 22.

27. A method for producing an antigen binding protein construct, the method comprising (a) culturing the cell according to any one of claims 24 to 26 under conditions sufficient for the cell to produce the antibody or antigen-binding fragment thereof or the antigen-binding protein construct; and (b) collecting the antibody or antigen-binding fragment thereof or the antigen-binding protein construct produced by the cell.

28. An antibody drug conjugate (ADC) comprising a therapeutic agent covalently bound to the antigen binding protein construct of any one of claims 1-21.

29. The antibody drug conjugate of claim 28, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

30. The antibody drug conjugate of claim 28 or 29, wherein the therapeutic agent is MMAE or MMAF.

31. The antibody drug conjugate of claim 28, wherein the therapeutic agent is selected from 32. The antibody drug conjugate of claim 28 or 31, wherein the therapeutic agent is linked to the antibody or antigen-binding fragment thereof or the antigen-binding protein construct via a linker.

33. The antibody drug conjugate according to any one of claims 28, 31 and 32, wherein the linker has the following structure:

34. The antibody drug conjugate according to any one of claims 28 and 31-33, wherein the antibody drug conjugate has the following structure: wherein n=1, 2, 3, 4, 5, 6, 7 or 8; wherein "Ab" represents the antibody or antigen-binding fragment thereof, or the antigen-binding protein construct.

35. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antigen binding protein construct according to any one of claims 1-21, or an antibody drug conjugate according to any one of claims 28-34.

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

37. The method of claim 35, wherein the cancer is a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung carcinoma), gastric cancer (e.g., gastric carcinoma), skin cancer (e.g., skin carcinoma), ampullary cancer (e.g., ampullary carcinoma), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, or brain cancer.

38. The method of any one of claims 35-37, wherein the subject is a human.

39. The method of any one of claims 35-38, wherein the method further comprises administering an anti-PD1 antibody to the subject.

40. The method of any one of claims 35-39, wherein the method further comprises administering chemotherapy to the subject.

41. A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising an antigen binding protein construct according to any one of claims 1-21, or an antibody drug conjugate according to any one of claims 28-34.

42. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising an antigen binding protein construct according to any one of claims 1-21, or an antibody drug conjugate according to any one of claims 28-34.

43. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) an antigen-binding protein construct according to any one of claims 1 to 21, and / or (b) The antibody drug conjugate according to any one of claims 28 to 34.

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